Section 1 Cell Death

**Chapter 1**

## Epi-Regulation of Cell Death in Cancer

*Antonio Beato, Laura Della Torre, Vincenza Capone, Daniela Carannante, Gregorio Favale, Giulia Verrilli, Lucia Altucci and Vincenzo Carafa*

### **Abstract**

How do organisms regulate the correct balance between the production of "new" cells and the elimination of the "old" ones, remains an important biology issue under investigation. Cell(s) death represents a fundamental process involved in organism development and cell homeostasis, whose alteration is considered one hallmark of cancer and lead to drug resistance and consequently treatment failure. The recent re-classification of cell death has identified new molecular programs in which several proteins have a pivotal role. Several studies have highlighted a direct link between epigenetic modifications and cell death mechanisms. Different epi-modifications have been described, capable of regulating diverse key players implicated in cell death, leading to uncontrolled proliferation of cancer cells. Scientific efforts are focused on the understanding the epigenetic regulation of cell death mechanisms by developing tools and/or new epi-molecules able to overcome cell death resistance. The development of new epi-molecular tools can overcome cell death deregulation thus potentially improving the sensitivity to the anti-tumor therapies. This chapter focuses on the main epigenetic deregulations in cell death mechanisms in cancer.

**Keywords:** epigenetics, cell death, cancer, apoptosis, necroptosis pyroptosis, Immunogenic cell death, NETosis, parthanatos

### **1. Introduction**

Epigenetics is the study of functionally heritable changes in the genome that occur without structural changes in the DNA sequence [1], characterizing cellular phenomena and molecular mechanisms responsible of the remodeling of a phenotype starting from a fixed structure that is determined by the genotype [2]. Epigenetic mechanisms can regulate gene expression through covalent chemical modifications, histone posttranslational modifications (PTMs), several RNA species or also through chromosomal superstructure modifications in which DNA is packaged without making any change in the DNA (in its) basic structure [3, 4]. During the past years, different types of epigenetic mechanisms have been identified (i) DNA methylation, (ii) histone modifications, and (iii) non-coding RNA (ncRNA),

able to modulate gene and protein expression [3]. Epigenetic changes are the results of the action of three different enzymatic classes, (i) *writers*, able to add chemical groups on DNA, histones and proteins; (ii) *readers,* which read and identify several "signals" through their structural domains, and (iii) *erasers* involved in the removal of chemical groups.

DNA methylation is one of the most known epigenetic modifications able to repress gene transcription and expression, especially when located near the transcription start sites of genes [5]. Well-known is the crucial role of both hypermethylation of tumor suppressor and global hypomethylation of oncogenes in tumor initiation and progression [6]. PTMs, changing the histone structure, are also able to alter gene expression. [7]. These alterations, mediated by the addition of chemical groups at the N-terminal tail of histones are covalent, reversible, and redundant creating a real "histone code" that regulates the chromatin structure, gene expression and the recruitment of different enzymes. These changes can include (i) acetylation of lysine residues; (ii) methylation of lysine and arginine residues; (iii) phosphorylation of serine residues; (iv) the binding of one or more monomers of ubiquitin (ubiquitination), (v) the binding of several polypeptide (SUMOylation), (vi) citrullination, consisting in the conversion of arginine residues into citrulline residues by specific enzymes [8]. Much progress has been made in understanding the roles of both microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in gene regulation which play an important part in several biological processes such as proliferation, differentiation, apoptosis [9].

Epigenetic changes are often associated to the genesis of many pathologies with a "high social impact" such as cancer. These modifications induce the blocking or definitive silencing of many cellular signal transduction pathways, the restoration of which today represents a promising therapeutic perspective. Particularly

**Figure 1.** *Epigenetic regulation in cell deaths.*

*Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*

studied are the cell death pathways, complex and finely regulated processes whose deregulation alters the correct cell homeostasis, responsible of the excessive cell proliferation. Cell death *phenomena* alteration represents one of the main markers of oncogenic cell transformation responsible for resistance to cancer and drug therapies failure [10]; indeed, a tumor cell still retains the ability to proliferate and/or to go into apoptosis but the pathways of regulation of these signals can be silenced and therefore inactive.

The aim of this chapter is to shed light on the epigenetic regulation of the molecular players involved in cell death pathways, whose alteration has a pivotal role in carcinogenesis. Considering the reversibility of the epigenetic modifications, they represent a promising target for anticancer therapy (**Figure 1**).

### **2. Cell death mechanisms**

For a long time, cell death was considered an inexorable event for cells, an indispensable cellular mechanism which allows the cell to die when it is damaged, altered or simply aged [11]. Cell death is to be considered not only as a destructive process for the organism but also as a defensive process that the cells put in place to preserve homeostasis [12]. Several studies have shown that cell death mechanisms not only allow the cell to die when it has reached the end of its life cycle, but they are useful events both during prenatal and the development for the removal of excess, damaged or altered formed cells [13]. Based on the severity of the insults, on the morphology and on the biological events that can be activated during the cell death process, we distinguish an accidental death cellular process (ACD), and a regulated cell death (RCD) [14].

Necrosis, a type of ACD, is an unordered and unscheduled death mechanism that cells puts in place in response to stimuli such as radiations, toxins, osmotic variations, viral or bacterial infections followed by a large immunogenic and inflammatory response. Some enzyme systems involved in this process are lytic enzymes called calpain and cathepsins as well as damage-associated molecular patterns (DAMPs) which can be DNA fragments, ATP, uric acid, inflammatory cytokines including High Mobility Group Box 1 (HMGB1), an inflammatory cytokine of great importance in the necrosis process [15].

On the other hand, RCD, is a controlled death process that can be genetically or pharmacologically regulated which is involved in two different scenarios. It acts as the main process in tissue development responsible for the cell's turnover in the absence of exogenous environmental perturbations [16, 17]. RCD can be also the result of prolonged intra and extracellular perturbations [18] and does not alter tissue homeostasis or cell development. When it occurs in physiological conditions it is called programmed cell death (PCD) [19–21]. Considering only morphological characteristics, it has been proposed a classification of several forms of cell death including: type I or apoptosis, type II or autophagy, type III or necrosis [14, 19].

In this last decade, a new subdivision of the various cell deaths has been proposed through essential and mechanistic aspects that distinguish them. Twelve types of cell deaths have been identified which are Necroptosis, Ferroptosis, Pyroptosis, Parthanatos, Entotic cell deah, NETotic cell death, Lysosome-dependent cell death (LDCD), Autophagy-dependent cell death (ADCD), Immunogenic cell death (ICD), Intrinsic apoptosis, Extrinsic apoptosis, Mithocondrial permeability transition-driven necrosis (MPT).

