Autophagy and Cell Death

**87**

**Chapter 5**

**Abstract**

**1. Introduction**

phenomenon.

autophagic activities.

Autophagy

*Hai Zhang and Zhinan Chen*

Autophagy and Cell Death:

Antitumor Drugs Targeting

efforts in developing cancer therapeutics targeting autophagy.

signaling transduction pathway, antitumor drugs

Autophagy, a degradation mechanism conserved among eukaryotes, plays an important role in cellular homeostasis by maintaining nutrients and energy balance. It is not surprising that autophagy has been associated with various pathological conditions such as neurodegeneration, aging, infection, and cancer. Its roles in cancer are complex and context-dependent. In this chapter, we will give an overview of regulation of autophagy with an emphasis in cancer and summarize the recent

**Keywords:** autophagy, autophagic cell death (ACD), autophagy-related genes (ATGs),

Autophagy derives from the Greek word "auto" and "phagein," auto means "self,"

The concept was first proposed in the 1960s, when de Duve observed that intracellular components are encased in membranes to form cystic structures and transported to a small compartment, the lysosome, which is responsible for recycling to degrade these ingredients [1]. Further studies show cell components, even organelles, are transported to the lysosome for degradation by cytoplasmic vesicles. Thus, the term autophagy was used for the first time to describe this process. But not until the early 1990s, the seminal work by Yoshinori Ohsumi on *Saccharomyces cerevisiae* resolved its molecular mechanism [2]. Now it is well-known that autophagy is the mechanism which maintains cellular homeostasis; damaged organelles or misfolded proteins are digested in lysosomes through autophagy for cellular energy recycling. However, autophagy is involved in the occurrence of cancer, neurodegenerative diseases, aging, and infection under pathological status. In this chapter, we focus on the formation and biological function of autophagy in cancer and discuss the application of antitumor drugs that target autophagy-related molecules or regulate

and phagein means "eating," so autophagy refers to a "self-eating" physiological

#### **Chapter 5**

## Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy

*Hai Zhang and Zhinan Chen*

#### **Abstract**

Autophagy, a degradation mechanism conserved among eukaryotes, plays an important role in cellular homeostasis by maintaining nutrients and energy balance. It is not surprising that autophagy has been associated with various pathological conditions such as neurodegeneration, aging, infection, and cancer. Its roles in cancer are complex and context-dependent. In this chapter, we will give an overview of regulation of autophagy with an emphasis in cancer and summarize the recent efforts in developing cancer therapeutics targeting autophagy.

**Keywords:** autophagy, autophagic cell death (ACD), autophagy-related genes (ATGs), signaling transduction pathway, antitumor drugs

#### **1. Introduction**

Autophagy derives from the Greek word "auto" and "phagein," auto means "self," and phagein means "eating," so autophagy refers to a "self-eating" physiological phenomenon.

The concept was first proposed in the 1960s, when de Duve observed that intracellular components are encased in membranes to form cystic structures and transported to a small compartment, the lysosome, which is responsible for recycling to degrade these ingredients [1]. Further studies show cell components, even organelles, are transported to the lysosome for degradation by cytoplasmic vesicles. Thus, the term autophagy was used for the first time to describe this process. But not until the early 1990s, the seminal work by Yoshinori Ohsumi on *Saccharomyces cerevisiae* resolved its molecular mechanism [2]. Now it is well-known that autophagy is the mechanism which maintains cellular homeostasis; damaged organelles or misfolded proteins are digested in lysosomes through autophagy for cellular energy recycling. However, autophagy is involved in the occurrence of cancer, neurodegenerative diseases, aging, and infection under pathological status. In this chapter, we focus on the formation and biological function of autophagy in cancer and discuss the application of antitumor drugs that target autophagy-related molecules or regulate autophagic activities.

### **2. Molecular mechanisms and biological functions of autophagy**

#### **2.1 Molecular mechanisms of autophagy**

Autophagy is a lysosomal-mediated self-degradation process of intracellular components. Under normal physiological conditions, autophagy degrades damaged organelles and nonfunctional proteins in cells to provide energy and nutrients to maintain intracellular homeostasis. However, autophagy may be induced to participate in disease processes during fasting, drug interactions, nutritional deficiencies, hypoxia, or other stress reactions. Autophagy is divided into three stages: initiation, phagophore membrane formation and extension, and maturation. When the cellular sensation is subjected to external pressure, the signal-transduction molecules are activated, which regulates autophagy-related genes (ATGs) and promotes autophagy. The molecular mechanisms of the autophagy process are summarized in **Figure 1** [3].

The energy receptor, AMP-activated protein kinase (AMPK), is activated to rapidly induce the upregulation of autophagy levels when cells are under nutrient and energy deficiency, protein accumulation, and stress. Autophagy induction is mainly achieved by the interaction between the Unc-51 like autophagy activating kinase 1 (ULK1) complex (including Atg1/ULK1, Atg17/FIP200, and Atg13) and mammalian target of rapamycin complex 1 (mTORC1) [4]. First, mTORC1 is activated when cellular energy is sufficient, which then phosphorylates ULK1 and Atg13 to inhibit autophagy. In contrast, mTORC1 activity is inhibited when cellular energy is deficient, and the resultant dephosphorylated Atg13 forms a complex with ULK1 and interacts with FIP200 to initiate autophagy [5]. The initiation of autophagy is closely related to the Class III PI3K (Vps34)-Atg6/Beclin1 complex [6, 7]. The Beclin1-PI3K complex recruits Atg12-Atg5 and Atg16L multimers and Atg8/microtubule-associated protein light chain 3 (LC3), which promotes the stretching and extension of autophagosome membranes [8]. The expansion of autophagosomes mainly depends on two ubiquitin-like conjugation systems: Atg12

**89**

**2.2 Autophagic cell death (ACD)**

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

the corresponding substrates and their degradation [14].

In addition to the direct involvement of the Atg gene in autophagy, some important signaling pathways also regulate autophagy. Among these pathways, the PI3k/Akt/mTOR pathway is a classical signal-transduction pathway associated with autophagy. Under nutrient-rich conditions, PI3k/Akt signaling activates the downstream mammalian target of rapamycin (mTOR) to form the mTOR complex consisting of ULK1/2, mATG13, FIP200, and Atg101. Once the mTOR complex is formed, phosphorylation of ULK1 and Atg13 results in ULK1 inactivation, which suppresses autophagy. In the absence of nutrients, the LKB/AMPK pathway is activated, and the binding of mTORC1 to ULK is blocked, thereby suppressing mTOR activity. Subsequently, ULK1 phosphorylates Atg13 and FIP200 to form autophagosomes, ultimately inducing autophagy [15]. The AMPK pathway is a cellular energy sensor, and it regulates the occurrence of autophagy. In the absence of nutrients, AMPK senses the changes in AMP levels and is activated, thereby phosphorylating tuberous sclerosis 2 (TSC2) and aggravating the inhibition of Rheb GTPase by TSC1/2, which ultimately inhibits mTOR activity and induces autophagy. In addition, AMPK activates ULK1 through phosphorylation, thereby promoting autophagy. Under nutrient-rich conditions, mTOR phosphorylates ULK1 to prevent AMPK from activating ULK1, which ultimately suppresses ULK1 activity and thereby inhibits autophagy [16]. The Hedgehog (Hh) signaling pathway also regulates autophagy. After Sonic hedgehog (SHh) in the Hh signaling pathway senses signaling stimulation, it transfers and binds to PATCHED-1 (PTCH) receptor, thereby releasing inhibition of the G-protein-coupled receptor (GPCR)-like protein Smoothened (Smo). The activation of Smo ultimately leads to the activation of the downstream Gli transcription factor. Gli has three homologues, of which Gli1 is involved in autophagy and inhibition of Gli1 induces autophagy. However, the mechanism by which Gli1 induces autophagy has not been well clarified [17, 18].

Programmed cell death is an orderly process, and, once this process is initiated, it proceeds automatically. Programmed cell death can be classified into at least three

binding and LC3-modification processes [9]. Atg12 binding is a ubiquitin-like process that requires the participation of ubiquitin-activating enzymes, E1 and E2. Atg12 is firstly activated by Atg7 (E1-like enzyme) and is then transported to Atg5 through Atg10 (E2-like enzyme), which subsequently binds to Atg16 to form a multibody complex and participates in the expansion of the autophagosome [10–12]. The LC3-modification process also requires the participation of ubiquitinactivating enzymes, E1 and E2. After the formation of the LC3 precursor, it is processed into cytosolic soluble LC3-I by Atg4 and is then covalently linked to phosphatidylethanolamine (PE) by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme) to form a fat-soluble LC3-II-PE that participates in the extension of the autophagosome membrane. LC3-II binds to the newly formed autophagosome membrane until the formation of the autolysosome. Therefore, LC3-II is often used as a marker for autophagy [11, 13]. After formation, autophagosomes fuse with lysosomes through the microtubule cytoskeleton under the action of the endosomal-sorting complex required for transport (ESCRT), as well as through monomeric GTPase (RabS), to form autolysosomes. The autophagy-receptor protein, P62/SQSTM1, recognizes and binds to autophagy-substrate proteins by binding to the ubiquitin-associated (UBA) domain, either dependently or independently. P62/SQSTM1 also anchors to the autophagosome membrane through the LC3/Atg8 interaction region (LIR); ultimately fusion between autophagosomes and lysosomes leads to P62/SQSTM1 entering into autolysosomes, together with

*DOI: http://dx.doi.org/10.5772/intechopen.88050*

**Figure 1.** *Schematic diagram of the molecular mechanisms of autophagy.*

#### *Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy DOI: http://dx.doi.org/10.5772/intechopen.88050*

*Programmed Cell Death*

**2.1 Molecular mechanisms of autophagy**

**2. Molecular mechanisms and biological functions of autophagy**

Autophagy is a lysosomal-mediated self-degradation process of intracellular components. Under normal physiological conditions, autophagy degrades damaged organelles and nonfunctional proteins in cells to provide energy and nutrients to maintain intracellular homeostasis. However, autophagy may be induced to participate in disease processes during fasting, drug interactions, nutritional deficiencies, hypoxia, or other stress reactions. Autophagy is divided into three stages: initiation, phagophore membrane formation and extension, and maturation. When the cellular sensation is subjected to external pressure, the signal-transduction molecules are activated, which regulates autophagy-related genes (ATGs) and promotes autophagy. The molecular mechanisms of the autophagy process are summarized in **Figure 1** [3]. The energy receptor, AMP-activated protein kinase (AMPK), is activated to rapidly induce the upregulation of autophagy levels when cells are under nutrient and energy deficiency, protein accumulation, and stress. Autophagy induction is mainly achieved by the interaction between the Unc-51 like autophagy activating kinase 1 (ULK1) complex (including Atg1/ULK1, Atg17/FIP200, and Atg13) and mammalian target of rapamycin complex 1 (mTORC1) [4]. First, mTORC1 is activated when cellular energy is sufficient, which then phosphorylates ULK1 and Atg13 to inhibit autophagy. In contrast, mTORC1 activity is inhibited when cellular energy is deficient, and the resultant dephosphorylated Atg13 forms a complex with ULK1 and interacts with FIP200 to initiate autophagy [5]. The initiation of autophagy is closely related to the Class III PI3K (Vps34)-Atg6/Beclin1 complex [6, 7]. The Beclin1-PI3K complex recruits Atg12-Atg5 and Atg16L multimers and Atg8/microtubule-associated protein light chain 3 (LC3), which promotes the stretching and extension of autophagosome membranes [8]. The expansion of autophagosomes mainly depends on two ubiquitin-like conjugation systems: Atg12

**88**

**Figure 1.**

*Schematic diagram of the molecular mechanisms of autophagy.*

binding and LC3-modification processes [9]. Atg12 binding is a ubiquitin-like process that requires the participation of ubiquitin-activating enzymes, E1 and E2. Atg12 is firstly activated by Atg7 (E1-like enzyme) and is then transported to Atg5 through Atg10 (E2-like enzyme), which subsequently binds to Atg16 to form a multibody complex and participates in the expansion of the autophagosome [10–12]. The LC3-modification process also requires the participation of ubiquitinactivating enzymes, E1 and E2. After the formation of the LC3 precursor, it is processed into cytosolic soluble LC3-I by Atg4 and is then covalently linked to phosphatidylethanolamine (PE) by Atg7 (E1-like enzyme) and Atg3 (E2-like enzyme) to form a fat-soluble LC3-II-PE that participates in the extension of the autophagosome membrane. LC3-II binds to the newly formed autophagosome membrane until the formation of the autolysosome. Therefore, LC3-II is often used as a marker for autophagy [11, 13]. After formation, autophagosomes fuse with lysosomes through the microtubule cytoskeleton under the action of the endosomal-sorting complex required for transport (ESCRT), as well as through monomeric GTPase (RabS), to form autolysosomes. The autophagy-receptor protein, P62/SQSTM1, recognizes and binds to autophagy-substrate proteins by binding to the ubiquitin-associated (UBA) domain, either dependently or independently. P62/SQSTM1 also anchors to the autophagosome membrane through the LC3/Atg8 interaction region (LIR); ultimately fusion between autophagosomes and lysosomes leads to P62/SQSTM1 entering into autolysosomes, together with the corresponding substrates and their degradation [14].

In addition to the direct involvement of the Atg gene in autophagy, some important signaling pathways also regulate autophagy. Among these pathways, the PI3k/Akt/mTOR pathway is a classical signal-transduction pathway associated with autophagy. Under nutrient-rich conditions, PI3k/Akt signaling activates the downstream mammalian target of rapamycin (mTOR) to form the mTOR complex consisting of ULK1/2, mATG13, FIP200, and Atg101. Once the mTOR complex is formed, phosphorylation of ULK1 and Atg13 results in ULK1 inactivation, which suppresses autophagy. In the absence of nutrients, the LKB/AMPK pathway is activated, and the binding of mTORC1 to ULK is blocked, thereby suppressing mTOR activity. Subsequently, ULK1 phosphorylates Atg13 and FIP200 to form autophagosomes, ultimately inducing autophagy [15]. The AMPK pathway is a cellular energy sensor, and it regulates the occurrence of autophagy. In the absence of nutrients, AMPK senses the changes in AMP levels and is activated, thereby phosphorylating tuberous sclerosis 2 (TSC2) and aggravating the inhibition of Rheb GTPase by TSC1/2, which ultimately inhibits mTOR activity and induces autophagy. In addition, AMPK activates ULK1 through phosphorylation, thereby promoting autophagy. Under nutrient-rich conditions, mTOR phosphorylates ULK1 to prevent AMPK from activating ULK1, which ultimately suppresses ULK1 activity and thereby inhibits autophagy [16]. The Hedgehog (Hh) signaling pathway also regulates autophagy. After Sonic hedgehog (SHh) in the Hh signaling pathway senses signaling stimulation, it transfers and binds to PATCHED-1 (PTCH) receptor, thereby releasing inhibition of the G-protein-coupled receptor (GPCR)-like protein Smoothened (Smo). The activation of Smo ultimately leads to the activation of the downstream Gli transcription factor. Gli has three homologues, of which Gli1 is involved in autophagy and inhibition of Gli1 induces autophagy. However, the mechanism by which Gli1 induces autophagy has not been well clarified [17, 18].

#### **2.2 Autophagic cell death (ACD)**

Programmed cell death is an orderly process, and, once this process is initiated, it proceeds automatically. Programmed cell death can be classified into at least three types: type I (apoptosis), type II (ACD), and type III (necrosis or lysosomal death). Type I programmed cell death is accompanied with typical morphological changes of the cells—such as cell shrinkage, chromatin condensation, and apoptotic-body formation—following the initiation of apoptosis (**Figure 2a**). The morphological features of cells undergoing type II programmed cell death are not as obvious, and only autophagic vacuoles can be observed under electron microscopy (**Figure 2b**). Necrosis is a form of cell death characterized by organelle swelling, cytoplasmicmembrane rupture, and leakage of cellular contents (**Figure 2c**). Therefore, ACD should be defined according to the following three features: (1) apoptosis is not triggered; (2) there is an increase in autophagic flux before or during cell death, rather than a mere elevation in autophagy marker; and (3) application of pharmaceutical inhibitor, like 3-MA, chloroquine, etc., or small interfering RNA (siRNA) against autophagy-related genes (Beclin1 or Atg5) inhibits cell death [19–21]. In short, ACD is a type of cell death caused by autophagy. Autophagy may also be involved in other types of cell death. Therefore, it is necessary to distinguish the difference between cell death that occurs with autophagy and cell death that is caused by autophagy. If inhibition of autophagy only changes cell morphologies but not the fate of cell death, then the death in question is cell death with autophagy. If inhibition of autophagy leads to alleviation or inhibition of cell death, then the death in question is cell death by autophagy [22].

The discovery of ACD has provided new ideas and strategies for anticancer therapy. It is well-known that autophagy plays a role as a "double-edged sword" in tumorigenesis (i.e., as a promoter and a suppressor of tumors). In terms of promoting tumorigenesis, autophagy is a protective mechanism that protects tumor cells from being killed. However, ACD breaks the protective mechanism of autophagy. Drug and hypoxia-mediated ACD converts the autophagy that originally benefits tumor-cell survival to become harmful to tumor cells and inhibits tumor-cell proliferation. In addition, there is a relationship between ACD and apoptosis, and autophagy has been considered to be essential for the occurrence of apoptosis. Autophagy usually occurs before apoptosis, with apoptosis being initiated after autophagy, which leads to accelerated cell death. Some researchers believe that the occurrence of autophagy inhibits apoptosis and, thus, protects tumor cells. In addition, apoptosis can coexist with autophagy to collectively promote cell death [23]. Therefore, regardless of the single role of ACD or the combined effect of ACD and apoptosis, the above studies provide an important theoretical basis for the development of antitumor drugs. In fact, many antitumor drugs, either newly developed ones or old drugs with a new target, share the same mechanism in anticancer therapy by targeting programmed cell death.

Since the discovery of autophagy, especially its role in tumorigenesis and tumor development, the use of autophagy to regulate the programmed cell death of tumor cells has become a useful tool for anticancer therapy. Drug-induced ACD is also a

**91**

treatments.

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

primary mechanism for antitumor drugs. Therefore, autophagy has been widely studied as a drug target in antitumor research. First, this new research strategy of old drugs provides ideas for autophagy-targeted drug discovery. Some repurposing drugs—such as the antimalarial drug chloroquine, the antifungal drug bafilomycin A1, and the immunosuppressant rapamycin—had not had their autophagy functions revealed when they were discovered. However, subsequent studies have revealed their other functions, which do not only induce autophagy but also inhibit tumor-cell proliferation through regulating autophagic activity. These old drugs are good candidates for being repurposed into antitumor drugs. Additionally, in the past few years with the development of omics technologies, a number of smallmolecule compounds targeting autophagy molecules or autophagy pathways have been discovered by high-throughput screening methods. These drugs also inhibit tumor-cell proliferation through the ACD pathway. The following section summarizes drugs that control tumor proliferation by regulating autophagy activity in cancer stem cells (CSCs) and tumor cells, as well as their mechanisms of action.

**3. Antitumor drugs that regulate autophagy to target cancer stem cells**

Cancer stem cells (CSCs) are a type of cell with stem cell characteristics in tumors that exhibit strong self-renewal and proliferative ability and play an important role in tumor survival, proliferation, metastasis, and recurrence. Taken together, CSCs maintain the vitality of tumor-cell populations through self-renewal

and infinite proliferation [24]. CSCs in tumor tissues in microenvironments with insufficient nutrients increase the recycling of intracellular substances by autophagy to continuously survive. Apoptosis, proliferation, and differentiation of CSCs are regulated by many factors, and autophagy and its signaling pathways play an important role in the apoptosis and differentiation of CSCs [25, 26]. A previous study has shown that the autophagy activity regulated by autophagy-related genes or autophagy pathways promotes the survival of CSCs. High Beclin1 expression promotes the survival and tumorigenicity of breast CSCs [27]. Chloroquine inhibits autophagy via the Janus kinase 2 (JAK2) and Hh pathways and kills breast and pancreatic CSCs [28, 29]. Nevertheless, another study has shown that autophagy also promotes the death of CSCs. CSCs from malignant gliomas cause cell death due to the accumulation of autophagy-related proteins and autophagosomes [30]. In addition, CSCs adapt to the changes of the microenvironment simultaneously through autophagy, and a large number of autophagosomes are found in damaged blood vessels and hypoxic parts of tumors. Autophagy induced by hypoxia-inducible factor (HIF) promotes metastasis of CD133+ pancreatic CSCs. Inhibition of autophagy reduces the viabilities of pancreatic cancer cells with stem cell characteristics and CD133+ liver CSCs under oxygen/nutrition deprivation [31]. Multi-regulation of autophagy on CSCs has prompted researchers to explore the feasibility of autophagy as a drug target for anticancer therapy. Numerous studies have demonstrated that some chemical synthetic drugs or natural extracts have a clear therapeutic effect on CSCs through regulating autophagy activity and may be used as novel antitumor

Small-molecule compounds induce programmed cell death in tumor cells through apoptosis and autophagy as their main antitumor mechanisms. After the concept of CSCs was proposed, researchers began to continuously explore small-molecule compounds and their effective targeting of CSCs. In 2009, Gupta et al. screened more than 16,000 compounds to discover that salinomycin has strong killing activity against breast CSCs and that the killing activity is >100-fold higher than that of paclitaxel [32]. Further studies have shown that salinomycin

*DOI: http://dx.doi.org/10.5772/intechopen.88050*

**Figure 2.** *Types of programmed cell death. a) apoptosis, b) autophagy and c) necrosis [22].*

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy DOI: http://dx.doi.org/10.5772/intechopen.88050*

*Programmed Cell Death*

question is cell death by autophagy [22].

ing programmed cell death.

types: type I (apoptosis), type II (ACD), and type III (necrosis or lysosomal death). Type I programmed cell death is accompanied with typical morphological changes of the cells—such as cell shrinkage, chromatin condensation, and apoptotic-body formation—following the initiation of apoptosis (**Figure 2a**). The morphological features of cells undergoing type II programmed cell death are not as obvious, and only autophagic vacuoles can be observed under electron microscopy (**Figure 2b**). Necrosis is a form of cell death characterized by organelle swelling, cytoplasmicmembrane rupture, and leakage of cellular contents (**Figure 2c**). Therefore, ACD should be defined according to the following three features: (1) apoptosis is not triggered; (2) there is an increase in autophagic flux before or during cell death, rather than a mere elevation in autophagy marker; and (3) application of pharmaceutical inhibitor, like 3-MA, chloroquine, etc., or small interfering RNA (siRNA) against autophagy-related genes (Beclin1 or Atg5) inhibits cell death [19–21]. In short, ACD is a type of cell death caused by autophagy. Autophagy may also be involved in other types of cell death. Therefore, it is necessary to distinguish the difference between cell death that occurs with autophagy and cell death that is caused by autophagy. If inhibition of autophagy only changes cell morphologies but not the fate of cell death, then the death in question is cell death with autophagy. If inhibition of autophagy leads to alleviation or inhibition of cell death, then the death in

The discovery of ACD has provided new ideas and strategies for anticancer therapy. It is well-known that autophagy plays a role as a "double-edged sword" in tumorigenesis (i.e., as a promoter and a suppressor of tumors). In terms of promoting tumorigenesis, autophagy is a protective mechanism that protects tumor cells from being killed. However, ACD breaks the protective mechanism of autophagy. Drug and hypoxia-mediated ACD converts the autophagy that originally benefits tumor-cell survival to become harmful to tumor cells and inhibits tumor-cell proliferation. In addition, there is a relationship between ACD and apoptosis, and autophagy has been considered to be essential for the occurrence of apoptosis. Autophagy usually occurs before apoptosis, with apoptosis being initiated after autophagy, which leads to accelerated cell death. Some researchers believe that the occurrence of autophagy inhibits apoptosis and, thus, protects tumor cells. In addition, apoptosis can coexist with autophagy to collectively promote cell death [23]. Therefore, regardless of the single role of ACD or the combined effect of ACD and apoptosis, the above studies provide an important theoretical basis for the development of antitumor drugs. In fact, many antitumor drugs, either newly developed ones or old drugs with a new target, share the same mechanism in anticancer therapy by target-

Since the discovery of autophagy, especially its role in tumorigenesis and tumor development, the use of autophagy to regulate the programmed cell death of tumor cells has become a useful tool for anticancer therapy. Drug-induced ACD is also a

*Types of programmed cell death. a) apoptosis, b) autophagy and c) necrosis [22].*

**90**

**Figure 2.**

primary mechanism for antitumor drugs. Therefore, autophagy has been widely studied as a drug target in antitumor research. First, this new research strategy of old drugs provides ideas for autophagy-targeted drug discovery. Some repurposing drugs—such as the antimalarial drug chloroquine, the antifungal drug bafilomycin A1, and the immunosuppressant rapamycin—had not had their autophagy functions revealed when they were discovered. However, subsequent studies have revealed their other functions, which do not only induce autophagy but also inhibit tumor-cell proliferation through regulating autophagic activity. These old drugs are good candidates for being repurposed into antitumor drugs. Additionally, in the past few years with the development of omics technologies, a number of smallmolecule compounds targeting autophagy molecules or autophagy pathways have been discovered by high-throughput screening methods. These drugs also inhibit tumor-cell proliferation through the ACD pathway. The following section summarizes drugs that control tumor proliferation by regulating autophagy activity in cancer stem cells (CSCs) and tumor cells, as well as their mechanisms of action.

#### **3. Antitumor drugs that regulate autophagy to target cancer stem cells**

Cancer stem cells (CSCs) are a type of cell with stem cell characteristics in tumors that exhibit strong self-renewal and proliferative ability and play an important role in tumor survival, proliferation, metastasis, and recurrence. Taken together, CSCs maintain the vitality of tumor-cell populations through self-renewal and infinite proliferation [24]. CSCs in tumor tissues in microenvironments with insufficient nutrients increase the recycling of intracellular substances by autophagy to continuously survive. Apoptosis, proliferation, and differentiation of CSCs are regulated by many factors, and autophagy and its signaling pathways play an important role in the apoptosis and differentiation of CSCs [25, 26]. A previous study has shown that the autophagy activity regulated by autophagy-related genes or autophagy pathways promotes the survival of CSCs. High Beclin1 expression promotes the survival and tumorigenicity of breast CSCs [27]. Chloroquine inhibits autophagy via the Janus kinase 2 (JAK2) and Hh pathways and kills breast and pancreatic CSCs [28, 29]. Nevertheless, another study has shown that autophagy also promotes the death of CSCs. CSCs from malignant gliomas cause cell death due to the accumulation of autophagy-related proteins and autophagosomes [30]. In addition, CSCs adapt to the changes of the microenvironment simultaneously through autophagy, and a large number of autophagosomes are found in damaged blood vessels and hypoxic parts of tumors. Autophagy induced by hypoxia-inducible factor (HIF) promotes metastasis of CD133+ pancreatic CSCs. Inhibition of autophagy reduces the viabilities of pancreatic cancer cells with stem cell characteristics and CD133+ liver CSCs under oxygen/nutrition deprivation [31]. Multi-regulation of autophagy on CSCs has prompted researchers to explore the feasibility of autophagy as a drug target for anticancer therapy. Numerous studies have demonstrated that some chemical synthetic drugs or natural extracts have a clear therapeutic effect on CSCs through regulating autophagy activity and may be used as novel antitumor treatments.

Small-molecule compounds induce programmed cell death in tumor cells through apoptosis and autophagy as their main antitumor mechanisms. After the concept of CSCs was proposed, researchers began to continuously explore small-molecule compounds and their effective targeting of CSCs. In 2009, Gupta et al. screened more than 16,000 compounds to discover that salinomycin has strong killing activity against breast CSCs and that the killing activity is >100-fold higher than that of paclitaxel [32]. Further studies have shown that salinomycin

has an inhibitory effect on a variety of CSCs. Salinomycin does not only induce apoptosis in CSCs but also inhibits DNA repair of CSCs. Interestingly, the proliferation of CSCs is inhibited by the increase or decrease of autophagic activity after the action of salinomycin [33]. On the one hand, after the action of salinomycin, the increase of reactive oxygen species (ROS) activates c-Jun N-terminal kinase (JNK) and AMPK signaling pathways to induce an increase of autophagy activity; on the other hand, salinomycin also affects the process of autophagosome and lysosome fusion and reduces the level of autophagic flux. These factors are the mechanisms by which salinomycin inhibits the proliferation of CSCs [34–36]. PI3k/ Akt/mTOR is a key pathway for autophagy regulation. When cells sense signs of starvation and hypoxia, they inhibit the activity of the PI3k/Akt/mTOR pathway and promote autophagy by phosphorylating Akt at amino-acid position 473 and mTOR at amino-acid position 2448. NVP-BEZ235 is a dual ATP-competitive PI3K and mTOR inhibitor, and its induced autophagy and apoptosis enhance the radiosensitivity of glioma stem cells [37]. Rottlerin, a small-molecule compound that acts as a protein kinase C-δ (PKC-δ) inhibitor, regulates the PI3k/Akt/mTOR pathway to activate autophagy. Rottlerin promotes the death of pancreatic CSCs through an endogenous apoptotic pathway after the activation of autophagy [38]. The same phenomenon is also observed in Rottlerin-treated prostate CSCs [39]. Nearly all malignant tumors have epigenetic abnormalities. Antitumor drugs targeting histone deacetylase (HDAC) are a primary focus in epigenetic research. Small-molecule inhibitors that target HDAC have a clear inhibitory effect on the proliferation of CSCs. Givinostat (ITF2357) and suberoylanilide hydroxamic acid (SAHA) are currently the most studied HDAC small-molecule inhibitors. Studies have shown that givinostat and SAHA can induce autophagy in lung and glioma CSCs, respectively, leading to cell death [40–42].

Autophagy is associated with drug resistance and metastasis of CSCs. Autophagy inhibitors or silencing of autophagy-related genes also affect the self-renewal and differentiation of CSCs or enhance the sensitivity of CSCs to chemotherapeutic drugs, thereby killing CSCs. The autophagy inhibitor, chloroquine, not only does inhibit autophagy activity of breast CSCs but also reduces the cell number and metastasis of breast CD44+/CD24−/low CSCs; this effect is damaging to the mitochondrial membrane structure, which leads to decreased cytochrome-c activity and increases oxidative stress. This process also leads to increased expression of the double-stranded DNA damage marker, γ-H2AX, which reduces the repair capacity of double-stranded DNA breaks, thereby inhibiting the proliferation of breast CSCs [43]. Drug resistance is a challenge for antitumor therapy. The small-molecule compound, baicalein competitively binds to the GTPase SAR1B protein, guanosine triphosphate, which is required for autophagy and inhibits autophagy activity, thereby selectively enhancing the sensitivity of mice liver CD133+ tumor-initiating stem cell-like cells (TICs) to mTORC1 inhibitors [44]. The autophagy inhibitor, quinacrine, which can pass through the blood-brain barrier, also increases the sensitivity of glioblastoma stem-like cells (GSCs) to the chemotherapeutic drug, temozolomide (TMZ). The combination of quinacrine and TMZ promotes iron-dependent cell death (ferroptosis) in GSCs [45]. The bromodomain and extra-terminal domain (BET) inhibitor, JQ1, is ineffective for drug-resistant CD34+ CD8- leukemia stem cells (LSCs) and hardly induces apoptosis in drug-resistant LSCs. However, JQ1 increases the expression of autophagy-related molecules, such as Beclin1 and LC3-II, through the AMPK-ULK1 pathway to promote autophagosome formation. The AMPK inhibitor, compound C, and AMPKα siRNA inhibit autophagy to further promote the apoptosis of drug-resistant CD34+CD8− LSCs [46].

In addition to small-molecule compounds, plant extracts also have an inhibitory effect on CSCs. Curcumin is a chemical component extracted from the roots

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Bitter-melon fruit extracts

**Table 1.**

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

**activity**

inhibition

unchanged

increasing

inhibition

induction

induction

induction

induction

induction

inhibition

induction

inhibition

inhibition

induction

induction

induction

induction

Autophagy induction

*Abbreviations: Jak2: Janus kinase 2; DNMT1: DNA (cytosine-5)-methyltransferase 1; CXCR4: C-X-C chemokine receptor type 4; ROS: reactive oxygen species; HDAC: histone deacetylases; DCLK1: Doublecortin like kinase 1; ULK1:* 

**Mechanism of autophagy regulation**

Jak2, DNMT1 [27, 42]

CXCR4 [28]

ROS [33]

Apoptotic pathway [35]

ROS [33]

PI3k/Akt/mTOR [36]

Apoptotic pathway [37]

Apoptotic pathway [38]

HDAC [39]

HDAC [40]

HDAC [41]

GTPase SAR1B protein [43]

Lipid peroxides [44]

AMPK-ULK1 pathway [45]

DCLK1 [46]

Wnt pathway [48]

AMPK pathway [49]

[47]

Wnt/β-catenin pathway

**References**

**Drugs Cancer stem cells Autophagy** 

Chloroquine Breast Autophagy

Salinomycin Breast Autophagy

NVP-BEZ235 Gliomas Autophagy

Rettlerin Pancreatic Autophagy

Givinostat Lung Autophagy

SAHA Glioblastoma Autophagy

Baicalein Mice liver tumor Autophagy

Quinacrine Glioblastoma Autophagy

JQ1 Leukemia Autophagy

Curcumin Colon Autophagy

Resveratrol Breast Autophagy

Colon and progenitor

*Antitumor drugs that target cancer stem cells by regulating autophagy.*

cells

*Unc-51 like autophagy activating kinase 1*

Pancreatic Autophagy

Breast Autophagy

Colon Autophagy

Prostate Autophagy

Glioblastoma Autophagy

Colon adenocarcinoma Autophagy

of some plants in the Zingiberaceae and Araceae families. Among the numerous biological activities of curcumin, its antitumor activity has aroused the attention of tumor biologists. Studies have shown that curcumin has an inhibitory effect on liver and ovarian CSCs. Autophagy induced by curcumin treatment in the DCLK1+ colon CSCs inhibits cell proliferation via apoptosis [47]. Resveratrol not only does inhibit the proliferation of breast CSCs by autophagy [48] but also increases the trans-differentiation of colon CSCs into endothelial cells [49]. Bitter-melon whole

*DOI: http://dx.doi.org/10.5772/intechopen.88050*


*Programmed Cell Death*

has an inhibitory effect on a variety of CSCs. Salinomycin does not only induce apoptosis in CSCs but also inhibits DNA repair of CSCs. Interestingly, the proliferation of CSCs is inhibited by the increase or decrease of autophagic activity after the action of salinomycin [33]. On the one hand, after the action of salinomycin, the increase of reactive oxygen species (ROS) activates c-Jun N-terminal kinase (JNK) and AMPK signaling pathways to induce an increase of autophagy activity; on the other hand, salinomycin also affects the process of autophagosome and lysosome fusion and reduces the level of autophagic flux. These factors are the mechanisms by which salinomycin inhibits the proliferation of CSCs [34–36]. PI3k/ Akt/mTOR is a key pathway for autophagy regulation. When cells sense signs of starvation and hypoxia, they inhibit the activity of the PI3k/Akt/mTOR pathway and promote autophagy by phosphorylating Akt at amino-acid position 473 and mTOR at amino-acid position 2448. NVP-BEZ235 is a dual ATP-competitive PI3K and mTOR inhibitor, and its induced autophagy and apoptosis enhance the radiosensitivity of glioma stem cells [37]. Rottlerin, a small-molecule compound that acts as a protein kinase C-δ (PKC-δ) inhibitor, regulates the PI3k/Akt/mTOR pathway to activate autophagy. Rottlerin promotes the death of pancreatic CSCs through an endogenous apoptotic pathway after the activation of autophagy [38]. The same phenomenon is also observed in Rottlerin-treated prostate CSCs [39]. Nearly all malignant tumors have epigenetic abnormalities. Antitumor drugs targeting histone deacetylase (HDAC) are a primary focus in epigenetic research. Small-molecule inhibitors that target HDAC have a clear inhibitory effect on the proliferation of CSCs. Givinostat (ITF2357) and suberoylanilide hydroxamic acid (SAHA) are currently the most studied HDAC small-molecule inhibitors. Studies have shown that givinostat and SAHA can induce autophagy in lung and glioma

Autophagy is associated with drug resistance and metastasis of CSCs. Autophagy inhibitors or silencing of autophagy-related genes also affect the self-renewal and differentiation of CSCs or enhance the sensitivity of CSCs to chemotherapeutic drugs, thereby killing CSCs. The autophagy inhibitor, chloroquine, not only does inhibit autophagy activity of breast CSCs but also reduces the cell number and metastasis of breast CD44+/CD24−/low CSCs; this effect is damaging to the

mitochondrial membrane structure, which leads to decreased cytochrome-c activity and increases oxidative stress. This process also leads to increased expression of the double-stranded DNA damage marker, γ-H2AX, which reduces the repair capacity of double-stranded DNA breaks, thereby inhibiting the proliferation of breast CSCs [43]. Drug resistance is a challenge for antitumor therapy. The small-molecule compound, baicalein competitively binds to the GTPase SAR1B protein, guanosine triphosphate, which is required for autophagy and inhibits autophagy activity, thereby selectively enhancing the sensitivity of mice liver CD133+ tumor-initiating stem cell-like cells (TICs) to mTORC1 inhibitors [44]. The autophagy inhibitor, quinacrine, which can pass through the blood-brain barrier, also increases the sensitivity of glioblastoma stem-like cells (GSCs) to the chemotherapeutic drug, temozolomide (TMZ). The combination of quinacrine and TMZ promotes iron-dependent cell death (ferroptosis) in GSCs [45]. The bromodomain and extra-terminal domain (BET) inhibitor, JQ1, is ineffective for drug-resistant CD34+ CD8- leukemia stem cells (LSCs) and hardly induces apoptosis in drug-resistant LSCs. However, JQ1 increases the expression of autophagy-related molecules, such as Beclin1 and LC3-II, through the AMPK-ULK1 pathway to promote autophagosome formation. The AMPK inhibitor, compound C, and AMPKα siRNA inhibit autophagy to further

promote the apoptosis of drug-resistant CD34+CD8− LSCs [46].

In addition to small-molecule compounds, plant extracts also have an inhibitory effect on CSCs. Curcumin is a chemical component extracted from the roots

CSCs, respectively, leading to cell death [40–42].

**92**

*Abbreviations: Jak2: Janus kinase 2; DNMT1: DNA (cytosine-5)-methyltransferase 1; CXCR4: C-X-C chemokine receptor type 4; ROS: reactive oxygen species; HDAC: histone deacetylases; DCLK1: Doublecortin like kinase 1; ULK1: Unc-51 like autophagy activating kinase 1*

#### **Table 1.**

*Antitumor drugs that target cancer stem cells by regulating autophagy.*

of some plants in the Zingiberaceae and Araceae families. Among the numerous biological activities of curcumin, its antitumor activity has aroused the attention of tumor biologists. Studies have shown that curcumin has an inhibitory effect on liver and ovarian CSCs. Autophagy induced by curcumin treatment in the DCLK1+ colon CSCs inhibits cell proliferation via apoptosis [47]. Resveratrol not only does inhibit the proliferation of breast CSCs by autophagy [48] but also increases the trans-differentiation of colon CSCs into endothelial cells [49]. Bitter-melon whole

fruit (BMW) and skin (BMSk) extracts significantly inhibit the proliferation and colonization of colon CSCs, and the BMW-treated colon CSCs also cause the upregulated expression of LC3-II, Beclin1, Atg7, and Atg12 to promote autophagy [50]. **Table 1** summarizes the recent progress of mechanism, targets, and references of drugs in anticancer stem cells.

### **4. Antitumor drugs that regulate autophagy to target tumor cells**

In the processes of tumorigenesis and tumor progression, autophagy has dual roles, and inhibition or promotion of autophagy is related to tumorigenesis. Some important signaling pathways—such as PI3K/Akt/mTOR, MAPK, JNK, and Hh pathways—do not only regulate tumor-cell growth and proliferation but also affect tumorigenesis by regulating autophagic activity. In addition, autophagy-related molecules—such as Atg5, Atg7, and Beclin1—are associated with tumorigenesis. Therefore, research in recent years regarding autophagy-targeted antitumor drugs includes not only autophagy-related signaling pathways but also autophagy-related molecules. In this section, antitumor drugs, like compounds and nature extracts, which target the signaling pathway (**Table 2**) or autophagyrelated molecules (**Table 3**), are introduced, and their antitumor mechanisms are discussed.