### **2.1 Epigenetic regulation in apoptosis**

Apoptosis, or type I PCD, is a finely regulated "molecular assisted suicide" mechanism, necessary for maintaining cellular homeostasis processes. It is the response to DNA damage (spontaneous apoptosis), or different conditions such as hypoxia, lack of growth factors and action of chemotherapeutic agents (induced apoptosis). It is also involved in physiological processes such as embryogenesis and differentiation. It is defined as a "clean" death mechanism since there is no release of waste elements: the apoptotic bodies - which contain cell fragments - are eliminated through the action of the immune system and more specifically through the action of macrophages [22]. The loss of apoptotic regulation causes uncontrolled cell proliferation leading to several human diseases such as cancer [23]. Apoptosis is the result of extrinsic or intrinsic signals, coming from outside and inside the cell, respectively. A pivotal role, in this pathway, is played by initiator and effector caspases synthesized as inactive zymogens and activated by a proteolytic cut [24]. The extrinsic apoptosis pathway is triggered by the link between death receptors of the Tumor Necrosis Factor (TNF) family with their specific pro-apoptotic ligands resulting in the activation of different molecular adapter able to cleave initiator caspases which in turn cleave and activate effector caspases [25] while the intrinsic pathway is triggered by mitochondrial dysfunction caused by cellular stress [26]. The main event is the release of cytochrome c from complex, called apoptosome, with other cytosolic proteins Apoptotic protease activating factor-1 (Apaf-1) and activates initiator and effector caspases [27].

Several epigenetic modifications have been identified as responsible for the evasion of the apoptotic process and carcinogenesis. As result of an alteration of DNA methyltransferases (DNMTs) functions, in cancer cells diffused events of hyper- and hypo-methylation, contributes to apoptosis resistance [28].

In several cancers, hypermethylation of the promoter region of tumor suppressor genes involved in the regulation of apoptotic processes leads to an uncontrollable proliferation contributing to apoptosis resistance of cancer cells [29].

Hypermethylation on FAS promoter region, is responsible of the suppression of its expression, leading to a Cutaneous T-cell lymphoma and neoplastic transformation of epithelial cells into colon cancer [30, 31]. In neuroblastoma, melanoma and ovarian cancer cells, the resistance to TRAIL-induced apoptosis is due to hypermethylation of the DR4 and DR5 promoters [32–34]. In other cancer types, such as hepatocellular carcinoma, bladder cancer, small-cell lung carcinoma, glioblastoma, retinoblastoma, and neuroblastoma, caspases 8 and 10 are silenced by the methylation on their promoters resulting in a block of apoptotic pathway [35–39]. Silencing of Apaf-1, as result of the block of intrinsic apoptotic pathway, is observed in leukemia and melanoma, as well as bladder and kidney cancers and is associated with therapeutic resistance [40–43].

Promoter hypermethylation of BAX, BAK, and PUMA in multiple myeloma and Burkitt's lymphoma cells, is responsible for the silencing of these genes and so of the abrogation of related death pathway while in prostate cancer patients, despite the hypermethylation of the Bcl-2 promoter, apoptotic pathways, particularly the extrinsic pathway, are largely preserved [44–46].

However, also a global genomic hypomethylation has a role for carcinogenesis [47]. In a variety of human cancers, including metastatic tumor, B-cell chronic lymphocytic leukemia, cervical, colorectal, hepatocellular and bladder cancer hypomethylation determine chromosomal instability and cancer transformation [48–52]. In addition to DNA methylation, other epigenetic modifications, such as histone modification and miRNA regulation, can alter apoptotic pathway. In Burkitt's lymphoma, a well-known repressive chromatin mark, the trimethylation of lysine 27 of histone H3 (H3K27me3), affects the expression levels of proapoptotic BIM protein [53]. In medulloblastoma patients, abnormal H3 and H4 acetylation patterns at the promoter region of DR4 gene expression, alter apoptosis [54]. Similarly, increased H3 and H4 acetylation induced by HDAC inhibitors affect the amounts of proapoptotic Bax protein in human colon cancer cells leading to cell cycle arrest and apoptosis [55].

An alteration of the balance between Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs) contributes to cancer promotion modulating the acetylation levels of several non-histone proteins involved in apoptotic cell death pathway such as Rb, E2F and ku70. Indeed, the involvement of Ku70 in promoting apoptosis, is strictly regulated by its acetylation level. Ku70, inhibits BAX activation, preventing its translocation to the mitochondrial membrane and suppressing apoptosis. Ku70 acetylation promoted by CBP and PCAF on two different lysine residues (K539 and K542), blocks Ku70-BAX connection and promotes apoptosis [56]. Acetylation of E2F1 is essential for the recruitment of several proteins that control the apoptotic response to DNA damage. In response to DNA damage, acetylated E2F1 interacts with Rb influencing the cellular response driven transcription of the proapoptotic target gene p73 [57, 58].

Through the regulation of gene expression, miRNAs are considered key regulators of several cellular processes such as apoptosis and have a pivotal role in cancer progression. A function in tumorigenesis was described for miR15/16 as well as for some miR-34 family members. In pituitary adenoma, B-cell chronic lymphocytic leukemia and prostate cancer, miR15/16 miRNAs down regulated or deleted led to overexpression of antiapoptotic BCL-2, as well as cyclin D1, MCL1, and WNT3A at the post-transcriptional level inducing cancer cell proliferation and invasiveness [59–62]. Further investigation indicated a positive feedback loop between p53 and miRNAs. P53 regulates miRNA expression at numerous levels and, as a transcription factor, p53 can affect the expression of individual miRNAs [63, 64].

MiR-34a and miR-34b/c, three members of the mir34 family, are direct p53 targets. MiR-34 family regulated SIRT1 mRNA leading to an increase in p53 acetylation levels which regulate cell-cycle and apoptosis [65]. MiR-34a is repressed via hypermethylation in different types of cancer such as gastric cancer, chronic lymphocytic leukemia, pancreatic, breast, colon, kidney cancer, and Burkitt's lymphoma [66], while miR-34b/c was down regulated in sarcoma, colon, and ovarian cancer [62]. Another miRNA, the miR-29b, able to target DNMT3b and MCL1 is significantly reduced in several cancers such as lung, pancreatic and ovarian [67–70]. An hypermethylation of miR-127 is characterized in cancers of the bladder, prostate, breast, and lung, as well as lymphoma [71]. This epi-modification is responsible of the miR-127 silencing, which in turn determines the hyperactivity of one of its molecular targets, the protooncogene BCL-6, in these cancers [72]. Other examples are miR-106b and miR-93, which are known to alter TGF-induced apoptosis in gastric cancer cells by inhibiting BIM expression while MiR-135a inhibits JAK2, resulting in a decrease in antiapoptotic Bcl-xL expression [73, 74]. MiR-135a expression is reduced in ovarian cancer, Hodgkin lymphoma, Acute Myeloid Leukemia (AML) (**Table 1**) [75, 76].

### **2.2 Epigenetic regulation in necroptosis**

Necroptosis, a form of regulated cell death independent from caspase activation, is regulated by specific death receptors, including (but not limited to) FAS/APO-1


#### **Table 1.**

*Epigenetic regulation in apoptosis.*

(CD95) and TNFR1, or pathogen recognition receptors (PRRs), including TLR3, TLR4, and Z-DNA binding protein 1 (ZBP1; also known as DAI) [77]. Necroptotic signaling pathway depends on the sequential activation of the receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain like pseudokinase (MLKL) and (at least in some settings) on the kinase activity of RIPK1, also called necrosome [19, 78]. Therefore, it is not surprising that necroptotic cell death signaling can also be regulated by epigenetic modifications at the necrosome components [79].

Necroptosis may represent a new therapeutic strategy to overcome resistance to apoptosis. In cancer, necroptosis has been defined as a *double-edged sword* for its pro- or anti-tumor effect [80]. Epigenetic alterations may modify the gene expression levels of the necroptosis regulators, affecting cancer initiation, promotion and progression [81]. Hypo- and hyper-methylation of key components of necroptosis existed in multiple tumors and could affect gene expression and prognosis of cancer patients [81]. A multi-omics approach identified promoter hypermethylation of (i)

MLKL in skin cutaneous melanoma (SKCM) and in colon adenocarcinoma (COAD); (ii) RIPK3 in adrenocortical carcinoma (ACC); (iii) RIPK1 in kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP). Differently, MLKL hypomethylation has been reported in low grade glioma (LGG) and uveal melanoma (UVM); RIPK3 hypomethylation in LGG, AML and KIRC; RIPK1 in LGG, thymoma (THYM), lung squamous cell carcinoma (LUSC), ACC, and SKCM [81]. Among the necrosome components, RIPK3 is often downregulated, in cancer which is why several studies focused on its epigenetic modifications, unlike RIPK1 or MLKL.

RIPK3 is normally expressed in normal tissues, but the genomic region near the *RIPK3* transcription start site (TSS) is highly methylated resulting in loss of RIPK3 expression in different types of primary cancers probably due to an adaptive process to evade necroptosis [82, 83]. In breast cancer, 85% of patients have reduced RIPK3 expression due to promoter hypermethylation [82]. However, robust re-expression of RIPK3 in recurrent breast tumor cells was unexpectedly noted. These data were confirmed by ChIP-Seq experiments in which RNA polymerase II occupies the promoter region of *RIPK3* and epigenetic histone markers, H3K9ac and H3K4me3, are enriched in the regulatory regions of the *RIPK3* gene adjacent to the RNA polymerase II binding site. Conversely, many of the cytosines in the *RIPK3* CpG island are methylated in primary tumors. However, treatment with HDAC inhibitors and/or hypomethylating agents, such as 5-azacytidine (5-AC), can restore RIPK3 expression and thus promotes sensitivity to chemotherapeutic agents in a RIPK3-dependent manner [82, 83].

As in breast cancer, RIPK3 expression is reduced also in lung cancer and this is associated with a poorer chemotherapy response. The promoter region of *RIPK3* being highly rich in CpG island is hypermethylated differently from primary human bronchial epithelial cells. The epigenetic silencing is responsible for RIPK3 and necroptotic cell death suppressions with worse response in non-small lung cancer (NSCLC) patients receiving chemotherapy. Therefore, demethylation treatments could improve the anticancer efficacy of chemotherapy [84]. A further study investigating the epigenetic landscape of necroptosis in lung adenocarcinoma (LUAD) did not identify any correlation between the levels of methylation in the *RIPK3* promoter and its mRNA expression [85].

The role of RIPK3 has also been discussed in malignant mesothelioma (MM) as downregulation at the transcriptional level consistent with epigenetic silencing via DNA methylation was observed in 62% of primary MMs. The high frequency of CpG methylation in the *RIPK3* promoter (22%) is mediated by DNA methyltransferase DNMT1 which contributes to a very poor overall survival (OS). In human pleural MM cells, *RIPK3* gene expression decrease both *in vitro* and in primary tumors, strengthening its pivotal role as tumor suppressor [86].

Some authors identified that the methylation carried out by DNMT1 in binding to the *RIPK3* promoter is stimulated by the oncometabolite in the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2-HG) produced by tumor-associated isocitrate dehydrogenases 1 (IDH1) mutation [86]. Tumorigenesis could be driven by IDH1 mutation at position 132 (R132) resulting in high levels of 2-HG production, which regulates DNMT1 activity by promoting its binding to specific DNA regions including the TSS of the *RIPK3* promoter. This phenomenon investigated in human brain cancers implies resistance to necroptosis and may support the survival of cancer cells, eventually leading to tumor formation [87].

Ten Eleven Translocation (TET) methylcytosine dioxygenases enzymes, using α-ketoglutarate (α-KG) as substrate, catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5-hmC), which is the first step for active DNA demethylation [88]. Some intermediates of the TCA cycle, including fumarate and α-KG, can competitively inhibit the enzymatic activity of TETs [89]. In Epstein–Barr virus-encoded latent membrane protein 1 (EBV-LMP1) positive cells, high levels of fumarate and low levels of α-KG, determine *RIPK3* silencing as the result of hypermethylation of its promoter region [89]. The oncomine database refers a significant downregulation of *RIPK3* in nasopharyngeal carcinoma (NPC), compared to nasopharyngitis tissues, as the result from the impairment of TETs' enzymatic activity in EBV-LMP1 positive cells [89]. From an epigenetic regulation/modification point of view, RIPK3 (among the necrosome components) was the most investigated and promising mediator. For instance, very little is known about the epigenetic regulation of MLKL which is essential for the execution of necroptosis. Interestingly, in Burkitt's lymphoma cell lines, MLKL expression levels correlate with the methylation status. As a result of the activity of the new DNA hypomethylating agent SGI-110, the silenced expression of MLKL is restored [90].

In conclusion, these studies indicate RIPK3 as a critical regulator of necroptosis, which is considered a tumor suppressor gene and whose low expression, also regulated at the epigenetic level, can be associated with poor prognosis in cancer. Hence, treatment with hypomethylating agents alone or in combination with chemotherapeutic agents facilitate the activation of necroptotic signaling (**Table 2**).

### **2.3 Epigenetic regulation in pyroptosis**

Pyroptosis is a form of inflammatory RCD induced by the activation of the NF-kB pathway followed by the triggering of intracellular sensors / receptors such as NLRP3, NLRC4 and AIM2, in response to DAMPs, Pathogen-associated molecular pattern (PAMPs) or different cytotoxic stimuli [91, 92]. Assembly of the inflammasome leads to pyroptotic cell death mediated by the cleavage of Gasdermin-D (GSDM-D), by caspases (caspase1 or caspase-4/5/11) and to the release of Interleukin-1*β (Il-1β)* and Il-18 in the microenvironment [93]. Pyroptosis can also occur with an alternative mechanism by which caspase-3 activates GSDM-E [94]. Recent studies have identified an epigenetic modulation of the pyroptotic process in cancer [95]. Among the


### **Table 2.** *Epigenetic regulation in necroptosis.*

### *Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*

proteins involved in the pyroptosis, epigenetic modification related to NLRP3, the sensor ASC, caspase-1 and GSDMs are the best characterized [96].