**95**

**4.1 Autophagy pathways**

**Table 2.**

*4.1.1 Drugs that target AMPK pathway*

AMPK is a metabolism and energy receptor of cells. The intracellular balance of energy and metabolism in tumor cells is often chaotic, leading to changes in AMPK activity to further cause activity changes of a series of downstream signaling pathways, which affect the autophagy process. In addition to its therapeutic effect on type II diabetes, metformin has also been found to have a therapeutic effect on tumors. Wang et al. has shown that metformin targets the molecules of AMPK/mTORC1 and mTORC2 pathways in the tumor cells of multiple myeloma. Activation of AMPK signaling inhibits mTORC1 and mTORC2 to further induce autophagy and inhibit myeloma cell proliferation [51]. In contrast, metformin also inhibits autophagy and promotes apoptosis through AMPK and p38/MAPK signaling pathways in the glucose-deprived H4IIE rat hepatocellular-carcinoma cell line [52]. Cannabinoids have a variety of biological activities, of which tumor inhibition has aroused attention from researchers. AMPK pathways activate autophagy after ROS-dependent activation in cannabinoid-treated pancreatic cancer cells, while ROS promotes GAPDH nuclear translation, leading to reduced glycolysis and NADH accumulation to block the Krebs cycle after blocking the respiratory-chain function. Simultaneously with the activation of the AMPK pathway, cannabinoids

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

**activity**

induction

Autophagy inhibition

inhibition

inhibition

Autophagic cell death

Autophagic cell death

Autophagy induction

Autophagy inhibition

Autophagy induction

induction

Autophagy inhibition

*Abbreviations: AMPK: 5' AMP-activated protein kinase; MAPK: mitogen-activated protein kinase; PEITC: phenethyl* 

*isothiocyanate; SAHA: Suberoylanilide hydroxamic acid; HDAC: histone deacetylases; Hh: Hedgehog*

**Mechanism of autophagy** 

Autophagosome-lysosome fusion inhibition

Autophagosome-lysosome fusion inhibition

Autophagosome-lysosome fusion inhibition

Hh [17]

Hh [17]

Hh [69]

Hh [70]

HDAC inhibitor [71]

HDAC inhibitor [74]

HDAC inhibitor [72, 73]

p53 [64]

**References**

[66]

[67]

[68]

**regulation**

**Drugs Cancer cell Autophagy** 

PEITC Liver cancer Autophagy

lymphoblastic leukemia cell

DQ661 Melanoma Autophagy

lys05 Glioblastoma Autophagy

rhabdomyosarcoma

cancer

cancer

lymphoma

Panobinostat Colon cancer Autophagy

lymphocytic leukemia

*Antitumor drugs that target cancer cells by regulating autophagy.*

cancer

*DOI: http://dx.doi.org/10.5772/intechopen.88050*

Bafilomycin A1 B-cell acute

Itraconazole Breast and liver

GANT61 Breast and liver

HhAntag Embryonal

Sonidegib Mantle cell

SAHA Colon and liver

MGCD0103 B-cell chronic


*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy DOI: http://dx.doi.org/10.5772/intechopen.88050*

*Abbreviations: AMPK: 5' AMP-activated protein kinase; MAPK: mitogen-activated protein kinase; PEITC: phenethyl isothiocyanate; SAHA: Suberoylanilide hydroxamic acid; HDAC: histone deacetylases; Hh: Hedgehog*

#### **Table 2.**

*Programmed Cell Death*

discussed.

*Ophiopogon japonicus*

of drugs in anticancer stem cells.

**Drugs Cancer cell Autophagy** 

Metformin Myeloma Autophagy

Cannabinoids Pancreatic cancer Autophagy

Safingol Colon carcinoma Autophagy

Compound 6 Lung carcinoma Autophagy

Cabazitaxel Lung carcinoma Autophagy

cancer cell

lymphoblastic leukemia

CYT-Rx20 Breast cancer Autophagy

AJ-5 Melanoma Autophagic

Arsenic-trioxide Glioma Autophagic

Fangchinoline Liver cancer Autophagic

Capsaicin Nasopharyngeal

NVP-BEZ235 T-cell acute

fruit (BMW) and skin (BMSk) extracts significantly inhibit the proliferation and colonization of colon CSCs, and the BMW-treated colon CSCs also cause the upregulated expression of LC3-II, Beclin1, Atg7, and Atg12 to promote autophagy [50]. **Table 1** summarizes the recent progress of mechanism, targets, and references

**4. Antitumor drugs that regulate autophagy to target tumor cells**

roles, and inhibition or promotion of autophagy is related to tumorigenesis. Some important signaling pathways—such as PI3K/Akt/mTOR, MAPK, JNK, and Hh pathways—do not only regulate tumor-cell growth and proliferation but also affect tumorigenesis by regulating autophagic activity. In addition, autophagy-related molecules—such as Atg5, Atg7, and Beclin1—are associated with tumorigenesis. Therefore, research in recent years regarding autophagy-targeted antitumor drugs includes not only autophagy-related signaling pathways but also autophagy-related molecules. In this section, antitumor drugs, like compounds and nature extracts, which target the signaling pathway (**Table 2**) or autophagyrelated molecules (**Table 3**), are introduced, and their antitumor mechanisms are

**activity**

induction

inhibition

induction

induction

induction

induction

cell death

Autophagy induction

Autophagy induction

induction

cell death

cell death

cell death

Rat liver carcinoma Autophagy

Lung carcinoma Autophagic

**Mechanism of autophagy** 

AMPK [50]

AMPK, p38/MAPK [51]

AMPK [52]

PI3K/Akt/mTOR [54]

PI3K/Akt/mTOR [55]

PI3K/Akt/mTOR [56]

PI3K/Akt/mTOR [57]

PI3K/Akt/mTOR [58]

PI3K/Akt/mTOR [59]

MAPK [60]

MAPK [61]

PI3K/Akt, ERK1/2 [62]

p53/sestrin2/AMPK [63]

**References**

**regulation**

In the processes of tumorigenesis and tumor progression, autophagy has dual

**94**

*Antitumor drugs that target cancer cells by regulating autophagy.*

#### **4.1 Autophagy pathways**

#### *4.1.1 Drugs that target AMPK pathway*

AMPK is a metabolism and energy receptor of cells. The intracellular balance of energy and metabolism in tumor cells is often chaotic, leading to changes in AMPK activity to further cause activity changes of a series of downstream signaling pathways, which affect the autophagy process. In addition to its therapeutic effect on type II diabetes, metformin has also been found to have a therapeutic effect on tumors. Wang et al. has shown that metformin targets the molecules of AMPK/mTORC1 and mTORC2 pathways in the tumor cells of multiple myeloma. Activation of AMPK signaling inhibits mTORC1 and mTORC2 to further induce autophagy and inhibit myeloma cell proliferation [51]. In contrast, metformin also inhibits autophagy and promotes apoptosis through AMPK and p38/MAPK signaling pathways in the glucose-deprived H4IIE rat hepatocellular-carcinoma cell line [52]. Cannabinoids have a variety of biological activities, of which tumor inhibition has aroused attention from researchers. AMPK pathways activate autophagy after ROS-dependent activation in cannabinoid-treated pancreatic cancer cells, while ROS promotes GAPDH nuclear translation, leading to reduced glycolysis and NADH accumulation to block the Krebs cycle after blocking the respiratory-chain function. Simultaneously with the activation of the AMPK pathway, cannabinoids

also inhibit the Akt/c-Myc pathway, resulting in the reduction of pyruvate kinase isoform M2 (PKM2) activity, glycolysis, and glutamine uptake. Therefore, cannabinoids inhibit the proliferation of pancreatic cancer cells through autophagy and cellular-metabolic pathways regulated by the AMPK pathway [53].

#### *4.1.2 Drugs that target PI3K/Akt/mTOR pathway*

The PI3K/Akt/mTOR pathway is directly involved in tumor-cell proliferation, and small-molecule drugs or natural extracts act on the PI3K/Akt/mTOR pathway to regulate tumor cell proliferation through autophagy. Specifically, 3-MA, LY294002, and wortmannin are classic inhibitors of the PI3K/Akt/mTOR pathway to suppress autophagy, which have killing effects on various tumor cells [54]. In addition, many compounds that regulate autophagy and inhibit tumor cells via the PI3K/Akt/mTOR pathway have been discovered in recent years. Protein kinase C and the sphingosine kinase inhibitor, Salingol, inhibit the phosphorylation level of key molecules of the PI3K/Akt/mTOR pathway—such as Akt, p70S6k, and rS6—to induce ACD in colon cancer cells [55]. A novel hybrid of the 3-benzyl coumarin seco-B-ring derivative and phenylsulfonylfuroxan (compound 6) reduces the phosphorylation levels of mTOR-S2448, Akt-S373, and the downstream p70s6K-S371 and 4EBP1-pT45 proteins in a concentration-/time-dependent manner to induce autophagy and apoptosis of A549 cells [56]. The antiprostate-cancer drug, cabazitaxel, also inhibits proliferation of A549 cells through autophagy which is regulated by the PI3K/Akt/mTOR pathway [57]. *Ophiopogon japonicus* is a traditional medicinal plant, and its main components are flavonoids and steroidal saponins. A previous study has shown that *O. japonicus* inhibits PI3K/Akt/mTOR signaling to induce ACD in A549 lung cancer cells [58]. Similarly, the inhibitory effect of capsaicin in the nasopharyngeal carcinoma cell line, NPC-TW01, is also achieved by autophagy regulated by the PI3K/Akt/mTOR pathway [59]. Specifically, mTORC1 is a key signaling molecule in the PI3K/Akt/mTOR pathway. Some small-molecule drugs, such as rapamycin and NVP-BEZ235, inhibit mTORC1 activity and induce autophagy and have a killing effect on T-cell acute lymphoblastic leukemia cells [60].

#### *4.1.3 Drugs that target the MAPK pathway*

The MAPK pathway plays an important role in the process of tumor-cell proliferation, growth, apoptosis, and cell-to-cell functional synchronization. The MAPK family members, ERK, p38 MAPK, JNK, and ERK5 are also involved in the regulation of autophagy. CYT-Rx20, a derivative of β-nitrostyrene, inhibits proliferation in the breast cancer cell lines, MDA-MB-231 and MCF-7 [61]. A new binuclear palladacycle complex, AJ-5, does not only induce apoptosis in melanoma cells but also inhibit Akt/mTOR activity and activate p38 and ERK1/2 MAPK signaling to induce ACD, with dual roles in apoptosis and ACD to inhibit the proliferation of melanoma cells [62]. Similar to AJ-5, arsenic-trioxide and ionizing-radiation combination treatment on glioma cells also inhibits Akt/mTOR activity and activates ERK1/2 MAPK signaling to promote ACD [63].

#### *4.1.4 Drugs that target the p53 pathway*

p53 is a tumor-suppressor protein that regulates the expression of multiple genes involved in apoptosis, growth inhibition, and DNA repair. Studies have shown that the regulation of autophagy by p53 is bidirectional depending on its localization. Cytoplasmic p53 inhibits autophagy in a transcriptional-independent manner, while nuclear p53 enhances autophagy by transactivation target gene.

**97**

**Table 3.**

undergo apoptosis [65].

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

**activity**

inhibition

Autophagy inhibition

Autophagy inhibition

induction

inhibition

inhibition

inhibition

inhibition

induction

inhibition

inhibition

death

death

**Mechanism of autophagy regulation**

ULK1 inhibitor [75]

ULK1 inhibitor [76]

ULK1 inhibitor [76]

ULK1 inhibitor [77]

Vps34 inhibitor [78]

Vps34 inhibitor [80]

Atg4B inhibitor [81]

Atg4B inhibitor [82]

Bcl-2 inhibitor [87]

Bcl-2 inhibitor [86]

Bcl-2 inhibitor [87]

Bcl-2 inhibitor [88,

p62/SQSTM1 inhibitor [90]

89]

**Refs.**

**Drugs Cancer cell Autophagy** 

SBI-0206965 Lung cancer cell Autophagy

LYN-1604 Breast cancer cell Autophagy

SAR405 Renal tumor Autophagy

Spautin-1 Cervical cancer Autophagy

S130 Colon cancer Autophagy

NSC185058 Saos-2 osteosarcoma cell Autophagy

ABT-737 Cervical cancer Autophagy

ABT-737 Colon cancer Autophagy

Verteporfin Prostate cancer cell Autophagy

*Small molecular inhibitors of autophagy-related molecules.*

gossypol Glioma Autophagic cell

Obatoclax Lymphoblastic leukemia Autophagic cell

P53 in human hepatocellular-carcinoma cells treated with fangchinoline is transported from the cytoplasm to the nucleus through nuclear translocation and selectively activates sestrin2 to initiate autophagy and promote ACD [64]. Numerous studies have shown that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) reactivates the normal activity of mutant p53 molecules in tumor cells and has an antitumor effect. PEITC has growth-inhibitory activity on tumor cells expressing p53R175H and restores the wide-type conformation and transcriptional activity of p53. In addition, PEITC enables p53R175H tumor cells to be sensitive to proteasome and autophagy-mediated degradation processes. Analysis of the mechanism showed that PEITC activates the classical p53 downstream target gene, causing tumor cells to arrest in the S phase and G2/M phase of the cell cycle and to

The fusion of autophagosomes and lysosomes forms autolysosomes, which degrade the encapsulated materials. The degradation activity can be evaluated by autophagic flux. A previous study has shown that antimalarial drugs—such as quinine, chloroquine, and its derivatives—block the fusion processes of autophagosomes and lysosomes to reduce the autophagic flux and kill tumor cells by inhibiting

*4.1.5 Drugs that target the autophagosome-lysosome fusion pathway*

*DOI: http://dx.doi.org/10.5772/intechopen.88050*

MRT67307 Mouse embryonic fibroblast

MRT68921 Mouse embryonic fibroblast


*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy DOI: http://dx.doi.org/10.5772/intechopen.88050*

**Table 3.**

*Programmed Cell Death*

also inhibit the Akt/c-Myc pathway, resulting in the reduction of pyruvate kinase isoform M2 (PKM2) activity, glycolysis, and glutamine uptake. Therefore, cannabinoids inhibit the proliferation of pancreatic cancer cells through autophagy and

The PI3K/Akt/mTOR pathway is directly involved in tumor-cell proliferation, and small-molecule drugs or natural extracts act on the PI3K/Akt/mTOR pathway to regulate tumor cell proliferation through autophagy. Specifically, 3-MA, LY294002, and wortmannin are classic inhibitors of the PI3K/Akt/mTOR pathway to suppress autophagy, which have killing effects on various tumor cells [54]. In addition, many compounds that regulate autophagy and inhibit tumor cells via the PI3K/Akt/mTOR pathway have been discovered in recent years. Protein kinase C and the sphingosine kinase inhibitor, Salingol, inhibit the phosphorylation level of key molecules of the PI3K/Akt/mTOR pathway—such as Akt, p70S6k, and rS6—to induce ACD in colon cancer cells [55]. A novel hybrid of the 3-benzyl coumarin seco-B-ring derivative and phenylsulfonylfuroxan (compound 6) reduces the phosphorylation levels of mTOR-S2448, Akt-S373, and the downstream p70s6K-S371 and 4EBP1-pT45 proteins in a concentration-/time-dependent manner to induce autophagy and apoptosis of A549 cells [56]. The antiprostate-cancer drug, cabazitaxel, also inhibits proliferation of A549 cells through autophagy which is regulated by the PI3K/Akt/mTOR pathway [57]. *Ophiopogon japonicus* is a traditional medicinal plant, and its main components are flavonoids and steroidal saponins. A previous study has shown that *O. japonicus* inhibits PI3K/Akt/mTOR signaling to induce ACD in A549 lung cancer cells [58]. Similarly, the inhibitory effect of capsaicin in the nasopharyngeal carcinoma cell line, NPC-TW01, is also achieved by autophagy regulated by the PI3K/Akt/mTOR pathway [59]. Specifically, mTORC1 is a key signaling molecule in the PI3K/Akt/mTOR pathway. Some small-molecule drugs, such as rapamycin and NVP-BEZ235, inhibit mTORC1 activity and induce autophagy and have a killing effect on T-cell acute lymphoblastic leukemia cells [60].

The MAPK pathway plays an important role in the process of tumor-cell proliferation, growth, apoptosis, and cell-to-cell functional synchronization. The MAPK family members, ERK, p38 MAPK, JNK, and ERK5 are also involved in the regulation of autophagy. CYT-Rx20, a derivative of β-nitrostyrene, inhibits proliferation in the breast cancer cell lines, MDA-MB-231 and MCF-7 [61]. A new binuclear palladacycle complex, AJ-5, does not only induce apoptosis in melanoma cells but also inhibit Akt/mTOR activity and activate p38 and ERK1/2 MAPK signaling to induce ACD, with dual roles in apoptosis and ACD to inhibit the proliferation of melanoma cells [62]. Similar to AJ-5, arsenic-trioxide and ionizing-radiation combination treatment on glioma cells also inhibits Akt/mTOR activity and activates ERK1/2

p53 is a tumor-suppressor protein that regulates the expression of multiple genes involved in apoptosis, growth inhibition, and DNA repair. Studies have shown that the regulation of autophagy by p53 is bidirectional depending on its localization. Cytoplasmic p53 inhibits autophagy in a transcriptional-independent manner, while nuclear p53 enhances autophagy by transactivation target gene.

cellular-metabolic pathways regulated by the AMPK pathway [53].

*4.1.2 Drugs that target PI3K/Akt/mTOR pathway*

*4.1.3 Drugs that target the MAPK pathway*

MAPK signaling to promote ACD [63].

*4.1.4 Drugs that target the p53 pathway*

**96**

*Small molecular inhibitors of autophagy-related molecules.*

P53 in human hepatocellular-carcinoma cells treated with fangchinoline is transported from the cytoplasm to the nucleus through nuclear translocation and selectively activates sestrin2 to initiate autophagy and promote ACD [64]. Numerous studies have shown that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) reactivates the normal activity of mutant p53 molecules in tumor cells and has an antitumor effect. PEITC has growth-inhibitory activity on tumor cells expressing p53R175H and restores the wide-type conformation and transcriptional activity of p53. In addition, PEITC enables p53R175H tumor cells to be sensitive to proteasome and autophagy-mediated degradation processes. Analysis of the mechanism showed that PEITC activates the classical p53 downstream target gene, causing tumor cells to arrest in the S phase and G2/M phase of the cell cycle and to undergo apoptosis [65].

#### *4.1.5 Drugs that target the autophagosome-lysosome fusion pathway*

The fusion of autophagosomes and lysosomes forms autolysosomes, which degrade the encapsulated materials. The degradation activity can be evaluated by autophagic flux. A previous study has shown that antimalarial drugs—such as quinine, chloroquine, and its derivatives—block the fusion processes of autophagosomes and lysosomes to reduce the autophagic flux and kill tumor cells by inhibiting autophagy activity [66]. Bafilomycin A1 is a vesicular H<sup>+</sup> -ATPase proton-pump inhibitor that inhibits autolysosome activity to promote tumor cell killing by inhibiting the activity of vacuolar V-ATPases [67]. In addition to the above drugs, studies in recent years have shown that some small-molecule drugs, such as lys05 and DQ661, share similar antitumor mechanisms [68, 69].

#### *4.1.6 Drugs that target the Hh pathway*

The Hh pathway plays an important role in embryonic development and tissue regeneration. Abnormal expression of SMO, PTCH, and Gli1 molecules in the Hh pathways is associated with tumorigenesis. The Gli1 small-molecule inhibitor, GANT61, does not only inhibit the proliferation of human hepatocellular-carcinoma cells but also synergize with itraconazole to kill breast cancer cells through the ACD pathway [18]. HhAntag, a small-molecule inhibitor of downstream element of Hh pathway, inhibits embryonal rhabdomyosarcoma (ERMS) proliferation through autophagy induction [70]. SMO antagonist, Sonidegib, selectively targets cell migration and adhesion of mantle cell lymphoma (MCL) and suppresses the proliferation of MCL via autophagy inhibition [71].