In gastric carcinoma, the loss of caspase-1 gene expression, which appears to be related to the worsening of the patient's prognosis, could be associated with methylation phenomena [97]. Indeed, anticancer therapy with the 5-aza-C hypomethylating agent, activates transcriptional mechanisms with expression of caspase-1 and the conclusion of the pyroptotic program [97]. In gastric, NPC and breast cancer, the hypermethylation at the promoter of the tumor suppressor ZDHHC1, induces pyroptosis by increasing the activation of caspase-1 in response to accumulated oxidative damage [98]. DNA methylation plays a crucial role in NLRP3-inflammasome activation in human monocytes, where, under physiological conditions, Cas-1a, ASC and Il-1β promoters are hypermethylated [95]. Furthermore, NF-kB and the demethylase TET2 are responsible for the hypomethylation and reactivation of ASC and Il-1β genes in differentiated monocytes and macrophages [95]. In lung, gastric and renal cancers, the hypermethylated state of ASC increased tumor growth and is associated with poor prognosis [99], indeed some studies report ASC demethylation as a possible strategy to induce selective cell death in cancer cells [100]. Conversely, reduced methylation in the ASC promoter, often associated with migration and invasion which are the basis of the metastatic process, is reported in patients with glioblastoma and squamous cell carcinoma [101, 102]. NLRP3-acetylation is fundamental for the assembly of the ASC domain and for its activation in response to exogenous stimuli in agingassociated inflammatory diseases as cancer; thus, the NLRP3 deacetylation mediated by SIRT2 represses its activity and inflammasome formation [103]. These evidences were confirmed in Aged SIRT2-deficient mice with a high-fat diet, which showed an increase in plasma Il-18 followed by an increase in NLRP3-inflammasome activity [103]. The epigenetic regulation of pyroptosis may also depend on the action of small non-coding RNAs. It is known that several miRNAs bind to 3′-untranslated NLRP3 gene region and degrade it [104]. To confirm these evidences, it was demonstrated that during myeloid differentiation, low levels of miR-233 increases NLRP3 inflammasome transcription, accompanied by the release of pro-inflammatory cytokines in activated macrophages [105]. In addition, XLOC\_000647 overexpression, an intergenic lncRNA, reduces the expression of NLRP3 in pancreatic cancer cells, playing a protective role against the starting of endothelial-mesenchymal transition (EndoMT), proliferation and metastasis formation, identifying a novel epigenetic mechanism involving the NLRP3-inflammasome in tumor progression of pancreatic cancer [106]. Additional research demonstrated the direct regulation of pro-caspase-1 by Neat1. This lncRNA can stabilize mature caspase-1 tetramers (p20: p10)2 and (p33: p10)2, promoting the assembly of the NLRP3-AIM2-inflammasomes, inducing a caspase-1-dependent pyroptosis [107]. The best characterized member of the GSDMs family, GSDM-D, appears to play a key role in NSCLC and can be regulated by methylation processes [108]. Elevated GSDM-D levels have been associated with unfavorable prognosis in lung cancer but favorable in skin cutaneous melanoma and its expression is regulated by the binding of Foxo1 on its promoter [109]. The hypermethylating activity of DNMT was also found at the GSDM-D promoter in lymphocytes natural killer, NK92 cells, in which it appears to be a critical checkpoint for the inhibition of the pyroptosis mechanism [110]. An indirect regulation occurs in colorectal cancer, where the rp1-85f18.6 knockout, a lncRNA highly expressed in CRC patients, leads to an increased pyroptosis through the cleavage of GSDM-D, suggesting a possible application of epigenetic modulators of inflammosomes for cancer therapy [111]. GSDM-E is found to be silenced in gastric, colorectal and breast cancer due to hypermethylation

of CpG islands within its promoter and appears to be related to an increased risk of metastasis [112]. Epigenetic regulation of GSDM-E may also depend on small noncoding RNA activity such as miR-155-5p, which can bind 3'-UTR reducing GSDM-E expression [113]. A further regulation takes place thanks to the presence of lncRNA which have been shown to be involved in pathological processes of various diseases including cancer by regulating directly or indirectly proteins involved in the main pyroptotic pathways [113]. Recent discoveries have identified new molecules, which in turn can activate or inhibit the expression of GSDMs, regulating pyroptosis at the epigenetic level [114]. One of the most important is Decitabine (DAC), a DNMT inhibitor used in hematological cancers therapy combined with chemotherapy, which can regulate the expression of several genes. In particular DAC treatment in several tumor cell lines induces DFN5 gene up-regulation leading to an increase of GSDM-E protein expression followed by pyroptosis activation. [115]. Moreover, treatment with methyltransferase inhibitors (e.g. 5-aza-C) increases the expression of GSDM-E in cancer cell lines and also improves the efficacy of chemotherapeutic agents (e.g. doxorubicin) to trigger pyroptosis [116]. Furthermore, anti-inflammatory drugs such as dimethyl fumarate (DMF) and monomethyl fumarate (MMF) have shown the ability to increase transcription levels of DNMT3a and DNMT3b, leading to GSDM-D silencing via its promoter hypermethylation (**Table 3**) [108].

### **2.4 Epigenetic regulation in immunogenic cell death**

Immunogenic Cell Death (ICD) is a process where dying cells activate an immunogenic response mediated by the release of DAMPs into the microenvironment, recognized by different immune cells and necessary for the immunological memory [117, 118].

DAMPs and nucleic acids released from dying cells, together with the release of chemo attractive agents in the microenvironment, contribute to increase the


### **Table 3.**

*Epigenetic regulation in pyroptosis.*

### *Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*

antigenicity of dying cells leading to the recruitment of innate immunity cells such as neutrophils and dendritic [119, 120].

Different molecular mechanisms are involved in this type of cell death, such as the UPR (Unfolded Protein Response) and autophagy as well as the release of many molecular players like Annexin 1, HMGB1, Interferons (IFNs) and different chemokines [121]. Under physiological stress, the endoplasmic reticulum (ER) activates the UPR, an evolutionarily conserved mechanism thanks to which ER chaperonins, Heat Shock Proteins (HSPs) such as HSP70 and Calreticulin (CALR) are translocated on the cell surface being an "eat me" signal for recognition by dendritic cells [122, 123].

Recently, it was demonstrated that epigenetic modifications can regulate several molecular players directly involved in ICD, supporting the idea for the development of new epigenetic drugs that can be used in cancer immunotherapy [121].

Histone and DNA methylation as well as ncRNAs are the main epigenetic modifications able to regulate targets that have a pivotal role in ICD such as HSPs, CALR, Annexin 1 and HMGB1 [121]. In lung cancer, inositol-requiring enzyme-1 (IRE1), an enzyme involved in UPR activation, is silenced by methylation. Indeed, treatment with Chaetocin, an Histone Lysine Methyltransferase (HKMT) inhibitor, determines an increment of the expression of this enzyme, suggesting that its regulation could be modulated via histone methylation (126,127). In colon and pancreatic cancer cell lines, the methylation at HSP90 promoter, related to an enhanced expression of DNA methyltransferase, inhibits its expression probably altering the immune response. The treatment with epigenetic modulators such as Zebularine, a DNMT inhibitor, can restore the immune response that leads to the induction of ICD [121, 124].

Different non-coding RNAs such as ncRNA-RB1, miR-27a and nc886, can modulate epigenetically CALR expression [121]. It has been shown that in A549 cell line (adenocarcinoma alveolar basal epithelial) the knockdown of ncRNA-RB1 reduces the expression of CALR, altering its translocation on the cell surface and probably influencing the fate of ICD [125]. Downregulation of Calreticulin was observed also in colorectal cancer by miR-27a action, resulting in a blocked Major Histocompatibility Complex (MHC) class I cell surface exposure [126]. In malignant gastric cancer cell lines, such as SNU-005, SNU-484 and MKN-01, the activity of the long non-coding RNA nc886, which has anti-proliferative and tumor suppressor roles [127, 128], has been found decreased compared to the non-malignant gastric cell line HFE-145, probably due to the CpG hypermethylation at the nc886 promoter region [128]. In nasopharyngeal carcinoma cell lines, both gene and protein expression of Annexin 1 are downregulated by methylation phenomena [129]. In head and neck squamous cell carcinoma, the presence of miRNA-196a/b epigenetically regulates Annexin 1, downregulating both mRNA and protein levels [130]. At the level of epigenetic regulation, it is thought that HMGB1 could act as an epigenetic modifier able to silence Tumor Necrosis Factor-alpha (TNF-α) and Il-1β [131]. miRNA-129-2, a tumor suppressor in glioma and hepatocellular carcinoma [132, 133], can inhibit the release of HMGB1. The regulatory region of this miRNA is strongly methylated in portions of its promoter region leading to its suppression and consequent expression of HMGB1 [134, 135]. Autophagy is essential for the ICD process as it promotes the synthesis and transport of ATP from the cell which is fundamental for an optimal immunogenic response [120, 136]. In submandibular carcinomas the expression of P2RX7 receptor is controlled by the methylation of its promoter and aberrant methylation phenomena may interfere with its expression and the related pathway [137]. Hypermethylation affects other autophagy players such as the Tensin Homolog (PTEN), as demonstrated in melanoma and in breast and stomach cancer [138, 139] and the Autophagy-Related

Protein 5 (ATG5), studied in melanoma and colorectal cancer. This epi-modification leads to a downregulation of PTEN and ATG5 protein expression during cancer progression [138–141]. The expression of CXCL10 in ovarian cancer cells, may depend on the methylation of its promoter, indeed the use of demethylating agents is able to increase its expression [142]. Acetylation can also modulate ICD [143], indeed Histone Deacetylase 3 (HDAC3)-deficient macrophages, stimulated with LPS, are unable to activate several genes involved in inflammation including IFNβ, demonstrating a main role for HDAC3 in controlling IFNβ expression. [144]. Furthermore, it has been shown that the use of caloric restriction mimetics (CRMs) may have a pivotal role in anticancer immunosurveillance [145]. CRMs stimulate ATP release by influencing acetylation of histone proteins showing a potential epigenetic mechanism able to induce or not autophagy during cancer (**Table 4**) [145, 146].

### **2.5 Epigenetic regulation in ferroptosis**

Ferroptosis is a newly discovered form of RCD reliant on iron-dependent lipid peroxidation [149]. The increase in free iron and the accumulation of lipid peroxides occurs through the action of a small molecule called erastin which can induce nonapoptotic cell death in an ST (Small T oncoproteins) and RASV12 (oncogenic allele of HRAS)-dependent way [150].

By the interaction with voltage-gated anion channels (VDAC), erastin can inhibit the cysteine/glutamate transport system Xc − (SLC7A11) leading to cysteine depletion, glutathione deficiency with excessive lipid peroxidation and consequently induction of ferroptotic cell death [151].

Some evidences highlight an epigenetic regulation of ferroptosis. For instance, ncRNAs regulate the progression of NSCLC mediating ferroptosis [152]. P53RRA, a cytosolic lncRNA, by interacting with G3BP1, promotes ferroptosis trough the


### **Table 4.**

*Epigenetic regulation in immunogenic cell death.*

*Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*

activation of p53 pathway and the transcription of some metabolic genes responsible of the increased intracellular concentration of iron and ROS lipids and of the inhibition of growth induced by erastine [153].

In lung cancer, the nuclear lncRNA LINC00336 is upregulated and, through the interaction with ELAV-like-RNA-binding protein 1 (ELAVL1), acts as an inhibitor of ferroptosis by decreasing the intracellular levels of iron and ROS lipids. Moreover, LINC00336 also acts as an endogenous sponge for another microRNA (miR-6852) which is a negative regulator of cystathionine-β-synthase (CBS) that has a pivotal role in ferroptosis [154].

Treatment of NSCLC cell line NCI-H1299 with XAV939, a Wnt/−catenin pathway inhibitor, resulted in a downregulated SLC7A11 expression that controls iron concentration and the activation of ferroptosis-mediated pathways responsible of the suppression of NSCLC progression [152]. Furthermore, the deubiquitinase DUB, a tumor suppressor inactivated in many types of tumors [155], after the assembly of the polycomb repressive deubiquitanase complex (PR-DUB) is able to inhibit the ubiquitinated histone H2A (H2Aub) placement on the SLCA711 promoter whose down-regulation blocks ferroptosis through the cysteine starvation and GSH depletion [156]. The monoubiquitination of H2B on lysine 120 (H2Bub1), a marker of transcriptional activation involved in the regulation of the Warburg effect and tumorigenesis [157], regulates both the expression of SLC7A11 and of a group of ion-binding genes linked to metabolism classifying this modification as a new epigenetic regulator of ferroptosis [158]. The activity of Lysine Demethylase 3B (KDM3B) inhibits erastin-induced ferroptosis through the activation of SLC7A11, cooperating with the transcription factor ATF4 [159]. In addition, BRD family proteins, including BRD4, can also participate in the epigenetic regulation of ferroptosis. The use of BRD4 inhibitor JQ1 has been shown to induce ferroptosis through the downregulation of GPX4, SLC7A11 and SLC3A2 expression in breast and lung cancer cells classifying it as a potential therapeutic agent in cancer treatment (**Table 5**) [160, 161].

### **2.6 Epigenetic regulation in NETosis**

NETosis is a form of cell death exclusive for neutrophils, caused by the uncontrolled production of netotic bodies, useful in physiological conditions for the neutralization of pathogens. The mechanism originates with the activation of ion channels associated with receptors able to modify the intracellular levels of calcium. Subsequent phosphorylation pathways lead to the production of mitochondrial ROS and the calcium-dependent activation of PAD4, responsible for the chromatin decondensation and the end of the NETotic process [162]. In breast cancer, the release of cancer extracellular chromatin networks (CECNs) into the microenvironment


**Table 5.** *Epigenetic regulation in ferroptosis.* appears to be related to the onset of lung metastases [163]. Among the key molecular processes of NETosis, the role played by the PAD4 enzyme is well known, which increases the levels of citrullination of histones in a calcium-dependent manner leading to chromatin decondensation and netotic nuclear collapse [164]. Several studies on patients with different tumors, such as breast, colorectal and lung cancer, have found an important increase in plasma levels of hypercitrullinated histone H3, suggesting it as a potential prognostic marker [165–167]. The hyper-citrullination of H3 is a widespread phenomenon during the formation of NETotic bodies as well as reduced levels of methylation of arginine 3 on histone H4 and high levels of acetylated lysine 16 on histone H4 as reported in breast cancer [163]. The increase in the enzymatic activity of PAD4 and in the netotic process is closely related to its epigenetic regulation. In MCF7 cancer cells, citrullination of the OKL38 promoter by PAD4 was described, suggesting a correlation between NETs formation and breast cancer [168]. Increased angiogenesis and deposition of fibrous material in malignant tumors also appears to be related to PAD4-mediated citrullination of antithrombin (cAT) [169]. In hepatocellular carcinomas, the global hypomethylated state of DNA and the hypermethylation of promoters of genes involved in tumorigenesis, such as p53 and p21, may partially depend on the reduced action of PAD4, on the expression and the enzymatic activity of DNMT3a [170].

In colon cancer, miR-155 can ensure the translation of PAD4 mRNA, inducing the netotic process and the tumor progression [171]. New evidence has proved the role played by miR-505 in breast and pancreatic cancer. It negatively regulates SIRT3 by altering mitochondrial metabolism and ROS production, triggering the production of NETs (**Table 6**) [172].