#### *4.1.7 Drugs that target the epigenetic pathways*

Epigenetic modification can regulate the occurrence of autophagy, including histone acetylation and DNA methylation, which play an important role in regulating the biological function of autophagy. Inhibition of HDAC activity to induce cell death has been the research strategy of antitumor drugs. Development of small-molecule inhibitors targeting HDAC has become a primary focus in this field. The small-molecule inhibitor, SAHA, does not only induce a killing effect on CSCs but also promote colon cancer and liver cancer cell death by FoxO1-dependent autophagy [72]. Panobinostat (LBH589) induces autophagy in a tumor-suppressor death-associated protein kinase (DAPK)-dependent manner and inhibits colon cancer cell proliferation. The combination of panobinostat and sorafenib significantly improves the therapeutic outcomes of liver cancer treatment [73, 74]. MGCD0103 not only induces apoptosis in B-cell chronic lymphocytic leukemia (CLL) but also inhibits tumor-cell protective autophagy and promotes CCL cell death through the activation of the PI3K/AKT/mTOR signaling molecules and caspase pathways [75].

#### **4.2 Small molecular inhibitors of autophagy-related molecules**

#### *4.2.1 Atg1/ULK1*

The autophagy-related gene, Atg1 homologue, ULK1, is an unc-51-like serine/threonine kinase that plays an important role in initiating autophagy. When cells are under nutrient deficiency, hypoxia, and other stresses, the upstream molecules of ULK1, mTORC1, and AMPK are activated, and the phosphorylation of ULK1 and Atg13 molecules regulates autophagy. In recent years, ULK1 has been a popular molecule in the research of autophagy-targeted drugs. ULK1 not only is a promoter of autophagy but is also an important kinase, which is more favorable for the study of small-molecule inhibitors. The high-throughput screening of Egan et al. [76] has shown that the small-molecule compound, SBI-0206965, inhibits ULK1 activity through the mTOR pathway, leading to a decrease in autophagy and inhibition of A549 cell proliferation. Unlike SBI-0206965, MRT67307 and MRT68921 inhibit both ULK1 and ULK2 activities and block autophagy [77]. The small-molecule compound, LYN-1604, and ULK1

**99**

*Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy*

reactive proteins—ATF3, RAD21, and caspase 3—activate autophagy to inhibit the

Vps34 is a Class III phosphoinositide-3 kinase (PI3K-III), and its 3-phosphophosphatidylinositol (PI3P), which catalyzes the formation of the substrate phosphatidylinositol (PI), is necessary for autophagosome formation. The smallmolecule inhibitor, SAR405, inhibits the catalytic activity of PIK3C3/Vps34, blocks the transport of vesicles from late endosomes to lysosomes, and inhibits autophagy, which suppresses tumor-cell proliferation [79]. In addition, PIK-III and Vps34-IN1 are also Vps34 inhibitors, which inhibit autophagy and have killing effects on tumor cells [80]. Spautin-1 is a potent and specific small-molecule inhibitor. It inhibits two ubiquitin-specific peptidases, USP10 and USP13, and promotes ubiquitination and degradation of the Vps34-PI3K complex, thereby inhibiting autophagy [81].

Atg4 is a key molecule in autophagy that cleaves the C-terminal arginine of Atg8/LC3 to expose the C-terminal glycine residue for covalent attachment to phosphatidylethanolamine (PE). This process induces Atg8/LC3-PE to be anchored to the membrane of autophagosomes to regulate autophagy. Fu et al. [82] identified a novel ATG4B small-molecule inhibitor, S130, through in silico screening and in vitro high-throughput screening system for fluorescence resonance-energy transfer and showed that S130 did not affect the autophagosome function and autophagosome fusion with lysosomes. Instead, S130 inhibited the delipidation process of LC3-PE and ultimately blocked autophagy. An antitumor study has shown that S130 effectively inhibits the growth of colon cancer cells, and nutrient deficiency further enhances the antitumor effect of S130; however, Atg4 overexpression partially counteracts the tumor-cell death process caused by S130. Similar to S130, NSC185058 is also a newly discovered small-molecule inhibitor that acts on the Atg4B target and inhibits autophagy after LC3B lipidation, leading to the death

Bcl-2 is an antiapoptotic protein containing multiple Bcl-2 homology (BH) domains. Under starvation conditions, activation of JNK1 phosphorylates Bcl-2 protein and causes the separation between Bcl-2 and Beclin1, thereby inducing autophagy [84]. In addition, multiple Bcl-2 homology 3 (BH3) mimetics can disrupt the interaction of Bcl-2/xl with Beclin1 to induce autophagy by competing for the BH3 domains [7]. Obatoclax (GX15-070) is a pan-Bcl2 inhibitor that induces apoptosis in various tumor cells. It also induces ACD in acute lymphoblastic leukemia cells in an Atg-dependent manner [85]. ABT-737, as a BH analogue, binds to the BH3 binding groove of Bcl-2 and Bcl-xl. A study by Maiuri et al. [86] has shown that the degree of polymerization of Beclin1, Bcl-2, and Bcl-xl is significantly reduced in ABT-737-treated cells, which enhances the autophagy level. However, ABT-737 also enhances the sensitivity of colon cancer cells to the chemotherapeutic drug, ixazomib, through downregulation of Mcl-1 and autophagy inhibition [87]. Similarly, gossypol is a small-molecule inhibitor of Bcl-2. Under natural conditions, gossypol is a mixture of (+)-gossypol and (−)-gossypol, and compared with (+)-gossypol, (−)-gossypol has higher antitumor activity. Gossypol and (−)-gossypol induce autophagy by inhibiting the response of Beclin1 and Bcl-2 in different tumor cells.

proliferation of MDA-MB-231 triple-negative breast cancer cells [78].

*DOI: http://dx.doi.org/10.5772/intechopen.88050*

of the Saos-2 osteosarcoma cells [83].

*4.2.4 B-cell lymphoma/leukemia-2 (Bcl-2)*

*4.2.2 Vps34*

*4.2.3 Atg4B*

reactive proteins—ATF3, RAD21, and caspase 3—activate autophagy to inhibit the proliferation of MDA-MB-231 triple-negative breast cancer cells [78].

#### *4.2.2 Vps34*

*Programmed Cell Death*

autophagy activity [66]. Bafilomycin A1 is a vesicular H<sup>+</sup>

and DQ661, share similar antitumor mechanisms [68, 69].

proliferation of MCL via autophagy inhibition [71].

*4.1.7 Drugs that target the epigenetic pathways*

molecules and caspase pathways [75].

*4.2.1 Atg1/ULK1*

**4.2 Small molecular inhibitors of autophagy-related molecules**

The autophagy-related gene, Atg1 homologue, ULK1, is an unc-51-like serine/threonine kinase that plays an important role in initiating autophagy. When cells are under nutrient deficiency, hypoxia, and other stresses, the upstream molecules of ULK1, mTORC1, and AMPK are activated, and the phosphorylation of ULK1 and Atg13 molecules regulates autophagy. In recent years, ULK1 has been a popular molecule in the research of autophagy-targeted drugs. ULK1 not only is a promoter of autophagy but is also an important kinase, which is more favorable for the study of small-molecule inhibitors. The high-throughput screening of Egan et al. [76] has shown that the small-molecule compound, SBI-0206965, inhibits ULK1 activity through the mTOR pathway, leading to a decrease in autophagy and inhibition of A549 cell proliferation. Unlike SBI-0206965, MRT67307 and MRT68921 inhibit both ULK1 and ULK2 activities and block autophagy [77]. The small-molecule compound, LYN-1604, and ULK1

*4.1.6 Drugs that target the Hh pathway*

inhibitor that inhibits autolysosome activity to promote tumor cell killing by inhibiting the activity of vacuolar V-ATPases [67]. In addition to the above drugs, studies in recent years have shown that some small-molecule drugs, such as lys05

The Hh pathway plays an important role in embryonic development and tissue regeneration. Abnormal expression of SMO, PTCH, and Gli1 molecules in the Hh pathways is associated with tumorigenesis. The Gli1 small-molecule inhibitor, GANT61, does not only inhibit the proliferation of human hepatocellular-carcinoma cells but also synergize with itraconazole to kill breast cancer cells through the ACD pathway [18]. HhAntag, a small-molecule inhibitor of downstream element of Hh pathway, inhibits embryonal rhabdomyosarcoma (ERMS) proliferation through autophagy induction [70]. SMO antagonist, Sonidegib, selectively targets cell migration and adhesion of mantle cell lymphoma (MCL) and suppresses the

Epigenetic modification can regulate the occurrence of autophagy, including histone acetylation and DNA methylation, which play an important role in regulating the biological function of autophagy. Inhibition of HDAC activity to induce cell death has been the research strategy of antitumor drugs. Development of small-molecule inhibitors targeting HDAC has become a primary focus in this field. The small-molecule inhibitor, SAHA, does not only induce a killing effect on CSCs but also promote colon cancer and liver cancer cell death by FoxO1-dependent autophagy [72]. Panobinostat (LBH589) induces autophagy in a tumor-suppressor death-associated protein kinase (DAPK)-dependent manner and inhibits colon cancer cell proliferation. The combination of panobinostat and sorafenib significantly improves the therapeutic outcomes of liver cancer treatment [73, 74]. MGCD0103 not only induces apoptosis in B-cell chronic lymphocytic leukemia (CLL) but also inhibits tumor-cell protective autophagy and promotes CCL cell death through the activation of the PI3K/AKT/mTOR signaling


**98**

Vps34 is a Class III phosphoinositide-3 kinase (PI3K-III), and its 3-phosphophosphatidylinositol (PI3P), which catalyzes the formation of the substrate phosphatidylinositol (PI), is necessary for autophagosome formation. The smallmolecule inhibitor, SAR405, inhibits the catalytic activity of PIK3C3/Vps34, blocks the transport of vesicles from late endosomes to lysosomes, and inhibits autophagy, which suppresses tumor-cell proliferation [79]. In addition, PIK-III and Vps34-IN1 are also Vps34 inhibitors, which inhibit autophagy and have killing effects on tumor cells [80]. Spautin-1 is a potent and specific small-molecule inhibitor. It inhibits two ubiquitin-specific peptidases, USP10 and USP13, and promotes ubiquitination and degradation of the Vps34-PI3K complex, thereby inhibiting autophagy [81].

#### *4.2.3 Atg4B*

Atg4 is a key molecule in autophagy that cleaves the C-terminal arginine of Atg8/LC3 to expose the C-terminal glycine residue for covalent attachment to phosphatidylethanolamine (PE). This process induces Atg8/LC3-PE to be anchored to the membrane of autophagosomes to regulate autophagy. Fu et al. [82] identified a novel ATG4B small-molecule inhibitor, S130, through in silico screening and in vitro high-throughput screening system for fluorescence resonance-energy transfer and showed that S130 did not affect the autophagosome function and autophagosome fusion with lysosomes. Instead, S130 inhibited the delipidation process of LC3-PE and ultimately blocked autophagy. An antitumor study has shown that S130 effectively inhibits the growth of colon cancer cells, and nutrient deficiency further enhances the antitumor effect of S130; however, Atg4 overexpression partially counteracts the tumor-cell death process caused by S130. Similar to S130, NSC185058 is also a newly discovered small-molecule inhibitor that acts on the Atg4B target and inhibits autophagy after LC3B lipidation, leading to the death of the Saos-2 osteosarcoma cells [83].

#### *4.2.4 B-cell lymphoma/leukemia-2 (Bcl-2)*

Bcl-2 is an antiapoptotic protein containing multiple Bcl-2 homology (BH) domains. Under starvation conditions, activation of JNK1 phosphorylates Bcl-2 protein and causes the separation between Bcl-2 and Beclin1, thereby inducing autophagy [84]. In addition, multiple Bcl-2 homology 3 (BH3) mimetics can disrupt the interaction of Bcl-2/xl with Beclin1 to induce autophagy by competing for the BH3 domains [7]. Obatoclax (GX15-070) is a pan-Bcl2 inhibitor that induces apoptosis in various tumor cells. It also induces ACD in acute lymphoblastic leukemia cells in an Atg-dependent manner [85]. ABT-737, as a BH analogue, binds to the BH3 binding groove of Bcl-2 and Bcl-xl. A study by Maiuri et al. [86] has shown that the degree of polymerization of Beclin1, Bcl-2, and Bcl-xl is significantly reduced in ABT-737-treated cells, which enhances the autophagy level. However, ABT-737 also enhances the sensitivity of colon cancer cells to the chemotherapeutic drug, ixazomib, through downregulation of Mcl-1 and autophagy inhibition [87]. Similarly, gossypol is a small-molecule inhibitor of Bcl-2. Under natural conditions, gossypol is a mixture of (+)-gossypol and (−)-gossypol, and compared with (+)-gossypol, (−)-gossypol has higher antitumor activity. Gossypol and (−)-gossypol induce autophagy by inhibiting the response of Beclin1 and Bcl-2 in different tumor cells.

They can inactivate cytoprotective autophagy to cause tumor cells to escape [88], promote pro-survival autophagy, and initiate ACD [89].

#### *4.2.5 p62*

p62, also known as SQSTM1 protein, plays an autophagy and apoptotic role in tumor cells. The LIR domain of four domains of the p62 protein is responsible for binding to the autophagy-receptor protein, Atg8/LC3, while the UBA domain can recruit ubiquitinated proteins to mediate autophagic degradation. Verteporfin is an inhibitor of p62 and has no effect on cell growth under normal conditions. However, verteporfin reduces the survival of MCF-7 cells during nutrient deprivation. In vivo experiments have shown that verteporfin also inhibits tumor formation frequency of PC-3 prostate cancer cells in a xenograft model [90].

In addition to small-molecule drugs and natural extracts, some macromolecularantibody drugs and noncoding miRNAs (miR)—such as anti-EGFR monoclonal antibody panitumumab, anti-20 monoclonal antibody rituximab, miR-22, and miR-101—have been reported to inhibit tumors in recent years through regulating autophagy activity [91, 92]. Although the emergence of autophagy provides a new idea for the development of antitumor drugs, anticancer treatments still encounter many challenges. Screening for drugs that target autophagy activity in cells may be the solution to some of these problems in the future.

#### **5. Conclusion**

The molecular mechanism and biological function of autophagy are now basically clear, and Janus role of autophagy determines that it plays an important role in the antitumor process. Some compounds, plant extracts killing tumor cells through the regulation of autophagy activity, especially induced autophagic cell death has also become an important strategy against tumor. However, there are still many obstacles to overcome in order to develop autophagic drugs; for example, there is a lack of specific biomarkers to distinguish autophagic cell death from other types of cell death. There is also a need to clarify which type of autophagy, cytoprotective, or cytotoxic should be targeted. Moreover it is difficult but important to determine to what extent autophagy should be induced before the cells reach the point of no return and undergo autophagic cell death.

#### **Author details**

Hai Zhang and Zhinan Chen\* Department of Cell Biology, National Translational Science Center for Molecular Medicine, The Fourth Military Medical University, Xi'an, China

\*Address all correspondence to: znchen@fmmu.edu.cn

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**101**

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[12] Suzuki NN, Yoshimoto K, Fujioka Y, et al. The crystal structure of plant ATG12 and its biological implication in autophagy. Autophagy. 2005;**1**:119-126.

[13] Kimura S, Fujita N, Noda T, et al. Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. Methods in Enzymology. 2009;**452**:1-12. DOI: 10.1016/ S0076-6879(08)03601-X

[14] Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. The Journal of Biological Chemistry. 2007;**282**:24131-24145. DOI:

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[16] Alers S, Loffler AS, Wesselborg S, et al. Role of AMPK-mTOR-Ulk1/2 in

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10.1007/82\_2017\_6

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*Programmed Cell Death*

*4.2.5 p62*

**5. Conclusion**

**Author details**

Hai Zhang and Zhinan Chen\*

They can inactivate cytoprotective autophagy to cause tumor cells to escape [88],

p62, also known as SQSTM1 protein, plays an autophagy and apoptotic role in tumor cells. The LIR domain of four domains of the p62 protein is responsible for binding to the autophagy-receptor protein, Atg8/LC3, while the UBA domain can recruit ubiquitinated proteins to mediate autophagic degradation. Verteporfin is an inhibitor of p62 and has no effect on cell growth under normal conditions. However, verteporfin reduces the survival of MCF-7 cells during nutrient deprivation. In vivo experiments have shown that verteporfin also inhibits tumor formation

In addition to small-molecule drugs and natural extracts, some macromolecularantibody drugs and noncoding miRNAs (miR)—such as anti-EGFR monoclonal antibody panitumumab, anti-20 monoclonal antibody rituximab, miR-22, and miR-101—have been reported to inhibit tumors in recent years through regulating autophagy activity [91, 92]. Although the emergence of autophagy provides a new idea for the development of antitumor drugs, anticancer treatments still encounter many challenges. Screening for drugs that target autophagy activity in cells may be

The molecular mechanism and biological function of autophagy are now basically clear, and Janus role of autophagy determines that it plays an important role in the antitumor process. Some compounds, plant extracts killing tumor cells through the regulation of autophagy activity, especially induced autophagic cell death has also become an important strategy against tumor. However, there are still many obstacles to overcome in order to develop autophagic drugs; for example, there is a lack of specific biomarkers to distinguish autophagic cell death from other types of cell death. There is also a need to clarify which type of autophagy, cytoprotective, or cytotoxic should be targeted. Moreover it is difficult but important to determine to what extent autophagy should be induced before the cells reach the point of no

Department of Cell Biology, National Translational Science Center for Molecular

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Medicine, The Fourth Military Medical University, Xi'an, China

\*Address all correspondence to: znchen@fmmu.edu.cn

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[90] Wang L, Kim D, Wise J, et al. p62 as a therapeutic target for inhibition of autophagy in prostate cancer. The Prostate. 2018;**78**:390-400. DOI: 10.1002/pros.23483

[91] Alinari L, Yu B, Christian BA, et al. Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has *in vitro* and *in vivo* activity in mantle cell lymphoma. Blood. 2011;**117**:4530-4541. DOI: 10.1182/ blood-2010-08-303354

[92] Zhang H, Tang J, Li C, et al. MiR-22 regulates 5-FU sensitivity by inhibiting autophagy and promoting apoptosis in colorectal cancer cells. Cancer Letters. 2015;**356**:781-790. DOI: 10.1016/j. canlet.2014.10.029

**109**

**Chapter 6**

**Abstract**

ling pathways.

**1. Introduction**

Diseases

Autophagy and Cell Death in

*Samo Ribarič and Irina Milisav Ribarič*

Alzheimer's, Parkinson's and Prion

Neurodegenerative brain disorders (NBD) impair brain cells' proteostasis with the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden of these proteins elicits the unfolded protein response (UPR) and stimulates autophagy (AUT). In the short term, UPR and AUT attenuate ER's burden. With prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the UPR, to meet the increased protein burden, leads to an increase in cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote cell death. This review describes the interlinked signalling pathways of AUT, apoptosis and necroptosis and their modulation by Alzheimer's, Parkinson's and prion diseases and outlines the pharmacological strategies for targeting AUT, apoptosis and necroptosis signal-

**Keywords:** Alzheimer's disease, apoptosis, autophagy, necroptosis,

**1.1 Proteostasis in neurodegenerative brain disorders (NBD)**

neurodegenerative brain disorders, Parkinson's disease, prion diseases, proteostasis

Proteostasis integrates synthesis, folding, trafficking and degradation of proteins. It is perturbed in the early stages of neurodegenerative brain disorders (NBD), before clinical manifestations [1–3]. Mutant, misfolded or unfolded proteins (P) or increased P production increases the endoplasmic reticulum (ER) protein burden in NBD such as Alzheimer's (AD), Parkinson's (PD) and prion diseases (PrD). This increased ER burden stimulates the unfolded protein response (UPR) and autophagy (AUT). The UPR response to ER stress is dichotomous [4–7]. During acute ER stress, UPR supports cell survival, by reducing ER's protein folding load and increasing ER's protein folding capacity. With prolonged ER stress, the UPR preferentially represses cell survival and triggers apoptosis. The failure of ER's stress responses (i.e. increased protein folding capacity and enhanced removal of mutant, misfolded or unfolded proteins by the UPR pathway) to attenuate the P burden leads to an increase in cytosolic P accumulation that further stimulates AUT. Over time, these P undergo post-translational changes and produce toxic monomers and

#### **Chapter 6**

## Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases

*Samo Ribarič and Irina Milisav Ribarič*

#### **Abstract**

Neurodegenerative brain disorders (NBD) impair brain cells' proteostasis with the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden of these proteins elicits the unfolded protein response (UPR) and stimulates autophagy (AUT). In the short term, UPR and AUT attenuate ER's burden. With prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the UPR, to meet the increased protein burden, leads to an increase in cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote cell death. This review describes the interlinked signalling pathways of AUT, apoptosis and necroptosis and their modulation by Alzheimer's, Parkinson's and prion diseases and outlines the pharmacological strategies for targeting AUT, apoptosis and necroptosis signalling pathways.

**Keywords:** Alzheimer's disease, apoptosis, autophagy, necroptosis, neurodegenerative brain disorders, Parkinson's disease, prion diseases, proteostasis

#### **1. Introduction**

#### **1.1 Proteostasis in neurodegenerative brain disorders (NBD)**

Proteostasis integrates synthesis, folding, trafficking and degradation of proteins. It is perturbed in the early stages of neurodegenerative brain disorders (NBD), before clinical manifestations [1–3]. Mutant, misfolded or unfolded proteins (P) or increased P production increases the endoplasmic reticulum (ER) protein burden in NBD such as Alzheimer's (AD), Parkinson's (PD) and prion diseases (PrD). This increased ER burden stimulates the unfolded protein response (UPR) and autophagy (AUT). The UPR response to ER stress is dichotomous [4–7]. During acute ER stress, UPR supports cell survival, by reducing ER's protein folding load and increasing ER's protein folding capacity. With prolonged ER stress, the UPR preferentially represses cell survival and triggers apoptosis. The failure of ER's stress responses (i.e. increased protein folding capacity and enhanced removal of mutant, misfolded or unfolded proteins by the UPR pathway) to attenuate the P burden leads to an increase in cytosolic P accumulation that further stimulates AUT. Over time, these P undergo post-translational changes and produce toxic monomers and

**Figure 1.**

*Proteostasis in human neurodegenerative brain disorders (NBD). Abbreviations: P (proteins), ER (endoplasmic reticulum), UPR (unfolded protein response), ROS (reactive oxygen species); red lines and arrows indicate progressive failure of proteostasis ultimately leading to NBD. Green arrows and lines indicate appropriate responses of proteostasis to altered P that prevent or slow down the progress of NBD.*

oligomers; their production is stimulated by chronic inflammation and increased reactive oxygen species (ROS) production. These monomers and oligomers repress AUT and trigger either apoptosis or necroptosis (**Figure 1**) [4, 6–8].

#### **1.2 Autophagy changes in selected NBD**

An efficient autophagy (AUT) delays or attenuates the progression of AD, PD and PrD [9–12]. A summary of AUT changes in selected NBD is shown in **Figure 2**. Post-translationally modified proteins (PTMP)—such as soluble amyloid β-peptide 42 with a single oxidised methionine residue at position 35 (Aβ42-MET35-OX) in Alzheimer's disease, alpha-synuclein oxidised on methionine residues (MET-OXαSYN) in Parkinson's disease and oxidised, self-propagating infectious isoforms of prion protein (MET-OX-PRPSc) in prion diseases (PrD)—inhibit (a) AUT, in AD, PD and PrD, and also (b) mitochondrial (MITO) function [13–23]. MET-OX-PRPSc indirectly damage MITO function. The normal prion protein (PrPc ) binds with

**111**

**Figure 2.**

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

a variety of molecules, including copper ions [24, 25], and PrPc

on copper binding for its antioxidant function. Loss of PrPc

oxidase and other MITO proteins [27–30].

correlate with Cu/Zn superoxide dismutase, glutathione reductase and cytochrome c oxidase activities [26]. These observations support the hypothesis that PrPc

*Summary of AUT changes in selected NBD. Abbreviations: AKT (protein kinase B), GSK3 (glycogen synthase kinase 3), JNK (c-Jun N-terminal kinase), TAU (TAU protein), TAU-P (phosphorylated TAU protein).*

an important endogenous scavenger, protecting structural and signalling proteins from oxidation, due to its high number of methionine residues, and (b) vital for the intracellular transport of copper to superoxide dismutase, which is dependent

PrPSc and MET-OX-PrPSc, which do not bind copper and have a reduced antioxidant activity, reduces the cell's intracellular antioxidant and copper transport capacity and precipitates MITO dysfunction, due to an increased oxidation of cytochrome c

AUT is inhibited at the stage of protein digestion (during autolysosome cargo degradation) by the undigestible PTMP and is diverted to the formation of large endocytic vacuoles that rupture and release the undigested PTMP into the cytosol, thus progressively increasing their intracellular concentration. PTMP of AD and PD accelerate microtube cytoskeletal depolarisation, thus blocking autolysosome retrograde trafficking and accelerating loss of neurites, synapses and synaptic transmission [31–39]. PTMP inhibition of MITO function leads to (a) a reduced ATP production and an increased MITO release of ROS and Ca2+ into the cytosol [38, 40–44] and

expression levels

, due to conversion to

is (a)

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

**Figure 2.**

*Programmed Cell Death*

**110**

**Figure 1.**

oligomers; their production is stimulated by chronic inflammation and increased reactive oxygen species (ROS) production. These monomers and oligomers repress

*Proteostasis in human neurodegenerative brain disorders (NBD). Abbreviations: P (proteins), ER (endoplasmic reticulum), UPR (unfolded protein response), ROS (reactive oxygen species); red lines and arrows indicate progressive failure of proteostasis ultimately leading to NBD. Green arrows and lines indicate* 

*appropriate responses of proteostasis to altered P that prevent or slow down the progress of NBD.*

An efficient autophagy (AUT) delays or attenuates the progression of AD, PD and PrD [9–12]. A summary of AUT changes in selected NBD is shown in **Figure 2**. Post-translationally modified proteins (PTMP)—such as soluble amyloid β-peptide 42 with a single oxidised methionine residue at position 35 (Aβ42-MET35-OX) in Alzheimer's disease, alpha-synuclein oxidised on methionine residues (MET-OXαSYN) in Parkinson's disease and oxidised, self-propagating infectious isoforms of prion protein (MET-OX-PRPSc) in prion diseases (PrD)—inhibit (a) AUT, in AD, PD and PrD, and also (b) mitochondrial (MITO) function [13–23]. MET-OX-PRPSc

) binds with

AUT and trigger either apoptosis or necroptosis (**Figure 1**) [4, 6–8].

indirectly damage MITO function. The normal prion protein (PrPc

**1.2 Autophagy changes in selected NBD**

*Summary of AUT changes in selected NBD. Abbreviations: AKT (protein kinase B), GSK3 (glycogen synthase kinase 3), JNK (c-Jun N-terminal kinase), TAU (TAU protein), TAU-P (phosphorylated TAU protein).*

a variety of molecules, including copper ions [24, 25], and PrPc expression levels correlate with Cu/Zn superoxide dismutase, glutathione reductase and cytochrome c oxidase activities [26]. These observations support the hypothesis that PrPc is (a) an important endogenous scavenger, protecting structural and signalling proteins from oxidation, due to its high number of methionine residues, and (b) vital for the intracellular transport of copper to superoxide dismutase, which is dependent on copper binding for its antioxidant function. Loss of PrPc , due to conversion to PrPSc and MET-OX-PrPSc, which do not bind copper and have a reduced antioxidant activity, reduces the cell's intracellular antioxidant and copper transport capacity and precipitates MITO dysfunction, due to an increased oxidation of cytochrome c oxidase and other MITO proteins [27–30].

AUT is inhibited at the stage of protein digestion (during autolysosome cargo degradation) by the undigestible PTMP and is diverted to the formation of large endocytic vacuoles that rupture and release the undigested PTMP into the cytosol, thus progressively increasing their intracellular concentration. PTMP of AD and PD accelerate microtube cytoskeletal depolarisation, thus blocking autolysosome retrograde trafficking and accelerating loss of neurites, synapses and synaptic transmission [31–39]. PTMP inhibition of MITO function leads to (a) a reduced ATP production and an increased MITO release of ROS and Ca2+ into the cytosol [38, 40–44] and

(b) activation of inflammasomes with an increased release of cytokines interleukin 1 (IL1), from microglia, and tumour necrosis factor alpha (TNFα), from astrocytes and neurons, and finally apoptosis or necroptosis [38, 45–52]. Apoptosis or necroptosis of nerve cells and astrocytes releases PTMP and their oligomers into the extracellular space, thus contributing to the spread of inflammation and neurodegenerative disorder in the brain. The physiological process of apoptosis that normally prevents the spill of cell's molecules to the extracellular space is perturbed by the altered proteostasis into a pathological one in NBD. This transformation is sustained by several intracellular processes including the accumulation of undigestible PTMP, increased oxidative stress, and distorted expression of apoptotic proteins [53–56].

The AUT capacity of brain cells is important in the regulation of immune responses and inflammation that occur in NBD [57, 58]. Protein aggregates (aggresomes), present in age-related NBD, activate inflammasomes. Activated inflammasomes lead to a low-grade inflammation associated with a declined autophagic capacity [59]. On the other hand, autophagy attenuation leads to inflammasome precipitated excessive caspase-1 activation and elevated IL-1β secretion in response to lipopolysaccharide (LPS) stimulation [10, 60, 61]. Also, ER stress and inflammation coexist in NBD, for example, in AD, and are intertwined [57]. Chronic neuroinflammation (CNI) develops into a self-damaging process and is an important factor in sustaining NBD including AD, PD and PRD. CNI includes activation of microglia and astrocytes and infiltration of peripheral immune cells. Transient activation of microglia, accompanied by the release of inflammatory cytokines that amplify the inflammatory response by activating and recruiting astrocytes and peripheral immune cells to the brain lesion, ensures the brain's integrity by removing foreign bodies and cell debris. CNI is toxic to neurons due to sustained release of inflammatory cytokines (e.g. ILs 1β and 6, TNFα) and ROS and microglial phagocytosis of neighbouring intact nerve cells, thus contributing to the development and progression of NBD. The progressive loss of neurons further contributes to generation of cell debris and sustains microglial hyperactivation [62].

The detrimental effects of PTMP, sustained inflammation and increased ROS production are further exacerbated by the formation of AUT-resistant soluble Aβ oligomers (AβO) in AD and AUT-resistant αSYN oligomers in PD that further stimulate chronic inflammation and increased cytosolic ROS, contributing to apoptosis or necroptosis of neurons. Therefore, activation of apoptosis or necroptosis in AD, PD or PrD is triggered by a positive feedback loop between chronic inflammation in the brain (to which astrocytes and microglia are the main contributor) and the production of PTMP. In addition to high levels of ROS, the production of PTMP in the cytosol is facilitated by copper ions in AD [63] and by iron ions, dopamine and accumulation of alpha-synuclein (the precursor of oxidised αSYN monomer) in PD [17]. Although chronic brain inflammation contributes to the process of PrPSc production, it is not necessary to sustain it, since the PrPSc only needs the PrPc molecules for its propagation [64].

#### **2. Crosstalk among AUT, apoptosis and necroptosis signalling pathways in selected NBD**

AUT, apoptosis and necroptosis have interlinked signalling pathways. Examples of key signalling molecules that regulate the transition among these three processes are presented in Section 2.1. The crosstalk among AUT, apoptosis and necroptosis signalling pathways, with the potential sites of modulation by Alzheimer's, Parkinson's and prion diseases (PrD), is summarised in **Figure 3**.

**113**

*kinase ULK1); XIAP (X-linked inhibitor of apoptosis protein).*

**Figure 3.**

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

*Crosstalk among AUT, apoptosis and necroptosis signalling pathways with the potential sites of modulation by AD, PD and PrD. Abbreviations: αSYN (alpha-synuclein); AβO (amyloid β oligomers); AβP (amyloid β monomers with 39 to 42 amino acid residues); AIF (apoptosis-inducing factor); AKT (protein kinase B); AMPK (5' AMPactivated protein kinase); AP-1 (activator protein 1); APAF-1 (apoptotic protease activating factor 1); APO-3L (APO3 ligand); ASK-1 (apoptosis signal-regulating kinase 1); ATG-5 (AUT-related 5); BAD (Bcl2-associated agonist of cell death); BAK (Bcl-2 homologous antagonist/killer); BAX (apoptosis regulator BAX); BCL-2 (B-cell lymphoma 2); BCL-XL (B-cell lymphoma-extra large); Beclin-1 (mammalian ortholog of the yeast AUT-related gene 6 (ATG-6)); BID (BH3 interacting domain death agonist); BIP-P (phosphorylated binding immunoglobulin protein); C-FLIP (FADD-like IL-1β-converting enzyme-inhibitory protein); calpain (proteolytic enzyme, a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases); CASP-3, CASP-8/10, CASP-9 (caspase-3, caspase-8/10, caspase-9); cIAP-1 (cellular inhibitor of apoptosis protein 1); CL-ATG5 (cleaved AUTrelated 5 (ATG5) protein); CL-BAX (cleaved apoptosis regulator BAX); CL-Beclin-1 (cleaved mammalian ortholog of the yeast AUT-related gene 6); Complex-I (TNFα bound to TNFα receptor that is associated with TRADD (tumour necrosis factor receptor type 1-associated death domain protein), RIPK1 (receptor-interacting serine/ threonine-protein kinase 1), TRAF2 (TNF receptor-associated factor 2) and cIAP-1/2 (cellular inhibitor of apoptosis protein 1 and 2)); Complex-IIa (pro-caspase-8, RIPK1, FADD (FAS-associated protein with death domain)); Complex-IIb (pro-caspase-8, RIPK1, RIPK3 (receptor-interacting serine/threonine-protein kinase 3), FADD, MLKL (mixed lineage kinase domain-like pseudokinase)); CYT-C (cytochrome c); DAMPs (damage-associated molecular patterns); DCAP-AKT (activation of toll/IL-1R (TIR) domain-containing adaptor proteins (e.g. mal, TRIF, TRIF-related adaptor molecule, IL-1R-associated kinase-1, IL-1R-associated kinase-M, MAPK, TNFR-associated factor 6, toll-interacting protein)); FAS (apoptosis antigen 1); HMGB-1 (high-mobility group box 1 protein); IKK (IκB kinase enzyme complex, part of the upstream NF-κB signal transduction cascade); IL-1β (interleukin-1 beta); JAK (Janus kinase); JNK (c-Jun N-terminal kinase); JNK-P (phosphorylated c-Jun N-terminal kinase); MITO (mitochondrial); MITO MP (mitochondrial membrane permeability); MITO ROS (mitochondrial reactive oxygen species); MKK7 (MAP kinase kinase 7); MLKL-O (MLKL oligomerisation with translocation and insertion into cell's and organelles' membranes with increased permeability); MLKL-P (phosphorylated pseudokinase mixed lineage kinase domain-like protein); mTORC1 (mammalian target of rapamycin complex 1); NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells protein complex); NOXA (adult T cell leukaemia-derived PMAresponsive); OMI alias HtrA2 (serine protease HTRA2); ORG (cell organelles); p53 (cellular tumour antigen p53); p62 (nucleoporin p62 protein complex associated with the nuclear envelope); PRO CASP 8/10 (pro-caspase-8/10); PRPC (normal form of prion protein); PRPSC (self-propagating, protease-resistant, infectious isoforms of prion protein); PTM (post-translationally modified); PUMA (p53 upregulated modulator of apoptosis); RIP-1 (receptorinteracting serine/threonine-protein kinase 1); ROS (reactive oxygen species, e.g. peroxides, superoxide, hydroxyl radical or singlet oxygen); SMAC (second mitochondria-derived activator of caspases); STAT (signal transducer and activator of transcription 3/5); tBID (truncated BID protein); TLR-4 (toll-like receptor 4, member of the pattern recognition receptor (PRR) family); TNFα (tumour necrosis factor alpha); TNFαR (tumour necrosis factor alpha receptor); TNFRS (tumour necrosis factor receptor superfamily); TRAF (TNF receptor-associated factor); TRAIL (TNF-related apoptosis-inducing ligand); UBIQ PROT (ubiquitinated proteins); ULK1 (serine/threonine-protein* 

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

#### **Figure 3.**

*Programmed Cell Death*

(b) activation of inflammasomes with an increased release of cytokines interleukin 1 (IL1), from microglia, and tumour necrosis factor alpha (TNFα), from astrocytes and neurons, and finally apoptosis or necroptosis [38, 45–52]. Apoptosis or necroptosis of nerve cells and astrocytes releases PTMP and their oligomers into the extracellular space, thus contributing to the spread of inflammation and neurodegenerative disorder in the brain. The physiological process of apoptosis that normally prevents the spill of cell's molecules to the extracellular space is perturbed by the altered proteostasis into a pathological one in NBD. This transformation is sustained by several intracellular processes including the accumulation of undigestible PTMP, increased oxidative stress,

The AUT capacity of brain cells is important in the regulation of immune responses and inflammation that occur in NBD [57, 58]. Protein aggregates (aggresomes), present in age-related NBD, activate inflammasomes. Activated inflammasomes lead to a low-grade inflammation associated with a declined autophagic capacity [59]. On the other hand, autophagy attenuation leads to inflammasome precipitated excessive caspase-1 activation and elevated IL-1β secretion in response to lipopolysaccharide (LPS) stimulation [10, 60, 61]. Also, ER stress and inflammation coexist in NBD, for example, in AD, and are intertwined [57]. Chronic neuroinflammation (CNI) develops into a self-damaging process and is an important factor in sustaining NBD including AD, PD and PRD. CNI includes activation of microglia and astrocytes and infiltration of peripheral immune cells. Transient activation of microglia, accompanied by the release of inflammatory cytokines that amplify the inflammatory response by activating and recruiting astrocytes and peripheral immune cells to the brain lesion, ensures the brain's integrity by removing foreign bodies and cell debris. CNI is toxic to neurons due to sustained release of inflammatory cytokines (e.g. ILs 1β and 6, TNFα) and ROS and microglial phagocytosis of neighbouring intact nerve cells, thus contributing to the development and progression of NBD. The progressive loss of neurons further contributes to generation of cell debris and

The detrimental effects of PTMP, sustained inflammation and increased ROS production are further exacerbated by the formation of AUT-resistant soluble Aβ oligomers (AβO) in AD and AUT-resistant αSYN oligomers in PD that further stimulate chronic inflammation and increased cytosolic ROS, contributing to apoptosis or necroptosis of neurons. Therefore, activation of apoptosis or necroptosis in AD, PD or PrD is triggered by a positive feedback loop between chronic inflammation in the brain (to which astrocytes and microglia are the main contributor) and the production of PTMP. In addition to high levels of ROS, the production of PTMP in the cytosol is facilitated by copper ions in AD [63] and by iron ions, dopamine and accumulation of alpha-synuclein (the precursor of oxidised αSYN monomer) in PD [17]. Although chronic brain inflammation contributes to the process of PrPSc production, it is not necessary to sustain it, since the PrPSc only needs the PrPc

**2. Crosstalk among AUT, apoptosis and necroptosis signalling pathways** 

Parkinson's and prion diseases (PrD), is summarised in **Figure 3**.

AUT, apoptosis and necroptosis have interlinked signalling pathways. Examples of key signalling molecules that regulate the transition among these three processes are presented in Section 2.1. The crosstalk among AUT, apoptosis and necroptosis signalling pathways, with the potential sites of modulation by Alzheimer's,

and distorted expression of apoptotic proteins [53–56].

sustains microglial hyperactivation [62].

molecules for its propagation [64].