### **2.7 Epigenetic regulation in parthanatos**

Parthanatos is a type of PCD characterized by hyperactivation of poly (ADP-ribose) polymerase 1 (PARP-1) followed by PAR accumulation and mitochondrial release of apoptosis inducing factor (AIF) [173]. The molecular interaction between AIF and the macrophage migration inhibitory factor (MIF) leads to massive DNA fragmentation and cell collapse [174]. The knowledge related to the epigenetic modification involved in parthanatic process and their role in tumors is currently poorly known. In liver cancer, the damage induced by


**Table 6.** *Epigenetic regulation in NETosis.* *Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*

UV rays causes the activation of PARP-1 and the PARylation of histones, with the consequent recall of ALC1 on chromatin and activation of DNA repair [175]. In fact, PARP-1 can facilitate the recruitment of repair systems through the decondensation of chromatin independently by ubiquitylation [176]. In breast cancer, the behavior and function of insulators is controlled by PARP-1, through conformational changes of chromatin. The increase in PARylation of CCCTCbinding factor (CTCF), triggers its functions as an insulator, activating mechanisms able to induce DNA hypomethylation, central feature of many forms of cancer [177, 178]. The correlation between PARP-1, chromatin opening and gene transcription activation is poorly explained in the literature. In breast cancer, PARP-1 allows chromatin access to RNA-pol II with the inhibition of demethylase activity of KDM5B by PARylation, leading to the global hypomethylation of H3K4 [179]. A very recent study identifies the lysine demethylase KDM6B as a key factor in the epigenetic control of parthanatos and in the response to antitumor therapy with alkylating agents. The reduction of KDM6B levels leads to the activation of DNA repair checkpoints mediated by MGMT, causing alkylating agents resistance. Conversely, the increase in KDM6B levels favors parthanatic cell death induced by alkylating agents [180]. These new insights open the window to understanding the epigenetic mechanisms underlying parthanatos and the epigenetic function of PARP-1 (**Table 7**).

### **3. Conclusions**

Epigenetics regulates several processes including differentiation, development, growth and cell death. Specifically, cell death controls various physiological and pathological phenomena that are crucial for life development. A deeper knowledge of both cell death and epigenetics, and their interconnections, might be the key to better understand how different processes in life are modulated and how to exploit them therapeutically.

The fact that epi-deregulation in cancer clearly also alters the main players of the different cell death pathways has important consequences. For examples, some epi compounds (i.e. HKMT, HDAC, HMT inhibitors) might be used also for regulation of the expression of the main players involved in cell death and might, in turn, help for cell death pathways reactivation in cancer or, also into the recognition of cancer cells by the immune system. In addition, the identification of possible epigenetic biomarkers linked to cell death players deregulation could be beneficial to contrast several cancers strengthening the well-known concept of "personalized therapy".


### **Table 7.** *Epigenetic regulation in parthanatos.*

### **Acknowledgements**

We thank D. Mancinelli for English language editing. We thank: MUR, Proof of Concept POC01\_00043; POR Campania FSE 2014e2020 ASSE III. Nuovi Farmaci e Biomarkers di risposta e Resistenza Farmacologica nel cancro del ColonRetto-Nabucco no.1682, MISE; Valere 2019:CAMPANIA. Ricerca Finalizzata of Italian Ministry of Health (GR-2018-12366268).

### **Conflict of interest**

The authors declare no conflict of interest.

### **Declaration**

Vincenza Capone and Giulia Verrilli hold a PhD fellowship "Dottorati Innovativi con Caratterizzazione Industriale" coded: B97J20000140006 (VC); B63D21008370006 (GV).

### **Abbreviations**


*Epi-Regulation of Cell Death in Cancer DOI: http://dx.doi.org/10.5772/intechopen.108919*


### **Author details**

Antonio Beato1 , Laura Della Torre1 , Vincenza Capone1 , Daniela Carannante1 , Gregorio Favale1 , Giulia Verrilli1 , Lucia Altucci1,2,3\* † and Vincenzo Carafa1 \* †

1 Università degli Studi della Campania "L. Vanvitelli", Naples, Italy

2 Biogem, Molecular Biology and Genetics Research Institute, Ariano Irpino, Italy

3 IEOS CNR, Napoli, Italy

\*Address all correspondence to: lucia.altucci@unicampania.it and vincenzo.carafa@unicampania.it

† Co-last authors.

© 2022 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.

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### **Chapter 2**

## Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death in Neurodegenerative and Cancer Diseases

*Silva Abrahamyan and Karina Galoian*

### **Abstract**

The proline-rich peptide (PRP-1) isolated from neurosecretory granules of the bovine neurohypophysis, produced by N.supraopticus and N.paraventricularis, has many potentially beneficial biological effects. PRP-1 has been shown to have the opposite effects on cell death in neurodegenerative and cancer diseases. It significantly reduces staurosporine-induced apoptosis of postnatal hippocampal cells, as well as doxorubicin-induced apoptosis of bone marrow monocytes and granulocytes, in both time- and dose-dependent manner. PRP-1 also exerts the opposite effect on the proliferation of bone marrow stromal cells obtained from normal humans and on the stromal cells isolated from human giant-cell tumor. PRP-1 cytostatically inhibits chondrosarcoma bulk tumor but exerts drastic cytotoxic effect on sarcomas cancer stem cells. The same peptide caused cell death through apoptosis in rats with Ehrlich Ascites Carcinoma model.

**Keywords:** proline-rich peptide (PRP-1), neurodegeneration, cancer diseases, ehrlich ascites carcinoma (EAC), chondrosarcoma

### **1. Introduction**

Our long-term scientific work has been aimed at studying the protective effects of certain physiologically active compounds, including proline-rich peptide (PRP-1, comprised of 15 amino acids residues, AGAPEPAEPAQPGVY, with an apparent molecular mass of 1475.25 Da) on brain plasticity in rats with different neurodegenerative models. While displaying neuroprotective role in those models, in cancer related studies, on the other hand, PRP-1 displayed its beneficial antiproliferative effect in cellular context dependent manner by triggering cell death leading to drastic decrease of cancer stem cell population responsible for disease relapse and drug resistance.

### **2. Effect of PRP-1 on cell death in the neurodegenerative diseases**

The protection of neurons from damage and death in neurodegenerative disorders, such as Alzheimer disease (AD), ischemic insults, Parkinson disease (PD), is a major challenge for neuroscientists in the twenty first century. Much attention is focused on the discovery of novel biomarkers for diagnosis and therapy of neurodegenerative diseases.

The new family of peptide neurohormones consisting of 10–15 amino acid residues isolated by prof. Galoyan and coworkers from bovine and human neurohypophysis neurosecretory granules are synthesized in the form of a common precursor protein (neurophysin-vasopressin associated glycoprotein) [1, 2]. These five peptides contain a high proportion of proline residues and, therefore, were designated as proline rich peptides (PRPs). The most studied is the bovine PRP-1 (also known as galarmin). The polypeptide is not species-specific; hence it is active in mice, humans, and rats. It has been shown that PRP-1, as well as its synthetic analogue, has many beneficial biological effects.