**in selected NBD**

**112**

*Crosstalk among AUT, apoptosis and necroptosis signalling pathways with the potential sites of modulation by AD, PD and PrD. Abbreviations: αSYN (alpha-synuclein); AβO (amyloid β oligomers); AβP (amyloid β monomers with 39 to 42 amino acid residues); AIF (apoptosis-inducing factor); AKT (protein kinase B); AMPK (5' AMPactivated protein kinase); AP-1 (activator protein 1); APAF-1 (apoptotic protease activating factor 1); APO-3L (APO3 ligand); ASK-1 (apoptosis signal-regulating kinase 1); ATG-5 (AUT-related 5); BAD (Bcl2-associated agonist of cell death); BAK (Bcl-2 homologous antagonist/killer); BAX (apoptosis regulator BAX); BCL-2 (B-cell lymphoma 2); BCL-XL (B-cell lymphoma-extra large); Beclin-1 (mammalian ortholog of the yeast AUT-related gene 6 (ATG-6)); BID (BH3 interacting domain death agonist); BIP-P (phosphorylated binding immunoglobulin protein); C-FLIP (FADD-like IL-1β-converting enzyme-inhibitory protein); calpain (proteolytic enzyme, a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases); CASP-3, CASP-8/10, CASP-9 (caspase-3, caspase-8/10, caspase-9); cIAP-1 (cellular inhibitor of apoptosis protein 1); CL-ATG5 (cleaved AUTrelated 5 (ATG5) protein); CL-BAX (cleaved apoptosis regulator BAX); CL-Beclin-1 (cleaved mammalian ortholog of the yeast AUT-related gene 6); Complex-I (TNFα bound to TNFα receptor that is associated with TRADD (tumour necrosis factor receptor type 1-associated death domain protein), RIPK1 (receptor-interacting serine/ threonine-protein kinase 1), TRAF2 (TNF receptor-associated factor 2) and cIAP-1/2 (cellular inhibitor of apoptosis protein 1 and 2)); Complex-IIa (pro-caspase-8, RIPK1, FADD (FAS-associated protein with death domain)); Complex-IIb (pro-caspase-8, RIPK1, RIPK3 (receptor-interacting serine/threonine-protein kinase 3), FADD, MLKL (mixed lineage kinase domain-like pseudokinase)); CYT-C (cytochrome c); DAMPs (damage-associated molecular patterns); DCAP-AKT (activation of toll/IL-1R (TIR) domain-containing adaptor proteins (e.g. mal, TRIF, TRIF-related adaptor molecule, IL-1R-associated kinase-1, IL-1R-associated kinase-M, MAPK, TNFR-associated factor 6, toll-interacting protein)); FAS (apoptosis antigen 1); HMGB-1 (high-mobility group box 1 protein); IKK (IκB kinase enzyme complex, part of the upstream NF-κB signal transduction cascade); IL-1β (interleukin-1 beta); JAK (Janus kinase); JNK (c-Jun N-terminal kinase); JNK-P (phosphorylated c-Jun N-terminal kinase); MITO (mitochondrial); MITO MP (mitochondrial membrane permeability); MITO ROS (mitochondrial reactive oxygen species); MKK7 (MAP kinase kinase 7); MLKL-O (MLKL oligomerisation with translocation and insertion into cell's and organelles' membranes with increased permeability); MLKL-P (phosphorylated pseudokinase mixed lineage kinase domain-like protein); mTORC1 (mammalian target of rapamycin complex 1); NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells protein complex); NOXA (adult T cell leukaemia-derived PMAresponsive); OMI alias HtrA2 (serine protease HTRA2); ORG (cell organelles); p53 (cellular tumour antigen p53); p62 (nucleoporin p62 protein complex associated with the nuclear envelope); PRO CASP 8/10 (pro-caspase-8/10); PRPC (normal form of prion protein); PRPSC (self-propagating, protease-resistant, infectious isoforms of prion protein); PTM (post-translationally modified); PUMA (p53 upregulated modulator of apoptosis); RIP-1 (receptorinteracting serine/threonine-protein kinase 1); ROS (reactive oxygen species, e.g. peroxides, superoxide, hydroxyl radical or singlet oxygen); SMAC (second mitochondria-derived activator of caspases); STAT (signal transducer and activator of transcription 3/5); tBID (truncated BID protein); TLR-4 (toll-like receptor 4, member of the pattern recognition receptor (PRR) family); TNFα (tumour necrosis factor alpha); TNFαR (tumour necrosis factor alpha receptor); TNFRS (tumour necrosis factor receptor superfamily); TRAF (TNF receptor-associated factor); TRAIL (TNF-related apoptosis-inducing ligand); UBIQ PROT (ubiquitinated proteins); ULK1 (serine/threonine-protein kinase ULK1); XIAP (X-linked inhibitor of apoptosis protein).*

#### **2.1 Examples of signalling molecules that regulate crosstalk among AUT, apoptosis and necroptosis pathways in selected NBD**

Intracellular *adenosine triphosphate* (ATP) promotes either apoptosis or necroptosis in a concentration-dependent manner; high ATP levels promote apoptosis, and low ATP levels promote necroptosis [65, 66]. Therefore, ATP production in the MITO determines the type of cell death. The best understood inflammation- and necroptosis-promoting cytokine that modulates mitochondrial ATP and ROS levels is TNFα [67]. As explained above, PTMP inhibition of MITO function leads to activation of inflammasomes with an increased release of tumour necrosis factor alpha (TNFα) from astrocytes and neurons [38, 45–52]. The sustained TNFα stimulation in NBD is the result of two mechanisms. (a) The PTMP of AD, PD and PrD are not digested by AUT; they accumulate in affected cells by their release into the cytosol from endolysosomal and autolysosomal compartment together with proteolytic enzymes [68]. (b) The PTMP in PD and PrD spread through the brain by a prion-like mechanism [69, 70]. The sustained TNFα stimulation can lead to over-activation of PARP1, a nuclear DNA repair enzyme that is activated by DNA damage, due to an increased MITO ROS production. PARP1 over-activation precipitates an acute depletion of NAD<sup>+</sup> , inhibition of oxidative phosphorylation with a severe drop in ATP production and a subsequent activation of necroptosis [65, 71–73].

*AUT-related 5* (Atg5) protein stimulates elongation of autophagosome membranes that envelope PTMP into autophagosomes [74–76] and also regulates the balance between AUT and apoptosis [77]. The neurons' cytosolic Ca2+ is increased in NBD due to the PTMP elicited (a) ER and MITO release of Ca2 into the cytosol [4–7, 78] and (b) an increased Ca2+ entry through the N-methyl-D-aspartate (NMDAR) glutamate receptor and ion channel proteins from the extracellular space [79–81]. Increased cytosolic Ca2+ promotes calpain-1- and calpain-2-mediated cleavage of ATG5, with a loss of pro-AUT function and concomitant triggering of cytochrome c-/caspase-mediated apoptosis due to the inhibition of Bcl-xL in the MITO by the cleaved ATG5 [82]. The calpain-1- and calpain-2-mediated cleavage of ATG5 is attenuated by decreased levels of cytosolic Ca2+ [83]. Cytosolic HMGB1 attenuates apoptosis by protecting the AUT proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation [84].

*Beclin 1* stimulates AUT [16, 85, 86]; an enhanced AUT has a concomitant antiapoptotic effect by clearing apoptosis-associated molecules, for example, active caspase-8 [87–89]. Beclin 1 is cleaved by caspases, thus losing its pro-AUT function, and the cleaved beclin 1 (i.e. C-terminal beclin 1 fragment) promotes apoptosis by triggering the release of MITO cytochrome c [90–92].

*B-cell lymphoma 2* (Bcl-2) family of proteins regulate MITO apoptotic pathway and also AUT; for example, Bcl-2 and Bcl-xL inhibit AUT and apoptosis [93, 94]. Bcl-2 and Bcl-xL proteins have an anti-apoptotic effect, whereas Bax, Bad, Bid, Bim, Bmf, PUMA and NOXA promote apoptosis. Calpain-mediated cleavage of Bax, induced by high cytosolic Ca2+, mediates apoptosis [82]. The interactions between anti-apoptotic and pro-apoptotic Bcl-2 family members determine the activation of apoptosis [95–104]. Bcl-2 and Bcl-xL associate with beclin 1 and supress the beclin 1-dependent autophagic activation [105]. This AUT suppression can be abolished by the pro-apoptotic Bcl-2 family proteins (e.g. Bad, Bid) [106]. The inhibition of Bcl-2 on beclin 1 is also attenuated by phosphorylation of Bcl-2 by JNK-1 or Beclin-1 by DAPK1, thus promoting AUT [107, 108]. Increased expression of Bak, Bad, Bcl-2 and Bcl-x was observed in AD [109]. Cytosolic PrPc protects human primary neurons from Bax-mediated apoptosis [110–112]; therefore the PrPsc-precipitated reduction should facilitate apoptosis in PrD.

**115**

of synaptic terminals [155–166].

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

*Caspase-8* activity is changed in NBD. It has been suggested that an increased caspase-8 activity, associated with an increased caspase-3 activity in the same hippocampal tissue sections from patients with AD, contributes to the development of AD in humans. Recently, two caspase-8 variants, with a reduced activity and associated with an increased risk for development of AD in human, were identified. This finding is consistent with the multiple AD-related changes in the human brain, including loss of synaptic plasticity and memory function and increased microglia pro-inflammatory activation [113]. Caspase-8, within the death-inducing complex II, triggers either apoptotic or necroptotic cell death. Activated caspase-8 promotes apoptosis and also inhibits necroptosis by cleaving RIPK1, RIPK3 and CYLD [114–116], thus preventing CYLD-mediated deubiquitylation of RIPK1 and subsequent RIPK1 kinase activation and necroptosis [117]. The association of caspase-8 with pseudo-caspase cFLIP suppresses apoptosis and also necroptosis, since the residual levels of caspase-8 activity are still sufficient to cleave and inactivate RIPK1

*c-Jun N-terminal kinase* (JNK) promotes either apoptosis or necroptosis, depending on its upstream signalling pathways. JNK is required for apoptosis of central nervous system neurons [119]. JNK promotes apoptosis by several signalling pathways that were characterised in different cell experimental models. It is unlikely that all of the observed JNK's pro-apoptotic effects are present in all of the cells at the same time [120]. However, it is important to be aware of the JNK's ability to modulate apoptosis at different levels. To summarise, the known pro-apoptotic effects of JNK are: (a) Activated MAP2Ks phosphorylate JNK and phosphorylated JNK translocates to the nucleus and phosphorylates c-Jun [121, 122] that promotes AP-1 expression; AP-1 promotes transcription of pro-apoptotic proteins TNF-α, Fas-L and Bak [123–125]. (b) JNK phosphorylates p53, enhancing the expression of pro-apoptotic genes Bax and PUMA [126–128]; the increased Bax expression and translocation to mitochondria is sufficient to promote MITO outer membrane permeabilization, the consequent release of cytochrome c and caspase-9 and caspase-3 activation [129–133]. (c) JNK phosphorylates 14-3-3-associated Bad, thus promoting its translocation into MITO and subsequent release of cytochrome c [134, 135]. (d) JNK phosphorylates pro-apoptotic proteins Bim and Bmf, and these phosphorylated proteins activate Bax and/or Bak [136–140]. (e) Phosphorylated Bim binds to and inhibits the Bcl2's anti-apoptotic activity, thus increasing the probability of MITO-activated apoptosis [141, 142]. (f) JNK inhibits the anti-apoptotic Bcl2 by phosphorylation, to induce apoptosis [143, 144]. (g) JNK has the ability to promote apoptosis by stimulating the activity of many pro-apoptotic signalling molecules. (h) Activation of TNFRS (e.g. TNFR1, DR3-6) can lead to apoptosis [144, 145]. (i) Activation of DRs and TNFα receptors stimulates JNK activation that promotes apoptosis by increased expression of DRs [146, 147]; increased expression of pro-apoptotic proteins Bak, Bim and Bax [148, 149]; inhibition of anti-apoptotic proteins XIAP (caspase-3, caspase-7 and caspase-9 inhibitor) and cIAP1 (caspase-8 inhibitor) [150, 151]. JNK's role in NBD is best understood in AD; JNK activation is positively correlated with AD progression [152]. Amyloid-β protein fragments activate JNK [153, 154]. Also, JNK phosphorylates tau, thus promoting (a) microtubule cytoskeleton breakdown, (b) attenuation of intracellular transport and (c) loss

*Activation of tumour necrosis factor receptor superfamily* (e.g. TLRs or TNFαR) or DNA damage can trigger necroptosis by activation of the Complex I-IIa-IIbphosphorylated pseudokinase mixed lineage kinase domain-like protein (MLKL) signalling pathway; the final steps are (a) RIP3-dependent phosphorylation of MITO proteins PGAM5 and Drp-1 (increasing MITO ROS production); (b) insertion of phosphorylated MLKL into the MITO membrane with the cumulative effects

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

and RIPK3 [118].

*Programmed Cell Death*

**2.1 Examples of signalling molecules that regulate crosstalk among AUT,** 

Intracellular *adenosine triphosphate* (ATP) promotes either apoptosis or necroptosis in a concentration-dependent manner; high ATP levels promote apoptosis, and low ATP levels promote necroptosis [65, 66]. Therefore, ATP production in the MITO determines the type of cell death. The best understood inflammation- and necroptosis-promoting cytokine that modulates mitochondrial ATP and ROS levels is TNFα [67]. As explained above, PTMP inhibition of MITO function leads to activation of inflammasomes with an increased release of tumour necrosis factor alpha (TNFα) from astrocytes and neurons [38, 45–52]. The sustained TNFα stimulation in NBD is the result of two mechanisms. (a) The PTMP of AD, PD and PrD are not digested by AUT; they accumulate in affected cells by their release into the cytosol from endolysosomal and autolysosomal compartment together with proteolytic enzymes [68]. (b) The PTMP in PD and PrD spread through the brain by a prion-like mechanism [69, 70]. The sustained TNFα stimulation can lead to over-activation of PARP1, a nuclear DNA repair enzyme that is activated by DNA damage, due to an increased MITO ROS produc-

tion. PARP1 over-activation precipitates an acute depletion of NAD<sup>+</sup>

in NBD due to the PTMP elicited (a) ER and MITO release of Ca2

calpain-mediated cleavage during inflammation [84].

triggering the release of MITO cytochrome c [90–92].

PrPsc-precipitated reduction should facilitate apoptosis in PrD.

activation of necroptosis [65, 71–73].

oxidative phosphorylation with a severe drop in ATP production and a subsequent

*AUT-related 5* (Atg5) protein stimulates elongation of autophagosome membranes that envelope PTMP into autophagosomes [74–76] and also regulates the balance between AUT and apoptosis [77]. The neurons' cytosolic Ca2+ is increased

(NMDAR) glutamate receptor and ion channel proteins from the extracellular space [79–81]. Increased cytosolic Ca2+ promotes calpain-1- and calpain-2-mediated cleavage of ATG5, with a loss of pro-AUT function and concomitant triggering of cytochrome c-/caspase-mediated apoptosis due to the inhibition of Bcl-xL in the MITO by the cleaved ATG5 [82]. The calpain-1- and calpain-2-mediated cleavage of ATG5 is attenuated by decreased levels of cytosolic Ca2+ [83]. Cytosolic HMGB1 attenuates apoptosis by protecting the AUT proteins beclin 1 and ATG5 from

*Beclin 1* stimulates AUT [16, 85, 86]; an enhanced AUT has a concomitant antiapoptotic effect by clearing apoptosis-associated molecules, for example, active caspase-8 [87–89]. Beclin 1 is cleaved by caspases, thus losing its pro-AUT function, and the cleaved beclin 1 (i.e. C-terminal beclin 1 fragment) promotes apoptosis by

*B-cell lymphoma 2* (Bcl-2) family of proteins regulate MITO apoptotic pathway and also AUT; for example, Bcl-2 and Bcl-xL inhibit AUT and apoptosis [93, 94]. Bcl-2 and Bcl-xL proteins have an anti-apoptotic effect, whereas Bax, Bad, Bid, Bim, Bmf, PUMA and NOXA promote apoptosis. Calpain-mediated cleavage of Bax, induced by high cytosolic Ca2+, mediates apoptosis [82]. The interactions between anti-apoptotic and pro-apoptotic Bcl-2 family members determine the activation of apoptosis [95–104]. Bcl-2 and Bcl-xL associate with beclin 1 and supress the beclin 1-dependent autophagic activation [105]. This AUT suppression can be abolished by the pro-apoptotic Bcl-2 family proteins (e.g. Bad, Bid) [106]. The inhibition of Bcl-2 on beclin 1 is also attenuated by phosphorylation of Bcl-2 by JNK-1 or Beclin-1 by DAPK1, thus promoting AUT [107, 108]. Increased expression of Bak, Bad, Bcl-2 and Bcl-x was observed in AD [109]. Cytosolic PrPc protects human primary neurons from Bax-mediated apoptosis [110–112]; therefore the

[4–7, 78] and (b) an increased Ca2+ entry through the N-methyl-D-aspartate

, inhibition of

into the cytosol

**apoptosis and necroptosis pathways in selected NBD**

**114**

*Caspase-8* activity is changed in NBD. It has been suggested that an increased caspase-8 activity, associated with an increased caspase-3 activity in the same hippocampal tissue sections from patients with AD, contributes to the development of AD in humans. Recently, two caspase-8 variants, with a reduced activity and associated with an increased risk for development of AD in human, were identified. This finding is consistent with the multiple AD-related changes in the human brain, including loss of synaptic plasticity and memory function and increased microglia pro-inflammatory activation [113]. Caspase-8, within the death-inducing complex II, triggers either apoptotic or necroptotic cell death. Activated caspase-8 promotes apoptosis and also inhibits necroptosis by cleaving RIPK1, RIPK3 and CYLD [114–116], thus preventing CYLD-mediated deubiquitylation of RIPK1 and subsequent RIPK1 kinase activation and necroptosis [117]. The association of caspase-8 with pseudo-caspase cFLIP suppresses apoptosis and also necroptosis, since the residual levels of caspase-8 activity are still sufficient to cleave and inactivate RIPK1 and RIPK3 [118].

*c-Jun N-terminal kinase* (JNK) promotes either apoptosis or necroptosis, depending on its upstream signalling pathways. JNK is required for apoptosis of central nervous system neurons [119]. JNK promotes apoptosis by several signalling pathways that were characterised in different cell experimental models. It is unlikely that all of the observed JNK's pro-apoptotic effects are present in all of the cells at the same time [120]. However, it is important to be aware of the JNK's ability to modulate apoptosis at different levels. To summarise, the known pro-apoptotic effects of JNK are: (a) Activated MAP2Ks phosphorylate JNK and phosphorylated JNK translocates to the nucleus and phosphorylates c-Jun [121, 122] that promotes AP-1 expression; AP-1 promotes transcription of pro-apoptotic proteins TNF-α, Fas-L and Bak [123–125]. (b) JNK phosphorylates p53, enhancing the expression of pro-apoptotic genes Bax and PUMA [126–128]; the increased Bax expression and translocation to mitochondria is sufficient to promote MITO outer membrane permeabilization, the consequent release of cytochrome c and caspase-9 and caspase-3 activation [129–133]. (c) JNK phosphorylates 14-3-3-associated Bad, thus promoting its translocation into MITO and subsequent release of cytochrome c [134, 135]. (d) JNK phosphorylates pro-apoptotic proteins Bim and Bmf, and these phosphorylated proteins activate Bax and/or Bak [136–140]. (e) Phosphorylated Bim binds to and inhibits the Bcl2's anti-apoptotic activity, thus increasing the probability of MITO-activated apoptosis [141, 142]. (f) JNK inhibits the anti-apoptotic Bcl2 by phosphorylation, to induce apoptosis [143, 144]. (g) JNK has the ability to promote apoptosis by stimulating the activity of many pro-apoptotic signalling molecules. (h) Activation of TNFRS (e.g. TNFR1, DR3-6) can lead to apoptosis [144, 145]. (i) Activation of DRs and TNFα receptors stimulates JNK activation that promotes apoptosis by increased expression of DRs [146, 147]; increased expression of pro-apoptotic proteins Bak, Bim and Bax [148, 149]; inhibition of anti-apoptotic proteins XIAP (caspase-3, caspase-7 and caspase-9 inhibitor) and cIAP1 (caspase-8 inhibitor) [150, 151]. JNK's role in NBD is best understood in AD; JNK activation is positively correlated with AD progression [152]. Amyloid-β protein fragments activate JNK [153, 154]. Also, JNK phosphorylates tau, thus promoting (a) microtubule cytoskeleton breakdown, (b) attenuation of intracellular transport and (c) loss of synaptic terminals [155–166].

*Activation of tumour necrosis factor receptor superfamily* (e.g. TLRs or TNFαR) or DNA damage can trigger necroptosis by activation of the Complex I-IIa-IIbphosphorylated pseudokinase mixed lineage kinase domain-like protein (MLKL) signalling pathway; the final steps are (a) RIP3-dependent phosphorylation of MITO proteins PGAM5 and Drp-1 (increasing MITO ROS production); (b) insertion of phosphorylated MLKL into the MITO membrane with the cumulative effects of increased MITO membrane permeability, loss of membrane potential, decreased ATP and increased ROS production [120, 167–169]; and (c) phosphorylated MLKL's translocation to the plasma membrane and activation of Ca2+ influx through plasma membrane channels with concomitant plasma membrane breakdown [169]. Increased cytosolic ROS production inactivates MAP kinase phosphatase 1, enabling sustained activation of phosphorylated JNK; phosphorylated JNK promotes necroptosis by (a) stimulating MLKL phosphorylation and by (b) promoting cytochrome c release from MITO via activation of BID [170, 171].