Neuronal injuries have been suggested to promote PRP-1 synthesis and its release as a messenger, modulating the signaling cascades and, therefore, contributing to protection, regeneration, and repair of the neurons.

The neuroprotective [3–7] and immunoregulatory [8–10] effects of PRP-1 through its involvement in the neuro-immuno-hematopoietic interaction have been demonstrated

### **Figure 1.**

*PRP-1-Ir Spinal Cord (SC) nerve structures in SC-hemisectioned rats with and without PRP-1 injection. Degenerative and strongly immunoreactive for PRP-1 motoneurons (MNs) with no processes are demonstrated in the spinal cord anterior horn, situated in the lightened pericellular area, perhaps, brain edema. After daily administration of PRP-1 to trauma-injured animals for 3 weeks, regeneration of strongly immunostained MNs and their processes was observed suggested the possibility of PRP-1 involvement in the mechanisms of neuronal repair: growth of nerve fibers and motoneurons survival. ABC immunohistochemical method.*

### *Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

in the following models of central nervous system damage: spinal cord hemisection; βA-peptide injection (model of Alzheimer disease); vestibular nuclei damage through vibration and unilateral labyrinthectomy; immobilization stress (IMO stress).

Our previous report on the effects of PRP-1 on SC-injured rats indicated the possibility of PRP-1 involvement in the mechanisms of neuronal repair [3]. Immunohistochemical study demonstrated that treatment with PRP-1 resulted in the recovery and growth of nerve fibers, glia proliferation, and motoneuron survival. Therefore, PRP-1 has been found to be a highly active neurotrophic-like substance (**Figure 1**).

PRP-1 participation in the regeneration of the nerve structures was also immunohistochemically demonstrated in the trauma-injured rats with SC hemisection treated

### **Figure 2.**

*SC nerve fibers in the SC hemisectioned rats regularly treated with NOX venom. Naja Naja Oxiana (NOX) snake venom prevented the scar formation, well observed two months after SC injury in the control rats (A) and resulted in the regeneration of nerve fibers growing through the trauma region (B, C). Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.*

### **Figure 3.**

*PRP-1-Ir structures in SC of injured rats treated with NOX venom. NOX increased the number of PRP-1-Ir nerve fibers (A) and astrocytes (B) in the SC lesion region and promoted the survival of the PRP-1-Ir motoneurons (C). In the boxed area the PRP-1-Ir astrocytes are seen migrating toward the injured side. ABC immunohistochemical method.*

by the administration of Naja Naja Oxiana (NOX) snake venom. NOX venom prevented the scar formation, well observed 2 months after the SC injury in the control rats, and resulted in the regeneration of the nerve fibers growing through the trauma region. It was suggested to exert the neuroprotective effect by involving the endogenous PRP-1 in the underlying mechanism of the neuronal recovery – based on the data regarding the survival of the immunoreactive to PRP-1 (PRP-1-Ir) motoneurons, the increased number of PRP-1-Ir nerve fibers in the SC lesion region, and appearance in the white matter of the PRP-1-Ir astrocytes migrating towards the injured side [7] (**Figures 2** and **3**).

Different forms of injury and types of stress induce different morphological responses. For example, 5-hour IMO stress induces deeper neurodegenerative changes, which is manifested by the histochemical method on detection of Ca2+-dependent acid phosphatase activity. The final product – phosphate precipitate of various sizes and shapes was localized in both the neurons and the extracellular region (**Figure 4**).

In the next neurodegenerative model, the vestibular nuclei injury caused by vibration and unilateral labyrinthectomy, in various brain regions of rats, including hypothalamus, hippocampus, brain stem (locus coureleus, nucleus hyppoglosus),

### **Figure 4.**

*Degenerative nerve structures in the hypothalamic SON and PVN of rats exposed to 5 h IMO stress. In the hypothalamic SON and PVN, the central chromatolysis and ectopied negative nuclei are revealed in the hypertrophied cells with no processes. High phosphatase activity is seen like a thich ring under the cellular membrane and extracellular area. Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.*

### *Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

cerebellum, the neurodegeneration was revealed by the presence of the hypertrophied cells situated in the tissue edema [11]. Single PRP-1 administration triggered regeneration and survival of neurons in the same brain regions. Interestingly, obvious regeneration of the structures is revealed in labyrinthectomized rats exposed to the 2 h daily vibration for 2 weeks.

Apart from this, in the distinct stress-related brain regions of labyrinthectomized rats, neurons, demonstrating PRP-1-immunoreactivity, (PRP-1-IR) were revealed in the cell nuclei, as opposed to the PRP-1-IR in the cytoplasm of the intact cells (**Figure 5**).

According to the literature, the nuclear localization of the proteins such as c-Fos, c-Jun, as well as the so-called Heat Shock factors with a molecular weight of 1–70 kD was detected in rats under stressful conditions (exposure to radiation, temperature, chemicals, etc.) [12, 13].

Using the immediate early gene c-fos-antibody, the stress-induced activation of neurons was immunohistochemically demonstrated by detection of the c-fos-immunoreactive (c-fos-Ir) nuclei in the distinct stress-related brain regions of labyrinthectomized rats. We assume that PRP-1 can control the DNA transcription and can function as a transcription factor similar to c-fos [14].

In the same stress-related brain regions of rats, high phosphatase (APh) activity was also detected in cellular nuclei in 15 minutes after stress, earlier than gene c-fos, which can be explained by the activation of cellular activators like c-fos through their phosphorylation (**Figure 6**).

#### **Figure 5.**

*PRP-1-Ir neural structures in the (A, B) supraoptic nucleus (SON), (C, D) locus coureleus (LC) and (E, F) nucleus Hyppoglosus (n.Hyp.) of intact and labyrintectomized rats brain. In the distinct stress-related brain regions of labyrintectomized rats, the stress-induced activation of neurons was found out by detection of the increased number of cell nuclei demonstrating PRP-1-IR and immediated early gene c-fos-IR. ABC immunohistochemical method.*

**Figure 6.**

*PRP-1-IR, transcription factor early gene c-fos-IR (c-fos-IR) and Acid Phospatase (APh) activity in the cortex of labyrintectomized rats. Nuclei of pyramidal cells in the brain cortex of labyrintectomized rats, demonstrated (A) PRP-1- and (B) c-fos-IR, as well as (C) high APh activity. Detection of APh activity in the injured cells nuclei can be explained by activating of cellular activators like c-fos through their phosphorylation. ABC immunohistochemical method and histochemical method on detection of Ca2+-dependent acidic phosphatase activity.*

Our results obtained in the mentioned models of the central nervous system injury indicate that PRP-1 therapy may protect against the neurodegeneration by enhancing the survival of the damaged neurons.

### **3. PRP-1 participation in the generating of new neurons**

Adult brain cell regeneration, also known as neurogenesis, demonstrated in many species, including rodents, is the process of generating new neurons, [15].