*FLICE inhibitory proteins* (FLIPs). Under stress-free conditions, FLIPs (FLICE inhibitory proteins) attenuate LC3's binding with ATG3, thus preventing ATG3 mediated elongation of autophagosomes and AUT. During stress, FLIPs allow for ATG3-LC3 interaction and stimulate AUT. Therefore, FLIPs (e.g. C-FLIP) can inhibit apoptosis and also AUT [172].

The *high-mobility group box protein 1* (HMGB1) is a nuclear protein released by glia and necrotic or hyper-excitatory neurons after inflammasome activation; it activates receptors for advanced glycation end products (RAGE) and the toll-like receptor (TLR) 4 on neurons and microglia [173, 174]. When HMGB1 binds to TLR4 on neurons, it phosphorylates MARCKS via MAP kinases and induces neurite degeneration, present in AD [173]. The disulphide form of HMGB1 potentiates the microglia pro-inflammatory response; therefore, repeated releases of HMGB from damaged nerve cells during chronic neuroinflammation in PD and AD could lead to an exacerbated neuroinflammatory response of microglia [175–177]. HMGB1, in a rat model of AD, caused (a) inhibition of microglial amyloid β-peptide 42 clearance and enhanced amyloid β-peptide 42 neurotoxicity [178] and (b) dysfunction of microglial amyloid β-peptide 40 phagocytosis [179].

The *nuclear factor kappa-light-chain-enhancer of activated B cells protein complex* (NF-κB) signalling pathway was repressed in a prion-infected cell line and animal brain tissues as evidenced by a decreased level of transcription factor p65/nuclear factor NF-kappa-B p65 subunit (p65) and downregulation of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) in both experimental models [180]. In AD cell models, the exposure to amyloid β-peptide or amyloid precursor protein induced NF-κB activation [181, 182], and inhibition of NF-κB transcriptional activity increased neuronal death in the presence of amyloid β-peptide [183]. NF-κB activation can protect neurons against amyloid β-peptide-induced cell death [184]. Patients with PD have an increased percentage of dopaminergic neurons in the substantia nigra with nuclear p65 immunoreactivity [185]. NF-κB is one of the several factors that regulate Beclin-1 expression; Beclin-1 promotes AUT by stimulating autophagosome formation [186–188]. Increased NF-κB activation in the brain, in addition to stimulating AUT, protects nerve cells against NBDs' mediated injury by several mechanisms including increased transcription of MITO antioxidant enzyme manganese superoxide dismutase (MnSOD) and Bcl-xL genes [189].

*Sirtuins* (SIRTs), NAD<sup>+</sup> -dependent protein deacetylases, modulate apoptosis and necroptosis [190]. For example, SIRT1 promotes AUT by deacetylation of ATG5, ATG7 and ATG8 [191]. Following TNFα receptor stimulation, SIRT2 promotes the association of RIP1 and RIP3, the subsequent formation of complex II and necroptosis [192]. In animal and cell culture models of AD, SIRT1 reduces neurodegeneration in mouse hippocampus and promotes primary neuronal survival [193]. The reduced SIRT1 mRNA and protein levels are associated with an accumulation of amyloid β-peptide 42 and tau in the brains of AD patients [194].

*Tumour protein p53* (p53) modulates AUT and apoptosis. It promotes apoptosis by Bax activation in the cytoplasm; BAX initiates apoptosis by triggering mitochondrial cyt c release and caspase-3 activation [195]. In the nucleus, p53 activates transcription of Bax, PUMA and Noxa [196]. PUMA displaces cytoplasmic p53 from

**117**

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

the Bcl-xL-p53 complex, promoting p53 activation of the apoptotic pathway [197]. In the nucleus, p53 also stimulates AUT through transcription activation of ULK1, sestrin1/2 and damage-regulated AUT modulator (DRAM) [198, 199]. Indirectly, p53 promotes AUT by mTOR inhibition, via activation of AMP-dependent kinase and tuberous sclerosis (TSC) 1/TSC2 complex pathway [200]. It was suggested that DRAM has a dual role of promoting either AUT- or p53-mediated apoptosis [201]. In a *Drosophila* model of AD tauopathy, p53 prevented neurodegeneration by increased expression of amphiphysin, clathrin light chain, clathrin heavy chain, RAS oncogene family and synaptotagmin β synaptic genes [202]. p53 levels are significantly increased in brains of patients with AD [203] and are correlated with brain MITO dysfunction [204]. Recently, it was suggested that tau oligomers sequester and downregulate functional phospho-p53 in an AD mouse model and in

*Ubiquitin-binding protein p62* (p62) modulates cell death switching between apoptosis and necroptosis. In a cell model, p62 promotes either necroptosis, when p62 is associated with the necrosome (i.e. complex II), or apoptosis when the P62 necrosome association is blocked [206]. The p62 regulates apoptotic and autophagic processes [207]. P62 mediates AUT degradation by first binding polyubiquitinated proteins with the ubiquitin-associated domain and then to autophagosomes through the LC3-interacting region [208, 209]. In response to tumour necrosis factor receptor stimulation, P62 promotes apoptosis by stimulating activation of caspase-8 [210, 211]. The levels of p62 are increased in NBD, for example, in PrD [212, 213]. Autophagy disposal of aberrant proteins is stimulated by the p62-Keap1-NRF2 signalling pathway [214]. For example, in a mouse model of AD, increased brain p62 expression improved cognition by an autophagy-mediated mechanism that reduced

**2.2 Summary of similarities/differences in the mechanistic pathways between** 

Beclin-1, ATG-5, NF-κB, JNK, p53, p62, HMGB1 and ROS are the key signalling molecules that mediate crosstalk among AUT, apoptosis and necroptosis. ATG5 and Beclin-1 in conjunction with ULK-1 and BAX promote AUT by initiating phagophore induction and nucleation steps. Cleavage of ATG-5 and Beclin-1 by calpain, caspase-3 or increased cytosolic free calcium changes their function from stimulating AUT to promoting apoptosis via increased MITO membrane permeability. Cleaved ATG5 inhibits the anti-apoptotic activity of BCL2 and BCL-XL on BAX and BAK, further promoting increased MITO membrane permeability and apoptosis. P53 activation plays a dual role by promoting apoptosis (via activation of PUMA and NOXA) and AUT by ULK1 activation. The JNK signalling kinase blocks the binding of BCL-2 to Beclin-1, thus enabling Beclin-1 to participate in AUT initiation, and also activates the apoptosis-triggering proteins BAX and BAK. Phosphorylated JNK promotes necroptosis by stimulating MLKL phosphorylation and apoptosis by caspase-8 activation. p62 promotes AUT and apoptosis. HMGB-1 is released during AUT, apoptosis and necroptosis, and by inhibiting the cleavage of ATG-5, BAX and Beclin-1 simultaneously promote AUT and inhibit apoptosis. Mild increases in cytosolic ROS act as signalling molecules that promote a physiological balance between AUT, apoptosis and necroptosis, which favour AUT; moderate and high increases in cytosolic ROS concentrations favour apoptosis and necroptosis over AUT. The products of post-translational protein modifications in AD, PD and PrD favour apoptosis and necroptosis over AUT by (a) increasing the activation of apoptosis (e.g. by increasing MITO membrane permeability) and necroptosis, by chronic activation of TRL4 and TNFα receptors [216–234],

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

patients with AD [205].

amyloid β-peptide 40/42 levels [215].

**selected NBD**

#### *Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

*Programmed Cell Death*

inhibit apoptosis and also AUT [172].

*Sirtuins* (SIRTs), NAD<sup>+</sup>

microglial amyloid β-peptide 40 phagocytosis [179].

of increased MITO membrane permeability, loss of membrane potential, decreased ATP and increased ROS production [120, 167–169]; and (c) phosphorylated MLKL's translocation to the plasma membrane and activation of Ca2+ influx through plasma membrane channels with concomitant plasma membrane breakdown [169]. Increased cytosolic ROS production inactivates MAP kinase phosphatase 1, enabling sustained activation of phosphorylated JNK; phosphorylated JNK promotes necroptosis by (a) stimulating MLKL phosphorylation and by (b) promoting

*FLICE inhibitory proteins* (FLIPs). Under stress-free conditions, FLIPs (FLICE inhibitory proteins) attenuate LC3's binding with ATG3, thus preventing ATG3 mediated elongation of autophagosomes and AUT. During stress, FLIPs allow for ATG3-LC3 interaction and stimulate AUT. Therefore, FLIPs (e.g. C-FLIP) can

The *high-mobility group box protein 1* (HMGB1) is a nuclear protein released by glia and necrotic or hyper-excitatory neurons after inflammasome activation; it activates receptors for advanced glycation end products (RAGE) and the toll-like receptor (TLR) 4 on neurons and microglia [173, 174]. When HMGB1 binds to TLR4 on neurons, it phosphorylates MARCKS via MAP kinases and induces neurite degeneration, present in AD [173]. The disulphide form of HMGB1 potentiates the microglia pro-inflammatory response; therefore, repeated releases of HMGB from damaged nerve cells during chronic neuroinflammation in PD and AD could lead to an exacerbated neuroinflammatory response of microglia [175–177]. HMGB1, in a rat model of AD, caused (a) inhibition of microglial amyloid β-peptide 42 clearance and enhanced amyloid β-peptide 42 neurotoxicity [178] and (b) dysfunction of

The *nuclear factor kappa-light-chain-enhancer of activated B cells protein complex* (NF-κB) signalling pathway was repressed in a prion-infected cell line and animal brain tissues as evidenced by a decreased level of transcription factor p65/nuclear factor NF-kappa-B p65 subunit (p65) and downregulation of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) in both experimental models [180]. In AD cell models, the exposure to amyloid β-peptide or amyloid precursor protein induced NF-κB activation [181, 182], and inhibition of NF-κB transcriptional activity increased neuronal death in the presence of amyloid β-peptide [183]. NF-κB activation can protect neurons against amyloid β-peptide-induced cell death [184]. Patients with PD have an increased percentage of dopaminergic neurons in the substantia nigra with nuclear p65 immunoreactivity [185]. NF-κB is one of the several factors that regulate Beclin-1 expression; Beclin-1 promotes AUT by stimulating autophagosome formation [186–188]. Increased NF-κB activation in the brain, in addition to stimulating AUT, protects nerve cells against NBDs' mediated injury by several mechanisms including increased transcription of MITO antioxidant enzyme manganese superoxide dismutase (MnSOD) and Bcl-xL genes [189].

necroptosis [190]. For example, SIRT1 promotes AUT by deacetylation of ATG5, ATG7 and ATG8 [191]. Following TNFα receptor stimulation, SIRT2 promotes the association of RIP1 and RIP3, the subsequent formation of complex II and necroptosis [192]. In animal and cell culture models of AD, SIRT1 reduces neurodegeneration in mouse hippocampus and promotes primary neuronal survival [193]. The reduced SIRT1 mRNA and protein levels are associated with an accumulation of

*Tumour protein p53* (p53) modulates AUT and apoptosis. It promotes apoptosis by Bax activation in the cytoplasm; BAX initiates apoptosis by triggering mitochondrial cyt c release and caspase-3 activation [195]. In the nucleus, p53 activates transcription of Bax, PUMA and Noxa [196]. PUMA displaces cytoplasmic p53 from

amyloid β-peptide 42 and tau in the brains of AD patients [194].


cytochrome c release from MITO via activation of BID [170, 171].

**116**

the Bcl-xL-p53 complex, promoting p53 activation of the apoptotic pathway [197]. In the nucleus, p53 also stimulates AUT through transcription activation of ULK1, sestrin1/2 and damage-regulated AUT modulator (DRAM) [198, 199]. Indirectly, p53 promotes AUT by mTOR inhibition, via activation of AMP-dependent kinase and tuberous sclerosis (TSC) 1/TSC2 complex pathway [200]. It was suggested that DRAM has a dual role of promoting either AUT- or p53-mediated apoptosis [201]. In a *Drosophila* model of AD tauopathy, p53 prevented neurodegeneration by increased expression of amphiphysin, clathrin light chain, clathrin heavy chain, RAS oncogene family and synaptotagmin β synaptic genes [202]. p53 levels are significantly increased in brains of patients with AD [203] and are correlated with brain MITO dysfunction [204]. Recently, it was suggested that tau oligomers sequester and downregulate functional phospho-p53 in an AD mouse model and in patients with AD [205].

*Ubiquitin-binding protein p62* (p62) modulates cell death switching between apoptosis and necroptosis. In a cell model, p62 promotes either necroptosis, when p62 is associated with the necrosome (i.e. complex II), or apoptosis when the P62 necrosome association is blocked [206]. The p62 regulates apoptotic and autophagic processes [207]. P62 mediates AUT degradation by first binding polyubiquitinated proteins with the ubiquitin-associated domain and then to autophagosomes through the LC3-interacting region [208, 209]. In response to tumour necrosis factor receptor stimulation, P62 promotes apoptosis by stimulating activation of caspase-8 [210, 211]. The levels of p62 are increased in NBD, for example, in PrD [212, 213]. Autophagy disposal of aberrant proteins is stimulated by the p62-Keap1-NRF2 signalling pathway [214]. For example, in a mouse model of AD, increased brain p62 expression improved cognition by an autophagy-mediated mechanism that reduced amyloid β-peptide 40/42 levels [215].

#### **2.2 Summary of similarities/differences in the mechanistic pathways between selected NBD**

Beclin-1, ATG-5, NF-κB, JNK, p53, p62, HMGB1 and ROS are the key signalling molecules that mediate crosstalk among AUT, apoptosis and necroptosis. ATG5 and Beclin-1 in conjunction with ULK-1 and BAX promote AUT by initiating phagophore induction and nucleation steps. Cleavage of ATG-5 and Beclin-1 by calpain, caspase-3 or increased cytosolic free calcium changes their function from stimulating AUT to promoting apoptosis via increased MITO membrane permeability. Cleaved ATG5 inhibits the anti-apoptotic activity of BCL2 and BCL-XL on BAX and BAK, further promoting increased MITO membrane permeability and apoptosis. P53 activation plays a dual role by promoting apoptosis (via activation of PUMA and NOXA) and AUT by ULK1 activation. The JNK signalling kinase blocks the binding of BCL-2 to Beclin-1, thus enabling Beclin-1 to participate in AUT initiation, and also activates the apoptosis-triggering proteins BAX and BAK. Phosphorylated JNK promotes necroptosis by stimulating MLKL phosphorylation and apoptosis by caspase-8 activation. p62 promotes AUT and apoptosis. HMGB-1 is released during AUT, apoptosis and necroptosis, and by inhibiting the cleavage of ATG-5, BAX and Beclin-1 simultaneously promote AUT and inhibit apoptosis. Mild increases in cytosolic ROS act as signalling molecules that promote a physiological balance between AUT, apoptosis and necroptosis, which favour AUT; moderate and high increases in cytosolic ROS concentrations favour apoptosis and necroptosis over AUT. The products of post-translational protein modifications in AD, PD and PrD favour apoptosis and necroptosis over AUT by (a) increasing the activation of apoptosis (e.g. by increasing MITO membrane permeability) and necroptosis, by chronic activation of TRL4 and TNFα receptors [216–234],

(b) promoting moderate to high increases in cytosolic ROS concentrations and (c) attenuating AUT [42, 62, 235–240]. In contrast to PD and AD, PrPSC-infected cells are more likely to respond with necroptosis and then apoptosis. For example, a significant upregulation of necroptosis signalling molecules phosphorylated MLKL, MLKL and receptor-interacting serine/threonine-protein kinase 3 (RIP3) was measured in the post-mortem cortical brains of patients with various types of human PRD [241].

#### **3. Pharmacological strategies targeting AUT, apoptosis and necroptosis signalling pathways**

At present, most of the studies, devoted to the development of pharmacological interventions for NBD, are focused on the crosstalk of AUT and apoptosis signalling pathways in neurons. Future research should also include development of pharmacological interventions that target other cells involved in the development of NBD, including microglia, astrocytes, endothelial cells and pericytes [242]. The development of pharmacological interventions for NBD should be guided by several key questions: (a) How to modulate the role of AUT from pro-death to pro-survival? (b) How is the information from the crosstalk among AUT, apoptosis and necroptosis integrated? (c) How to modulate the crosstalk among AUT, apoptosis and necroptosis? and (d) How is the information from the crosstalk among AUT, apoptosis and necroptosis (e.g. inflammation-promoting molecules) shared among different cells involved in the development of NBD? [242]. Examples of pharmacological strategies are given below:

Pharmacological strategies to ameliorate MITO dysfunction include:

	- (a1) Mercaptamine that increases levels of glutathione in human [78].
	- (a2) Antioxidant vatiquinone used in clinical trials [243].
	- (a3) RTA-308 stimulates Nrf2 to enhance the expression of pro-oxidant genes and to repress inflammatory genes in an animal model [244].
	- (a4) Antioxidants coenzyme Q, lipoic acid and green tea polyphenol epigallocatechin gallate attenuate the effects of NBD in animal models [245–248].
	- (a5) Ceria nanoparticles are ROS scavengers that localise in MITO and suppress neuronal death in an AD mouse model [249].

AUT inducers are (a) mTOR inhibitors, either ATP-competitive inhibitors (e.g. Torin1 and related compounds) or non-ATP-competitive inhibitors (e.g., rapamycin and rapalogs), and (b) acting by mTOR-independent targets [238]. The most promising AUT inducers, acting by mTOR inhibition, are the non-ATP-competitive inhibitors rapamycin and rapalogs that are mTORC1 selective and induced AUT in animal models of AD, PD and PrD [251–258]. The AMPK signalling pathway is activated by mTOR-independent AUT activators, for example, by trehalose. Trehalose inhibits GLUT proteins, thus eliciting AMPK activation [259]. Trehalose-induced

**119**

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

models of NBD, including AD, PD and PrD [260–265].

of new therapeutic interventions for these NBD.

The author declares no conflict of interest.

**Acknowledgements**

figures is acknowledged.

**Conflicts of interest**

AUT induction, with concomitant therapeutic effects, was demonstrated in mouse

Neurodegenerative brain disorders (NBD) change brain cell proteostasis due to the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden elicits the unfolded protein response (UPR) and stimulates AUT. In the short term, these responses tend to attenuate ER's stress, by reducing the ER's protein load and increasing the ER's folding capacity. In the long term, with prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the ER stress response to meet the increased protein burden is reflected in an increased cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote either apoptosis or necroptosis. Apoptosis and necroptosis of the affected cells lead to the release of toxic proteins into the surrounding tissue and trigger the response of microglia and astrocytes. Chronic neuroinflammation, sustained by the spread of progressive failure of AUT among brain cells, due to the release of toxic monomers and oligomers from dying cells and their uptake by initially healthy cells and by the persistent activation of microglia and astrocytes by toxic monomers and oligomers, also contributes to nerve apoptosis or necroptosis. The signalling pathways of apoptosis, AUT and necroptosis are interlinked. A better understanding on how chronic neuroinflammation, Alzheimer's, Parkinson's and prion diseases modulate the crosstalk among these signalling pathways could contribute to the development

The author thanks Professor Irina Milisav for reviewing the manuscript and suggesting improvements. The assistance of Ms. Vanja Mavrin in drawing the final

TNFα signalling pathway is the focus of pharmacological interventions targeting neuroinflammation in NBD with a variety of compounds [57]: (a) serotonin binds to microglial receptors and has anti-inflammatory effects; serotonin treatment reduced TNFα release in cultured primary microglia cells exposed to AβO and in mouse brains infused with AβO and also prevented AD-associated behavioural changes [266]; (b) etanercept, a decoy TNF receptor and IgG1 Fc fusion protein that inhibits the binding of soluble TNF to cell-surface TNF receptors, was evaluated in several clinical trials on patients with AD; no statistically significant results were reported; however, the drug was well tolerated, and largescale trials are expected [57]; and (c) infliximab, a human monoclonal antibody that binds TNFα and was used to treat human auto-immune and inflammatory diseases, prevented eIF2a phosphorylation and long-term memory loss in a mouse

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

model of AD [7, 267].

**4. Conclusions**

#### *Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

AUT induction, with concomitant therapeutic effects, was demonstrated in mouse models of NBD, including AD, PD and PrD [260–265].

TNFα signalling pathway is the focus of pharmacological interventions targeting neuroinflammation in NBD with a variety of compounds [57]: (a) serotonin binds to microglial receptors and has anti-inflammatory effects; serotonin treatment reduced TNFα release in cultured primary microglia cells exposed to AβO and in mouse brains infused with AβO and also prevented AD-associated behavioural changes [266]; (b) etanercept, a decoy TNF receptor and IgG1 Fc fusion protein that inhibits the binding of soluble TNF to cell-surface TNF receptors, was evaluated in several clinical trials on patients with AD; no statistically significant results were reported; however, the drug was well tolerated, and largescale trials are expected [57]; and (c) infliximab, a human monoclonal antibody that binds TNFα and was used to treat human auto-immune and inflammatory diseases, prevented eIF2a phosphorylation and long-term memory loss in a mouse model of AD [7, 267].

#### **4. Conclusions**

*Programmed Cell Death*

human PRD [241].

**signalling pathways**

logical strategies are given below:

(a) Targeting excessive ROS production:

(b) promoting moderate to high increases in cytosolic ROS concentrations and (c) attenuating AUT [42, 62, 235–240]. In contrast to PD and AD, PrPSC-infected cells are more likely to respond with necroptosis and then apoptosis. For example, a significant upregulation of necroptosis signalling molecules phosphorylated MLKL, MLKL and receptor-interacting serine/threonine-protein kinase 3 (RIP3) was measured in the post-mortem cortical brains of patients with various types of

**3. Pharmacological strategies targeting AUT, apoptosis and necroptosis** 

Pharmacological strategies to ameliorate MITO dysfunction include:

(a2) Antioxidant vatiquinone used in clinical trials [243].

press neuronal death in an AD mouse model [249].

(a1) Mercaptamine that increases levels of glutathione in human [78].

(a3) RTA-308 stimulates Nrf2 to enhance the expression of pro-oxidant genes and to repress inflammatory genes in an animal model [244].

(a4) Antioxidants coenzyme Q, lipoic acid and green tea polyphenol epigallocatechin gallate attenuate the effects of NBD in animal models [245–248].

(a5) Ceria nanoparticles are ROS scavengers that localise in MITO and sup-

(b) Targeting mitochondrial biogenesis: stimulation of PGC1-α's ROS scavenging activity with SIRT1 could attenuate ROS-induced damage in AD [250].

AUT inducers are (a) mTOR inhibitors, either ATP-competitive inhibitors (e.g. Torin1 and related compounds) or non-ATP-competitive inhibitors (e.g., rapamycin and rapalogs), and (b) acting by mTOR-independent targets [238]. The most promising AUT inducers, acting by mTOR inhibition, are the non-ATP-competitive inhibitors rapamycin and rapalogs that are mTORC1 selective and induced AUT in animal models of AD, PD and PrD [251–258]. The AMPK signalling pathway is activated by mTOR-independent AUT activators, for example, by trehalose. Trehalose inhibits GLUT proteins, thus eliciting AMPK activation [259]. Trehalose-induced

At present, most of the studies, devoted to the development of pharmacological interventions for NBD, are focused on the crosstalk of AUT and apoptosis signalling pathways in neurons. Future research should also include development of pharmacological interventions that target other cells involved in the development of NBD, including microglia, astrocytes, endothelial cells and pericytes [242]. The development of pharmacological interventions for NBD should be guided by several key questions: (a) How to modulate the role of AUT from pro-death to pro-survival? (b) How is the information from the crosstalk among AUT, apoptosis and necroptosis integrated? (c) How to modulate the crosstalk among AUT, apoptosis and necroptosis? and (d) How is the information from the crosstalk among AUT, apoptosis and necroptosis (e.g. inflammation-promoting molecules) shared among different cells involved in the development of NBD? [242]. Examples of pharmaco-

**118**

Neurodegenerative brain disorders (NBD) change brain cell proteostasis due to the accumulation of normal, mutant, misfolded or unfolded proteins in the endoplasmic reticulum (ER). The increased ER burden elicits the unfolded protein response (UPR) and stimulates AUT. In the short term, these responses tend to attenuate ER's stress, by reducing the ER's protein load and increasing the ER's folding capacity. In the long term, with prolonged ER stress, the UPR changes from supporting cell survival to promoting apoptosis. The failure of the ER stress response to meet the increased protein burden is reflected in an increased cytosolic protein accumulation that initially further stimulates AUT. Over time, the accumulated proteins in the cytosol undergo post-translational changes into toxic monomers and oligomers that repress AUT at multiple levels and promote either apoptosis or necroptosis. Apoptosis and necroptosis of the affected cells lead to the release of toxic proteins into the surrounding tissue and trigger the response of microglia and astrocytes. Chronic neuroinflammation, sustained by the spread of progressive failure of AUT among brain cells, due to the release of toxic monomers and oligomers from dying cells and their uptake by initially healthy cells and by the persistent activation of microglia and astrocytes by toxic monomers and oligomers, also contributes to nerve apoptosis or necroptosis. The signalling pathways of apoptosis, AUT and necroptosis are interlinked. A better understanding on how chronic neuroinflammation, Alzheimer's, Parkinson's and prion diseases modulate the crosstalk among these signalling pathways could contribute to the development of new therapeutic interventions for these NBD.

#### **Acknowledgements**

The author thanks Professor Irina Milisav for reviewing the manuscript and suggesting improvements. The assistance of Ms. Vanja Mavrin in drawing the final figures is acknowledged.

#### **Conflicts of interest**

The author declares no conflict of interest.

*Programmed Cell Death*

#### **Funding**

This work was supported by ARRS grant number P3-0171.

#### **Author details**

Samo Ribarič\* and Irina Milisav Ribarič Faculty of Medicine, Institute of Pathophysiology, University of Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to: samo.ribaric@mf.uni-lj.si

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**121**

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases*

prion diseases. Frontiers in Aging Neuroscience. 2017;**9**:120. DOI: 10.3389/

[9] Bingol B. Autophagy and lysosomal pathways in nervous system disorders. Molecular and Cellular Neurosciences. 2018;**91**:167-208. DOI: 10.1016/j.

[10] Lai M, Yao H, Shah SZA, Wu W, Wang D, Zhao Y, et al. The NLRP3 caspase 1 inflammasome negatively regulates autophagy via TLR4-TRIF in prion peptide-infected microglia. Frontiers in Aging Neuroscience. 2018;**10**:116. DOI: 10.3389/

[11] Fan XY, Tian C, Wang H, Xu Y, Ren K, Zhang BY, et al. Activation of the AMPK-ULK1 pathway plays an important role in autophagy during prion infection. Scientific Reports. 2015;**5**:14728. DOI: 10.1038/srep14728

[12] Wang H, Tian C, Sun J, Chen LN, Lv Y, Yang XD, et al. Overexpression of PLK3 mediates the degradation of abnormal prion proteins

dependent on chaperone-mediated autophagy. Molecular Neurobiology. 2017;**54**(6):4401-4413. DOI: 10.1007/

[13] Grant CM. Sup35 methionine oxidation is a trigger for de novo [PSI(+)] prion formation. Prion. 2015;**9**(4):257-265. DOI: 10.1080/19336896.2015.1065372

[14] Nixon RA. Autophagy,

amyloidogenesis and Alzheimer disease. Journal of Cell Science. 2007;**120**(Pt 23):4081-4091. DOI: 10.1242/jcs.019265

[15] Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, et al. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates

s12035-016-9985-0

fnagi.2017.00120

mcn.2018.04.009

fnagi.2018.00116

*DOI: http://dx.doi.org/10.5772/intechopen.86706*

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[2] Dickson DW. Parkinson's disease and parkinsonism: Neuropathology. Cold Spring Harbor Perspectives in Medicine. 2012;**2**(8):1-15. DOI: 10.1101/

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[4] Martin-Jimenez CA, Garcia-Vega A, Cabezas R, Aliev G, Echeverria V, Gonzalez J, et al. Astrocytes and endoplasmic reticulum stress: A bridge between obesity and neurodegenerative diseases. Progress in Neurobiology. 2017;**158**:45-68. DOI: 10.1016/j.

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[6] Gerakis Y, Hetz C. Emerging roles of ER stress in the etiology and pathogenesis of Alzheimer's disease. The FEBS Journal. 2018;**285**(6):995-1011.

[7] Freeman OJ, Mallucci GR. The UPR and synaptic dysfunction in neurodegeneration. Brain Research. 2016;**1648**(Pt B):530-537. DOI: 10.1016/j.

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*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

#### **References**

*Programmed Cell Death*

This work was supported by ARRS grant number P3-0171.

**Funding**

**120**

**Author details**

Ljubljana, Slovenia

Samo Ribarič\* and Irina Milisav Ribarič

provided the original work is properly cited.

Faculty of Medicine, Institute of Pathophysiology, University of Ljubljana,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: samo.ribaric@mf.uni-lj.si

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[19] Elmallah MI, Borgmeyer U, Betzel C, Redecke L. Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion. Prion. 2013;**7**(5):404-411. DOI: 10.4161/pri.26745

[20] Younan ND, Nadal RC, Davies P, Brown DR, Viles JH. Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway. The Journal of Biological Chemistry. 2012;**287**(34):28263-28275. DOI: 10.1074/jbc.M112.354779

[21] Coskun P, Wyrembak J, Schriner SE, Chen HW, Marciniack C, Laferla F, et al. A mitochondrial etiology of Alzheimer and Parkinson disease. Biochimica et Biophysica Acta. 2012;**1820**(5):553-564. DOI: 10.1016/j.bbagen.2011.08.008

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[23] Siskova Z, Mahad DJ, Pudney C, Campbell G, Cadogan M, Asuni A, et al. Morphological and functional abnormalities in mitochondria associated with synaptic degeneration in prion disease. The American Journal of Pathology. 2010;**177**(3):1411-1421. DOI: 10.2353/ajpath.2010.091037

[24] Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, et al. The cellular prion protein binds copper in vivo. Nature. 1997;**390**(6661):684- 687. DOI: 10.1038/37783

[25] Jones CE, Abdelraheim SR, Brown DR, Viles JH. Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein. The Journal of Biological Chemistry. 2004;**279**(31):32018-32027. DOI: 10.1074/jbc.M403467200

[26] Zeng L, Zou W, Wang G. Cellular prion protein (PrP(C)) and its role in stress responses. International Journal of Clinical and Experimental Medicine. 2015;**8**(5):8042-8050

[27] Thackray AM, Knight R, Haswell SJ, Bujdoso R, Brown DR. Metal imbalance and compromised antioxidant function are early changes in prion disease. The Biochemical Journal. 2002;**362** (Pt 1):253-258

[28] Brown DR. Neurodegeneration and oxidative stress: Prion disease results from loss of antioxidant defence. Folia Neuropathologica. 2005;**43**(4):229-243

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10.1007/s11033-009-9899-2

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[39] Prots I, Veber V, Brey S, Campioni S,

synuclein oligomers impair neuronal microtubule-kinesin interplay. The Journal of Biological Chemistry. 2013;**288**(30):21742-21754. DOI:

[40] Schapira AH. Mitochondria in the aetiology and pathogenesis of Parkinson's disease. Lancet Neurology. 2008;**7**(1):97-109. DOI: 10.1016/

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*DOI: http://dx.doi.org/10.5772/intechopen.86706*

encephalopathies. Brain Research Brain Research Reviews. 2002;**38**(3):328-339

[30] Nadal RC, Abdelraheim SR, Brazier MW, Rigby SE, Brown DR, Viles JH. Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals. Free Radical Biology & Medicine. 2007;**42**(1):79-89. DOI: 10.1016/j.freeradbiomed.2006.09.019

[31] Alim MA, Hossain MS, Arima K, Takeda K, Izumiyama Y, Nakamura M, et al. Tubulin seeds alpha-synuclein fibril formation. The Journal of Biological Chemistry. 2002;**277**(3): 2112-2117. DOI: 10.1074/jbc.M102981200

[32] Esteves AR, Arduino DM, Swerdlow RH, Oliveira CR, Cardoso SM. Microtubule depolymerization

potentiates alpha-synuclein

neuro.24.005.2009

oligomerization. Frontiers in Aging Neuroscience. 2010;**1**:5. DOI: 10.3389/

[33] Kim M, Jung W, Lee IH, Bhak G, Paik SR, Hahn JS. Impairment of microtubule system increases alphasynuclein aggregation and toxicity. Biochemical and Biophysical Research Communications. 2008;**365**(4):628-635.

DOI: 10.1016/j.bbrc.2007.11.020

[34] Lee HJ, Khoshaghideh F, Lee S, Lee SJ. Impairment of microtubule-dependent trafficking by overexpression of alpha-synuclein. The European Journal of Neuroscience.

2006;**24**(11):3153-3162. DOI: 10.1111/j.1460-9568.2006.05210.x

[35] Nakayama K, Suzuki Y, Yazawa I. Microtubule depolymerization suppresses alpha-synuclein accumulation in a mouse model of multiple system atrophy. The American Journal of Pathology. 2009;**174**(4):1471- 1480. DOI: 10.2353/ajpath.2009.080503

[36] Nakayama K, Suzuki Y, Yazawa I. Binding of neuronal alpha-synuclein to beta-III tubulin and accumulation

*Autophagy and Cell Death in Alzheimer's, Parkinson's and Prion Diseases DOI: http://dx.doi.org/10.5772/intechopen.86706*

encephalopathies. Brain Research Brain Research Reviews. 2002;**38**(3):328-339

*Programmed Cell Death*

JCI33585

bi049979h

amyloid beta accumulation in mice. The Journal of Clinical Investigation. 2008;**118**(6):2190-2199. DOI: 10.1172/ [22] Borger E, Aitken L, Muirhead KE, Allen ZE, Ainge JA, Conway SJ, et al. Mitochondrial beta-amyloid in Alzheimer's disease. Biochemical Society Transactions. 2011;**39**(4):868- 873. DOI: 10.1042/BST0390868

[23] Siskova Z, Mahad DJ, Pudney C, Campbell G, Cadogan M, Asuni A, et al. Morphological and functional abnormalities in mitochondria

associated with synaptic degeneration in prion disease. The American Journal of Pathology. 2010;**177**(3):1411-1421. DOI:

10.2353/ajpath.2010.091037

687. DOI: 10.1038/37783

10.1074/jbc.M403467200

2015;**8**(5):8042-8050

(Pt 1):253-258

[26] Zeng L, Zou W, Wang G. Cellular prion protein (PrP(C)) and its role in stress responses. International Journal of Clinical and Experimental Medicine.

[27] Thackray AM, Knight R, Haswell SJ, Bujdoso R, Brown DR. Metal imbalance and compromised antioxidant function are early changes in prion disease. The

[28] Brown DR. Neurodegeneration and oxidative stress: Prion disease results from loss of antioxidant defence. Folia Neuropathologica. 2005;**43**(4):229-243

Biochemical Journal. 2002;**362**

[29] Milhavet O, Lehmann S. Oxidative stress and the prion protein in transmissible spongiform

[24] Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, et al. The cellular prion protein binds copper in vivo. Nature. 1997;**390**(6661):684-

[25] Jones CE, Abdelraheim SR, Brown DR, Viles JH. Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein. The Journal of Biological Chemistry. 2004;**279**(31):32018-32027. DOI:

[16] Salminen A, Kaarniranta K, Kauppinen A, Ojala J, Haapasalo A, Soininen H, et al. Impaired autophagy and APP processing in Alzheimer's disease: The potential role of Beclin 1 interactome. Progress in Neurobiology. 2013;**106-107**:33-54. DOI: 10.1016/j.

[17] Hokenson MJ, Uversky VN, Goers J,

Yamin G, Munishkina LA, Fink AL. Role of individual methionines in the fibrillation of methionine-oxidized

alpha-synuclein. Biochemistry. 2004;**43**(15):4621-4633. DOI: 10.1021/

[18] Leong SL, Pham CL, Galatis D, Fodero-Tavoletti MT, Perez K, Hill AF, et al. Formation of dopamine-mediated alpha-synuclein-soluble oligomers requires methionine oxidation. Free Radical Biology & Medicine. 2009;**46**(10):1328-1337. DOI: 10.1016/j.

freeradbiomed.2009.02.009

DOI: 10.4161/pri.26745

10.1074/jbc.M112.354779

[19] Elmallah MI, Borgmeyer U, Betzel C, Redecke L. Impact of

methionine oxidation as an initial event on the pathway of human prion protein conversion. Prion. 2013;**7**(5):404-411.

[20] Younan ND, Nadal RC, Davies P, Brown DR, Viles JH. Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway. The Journal of Biological Chemistry. 2012;**287**(34):28263-28275. DOI:

[21] Coskun P, Wyrembak J, Schriner SE, Chen HW, Marciniack C, Laferla F, et al. A mitochondrial etiology of Alzheimer and Parkinson disease. Biochimica et Biophysica Acta. 2012;**1820**(5):553-564. DOI: 10.1016/j.bbagen.2011.08.008

pneurobio.2013.06.002

**122**

[30] Nadal RC, Abdelraheim SR, Brazier MW, Rigby SE, Brown DR, Viles JH. Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals. Free Radical Biology & Medicine. 2007;**42**(1):79-89. DOI: 10.1016/j.freeradbiomed.2006.09.019

[31] Alim MA, Hossain MS, Arima K, Takeda K, Izumiyama Y, Nakamura M, et al. Tubulin seeds alpha-synuclein fibril formation. The Journal of Biological Chemistry. 2002;**277**(3): 2112-2117. DOI: 10.1074/jbc.M102981200

[32] Esteves AR, Arduino DM, Swerdlow RH, Oliveira CR, Cardoso SM. Microtubule depolymerization potentiates alpha-synuclein oligomerization. Frontiers in Aging Neuroscience. 2010;**1**:5. DOI: 10.3389/ neuro.24.005.2009

[33] Kim M, Jung W, Lee IH, Bhak G, Paik SR, Hahn JS. Impairment of microtubule system increases alphasynuclein aggregation and toxicity. Biochemical and Biophysical Research Communications. 2008;**365**(4):628-635. DOI: 10.1016/j.bbrc.2007.11.020

[34] Lee HJ, Khoshaghideh F, Lee S, Lee SJ. Impairment of microtubule-dependent trafficking by overexpression of alpha-synuclein. The European Journal of Neuroscience. 2006;**24**(11):3153-3162. DOI: 10.1111/j.1460-9568.2006.05210.x

[35] Nakayama K, Suzuki Y, Yazawa I. Microtubule depolymerization suppresses alpha-synuclein accumulation in a mouse model of multiple system atrophy. The American Journal of Pathology. 2009;**174**(4):1471- 1480. DOI: 10.2353/ajpath.2009.080503

[36] Nakayama K, Suzuki Y, Yazawa I. Binding of neuronal alpha-synuclein to beta-III tubulin and accumulation

in a model of multiple system atrophy. Biochemical and Biophysical Research Communications. 2012;**417**(4):1170- 1175. DOI: 10.1016/j.bbrc.2011.12.092

[37] Zhou RM, Huang YX, Li XL, Chen C, Shi Q, Wang GR, et al. Molecular interaction of alpha-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells. Molecular Biology Reports. 2010;**37**(7):3183-3192. DOI: 10.1007/s11033-009-9899-2

[38] Eckert A, Nisbet R, Grimm A, Gotz J. March separate, strike together-role of phosphorylated TAU in mitochondrial dysfunction in Alzheimer's disease. Biochimica et Biophysica Acta. 2014;**1842**(8):1258- 1266. DOI: 10.1016/j.bbadis.2013.08.013

[39] Prots I, Veber V, Brey S, Campioni S, Buder K, Riek R, et al. Alphasynuclein oligomers impair neuronal microtubule-kinesin interplay. The Journal of Biological Chemistry. 2013;**288**(30):21742-21754. DOI: 10.1074/jbc.M113.451815

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**140**

### *Edited by Hala Gali-Muhtasib and Omar Nasser Rahal*

This book incorporates developments in our understanding of cell death mechanisms and highlights recent advances in programmed cell death regulation processes. It provides the reader with the network of pathways targeted by herbal anticancer drugs and discusses the role of endoplasmic reticulum stress in cell death mechanisms in addition to highlighting the mechanisms of autophagy and its role in diseases. This book provides valuable material for researchers and for teaching postgraduate students. Emphasis on recent advances and their clinical applications offers insights to researchers that will likely lead to the development of novel therapeutic approaches.

Published in London, UK © 2020 IntechOpen © tonaquatic / iStock

Programmed Cell Death

Programmed Cell Death

*Edited by Hala Gali-Muhtasib* 

*and Omar Nasser Rahal*