### **Figure 7.**

*GFAP-, Nestin- and PRP-1-Immunoreactive structures in the brain of 45-days aged rats exposed to prenatal IMO stress. Using markers against the neural progenitor cells, GFAP- and nestin-Ir radial astrocytes (A, B, D), nestinand PRP-1-Ir cell structures of different sizes and forms (C, F), as well as PRP-1-Ir varicose nerve fibers and varicosities (E) were detected in the distinct stress-related brain regions. Taking into account the results obtained, we suggest that they could be the intermediate neural progenitor cells and that PRP-1 could participate in the generation of new functional neurons following the injury. ABC immunohistochemical method.*

### *Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

Today, there is scientific evidence of the stem cells presence in many more tissues and organs. One of their characteristics is ability to self-renew and to differentiate, to secure primary steady state functioning of a cell, called homeostasis, and, with limitations, to replace cells that die because of injury or disease [16, 17]. The list of adult tissues reported to contain stem cells is growing and includes bone marrow, peripheral blood, brain, spinal cord, muscle, and other tissues. Multipotent adult progenitor cells (MAPCs), derived from pluripotent mesenchymal stem cells, purified and isolated by Jiang et al. [18], could differentiate both into mesenchymal and neural cells.

Our recent histochemical and immunohistochemical studies in newborn rats exposed to the acute prenatal immobilization (psychogenic) stress brought new information about PRP-1 participation in the brain recovery process through generating new neurons [19].

To differentiate PRP-1-immunoreactive (PRP-1-Ir) glial cells and small undifferentiated cells found in the injured brain, we used antibodies against the astrocyte marker GFAP, neuroepithelial stem cells marker gene nestin [20, 21], and mouse stem cells. Considering the obtained results regarding the detection of GFAP-, nestin-, and

### **Figure 8.**

*Cellular structures resembling mesenchymal cells in the bone marrow (BM) and brain of the immobilized rats. (A-C) numerous round in shape and fusiform small cells with short axon-like extensions, being in the various proliferative stages (arrows), are visible in the BM stroma (B). (D) small cells (arrow heads) and (E, F) dark-colored fusiform cells with processes are detected in the cerebellum (G-I). Using antibodies against the mouse stem cells (MSCs), nestin and PRP-1, in the spinal cord of the injured rats, (G) MSCs-Ir, (H) PRP-1-Ir and (I) nestin-Ir cellular structures are revealed. Histochemical method on detection of Ca+2-dependant acid phosphatase activity (A-E) and ABC Immunohistochemical method (G-I).*

PRP-1-immunoreactive radial astrocytes and cell structures of different sizes and forms, we suggest that they could be the intermediate neural progenitor cells, and that PRP-1 could participate in the generation of new functional neurons following the injury (**Figure 7**).

We succeeded also in detecting the cellular structures resembling the mesenchymal cells in both bone marrow and different stress-related brain regions of the immobilized rats some of which expressed immunoreactivity against MSCs, nestin and PRP-1 (**Figure 8**).

In addition to the established functions of PRP-1 for cell survival and neurogenesis, using the PRP-1-antiserum, as well as antiserum against the synaptophysin (presynaptic vesicle protein, marker for the functional synapsis), we also suggested that PRP-1 could mediate higher brain activity through the formation of new synapses, thus, increasing the number of connections between the neurons (**Figure 9**).

We suggest that the process of generating new neurons occurs in injured brain, most probably proceeding from BM-derived cells that migrate into the brain and

### **Figure 9.**

*Synaptophysine-Ir (Syn-Ir) and PRP-1-Ir varicose nerve fibers and varicosities in the brain of 75-days aged rats exposed to prenatal IMO stress and injected with PRP-1. Syn-Ir (A-C) and PRP-1-Ir (D-F) nerve fibers and varicosities, possibly synapses, scattered in the brain, are well demonstrated. ABC immunohistochemical method.*

*Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

express neuronal marker genes, or from the neural stem/progenitor cells (NPCs) in non-neurogenic regions giving rise to the neurons mediated by the local astrocyte populations.

### **4. Protective effect of PRP-1 on the immune system cells death**

Histochemical and immunohistochemical studies were also carried out to investigate the morpho-functional states of bone marrow (BM) structures of intact rats, rats injected with PRP-1, and rats exposed to immobilization stress and to bilateral electro-stimulation of the hypothalamic paraventricular nucleus [11].

The increased number of PRP-1-Ir blood-forming cells were observed in BM stroma and sinusoidal capillaries after PRP-1-antiserum was applied (**Figure 10**).

In addition, PRP-1-Ir varicose nerve fibers and islands of PRP-1-Ir immune system cells were detected in the surrounding areas of sinusoids (**Figure 11**).

The possibility that the PRP-1 synthesis takes place in the immune cells exists due to the evidence of distinct neuropeptides biosynthesis in the lymphocytes. Based on this, we conducted in vitro experiments in intact lymphocytes isolated from the rat bone marrow. By using PRP-1-antiserum and the flow cytofluorimetric analysis, about 4–5% PRP-1 was detected in the lymphocytes in the presence of some

#### **Figure 10.**

*Hematopoiesis in the bone marrow of rats injected with PRP-1. (A) sinusoidal capillary in the intact rat BM appeared to be empty, in general (black asterisk). Increased number of immune system cells both in sinusoid (asterisk) (B) and stroma (B-D) are well seen in the BM of injected with PRP-1 rats. (B) in the sinusoid, a megakaryocyte (arrow) with the homogenously and densely stained nuclei is demonstrated in the stage of platelets release. Histochemical method on detection of Ca2+-dependent acidic phosphatase activity.*

### **Figure 11.**

*PRP-1-Ir structures in bone marrow of the immobilized rats. The increased number of PRP-1-Ir blood-formed cells is detected in BM sinusoidal capillaries and stroma. Among these cells, islands of PRP-1-Ir immune system cells (white asterisks) were found inside and around the sinusoids (black asterisks). A PRP-1-Ir capillary (arrow) (A) and a few single PRP-1-Ir varicose nerve fibers (arrows) (C) are seen. (D): fragment of 10C. ABC immunohistochemical method.*

activators, such as phytohemagglutinin (FGA), phorbol miristil acetate (FMA), and concanavaline A (ConA), compared to near 0% in the intact immune cells. Data obtained indicate the possible synthesis of PRP-1 in the immune system cells in vivo.

Thus, adult stem cells plasticity in response to the immobilization stress is assumed in some of the studied brain regions. However, whether these cells are indeed bone marrow-derived stem cells circulating in the blood is a question to be answered. In regards to the PRP-1-Ir migrating cells from the SC central canal, we assumed that they could be the SC stem cell-derived structures.

### **5. Effect of PRP-1 on inflammation**

The inflammation, a physiological response to a variety of tissue damages, is made up of multiple related chains of cellular and chemical reactions. This cascade of responses activates the localized production of cytokines implicated in the cell recruitment and differentiation through specific gene expression. Though the processes behind the acute neuroinflammation following trauma or stroke may worsen the initial lesion through the increased neuronal loss, they stimulate the subsequent functional recovery through promoting neuronal plasticity. However, the consequences of chronic neuroinflammation are suspected to include neuronal loss in pathological conditions including neurodegenerative and autoimmune disorders and diseases [22].

Inflammation of the brain is linked to the biosynthesis and secretion of several neuroactive molecules, such as oxygen and nitrogen free radicals, cytokines, excitatory amino acids, proteases, complement proteins, and others, by the activated glial cells [23]. Though chronic, unregulated, and ongoing inflammation is highly prejudicial, inflammation is generally a beneficial process for the organisms due to its role in containing and curbing the survival and the spread of pathogens, as well as energy conservation and tissue recovery [24].

Beta Amyloid peptide (Aβ) (1–42) by its nature is neurotoxic and is linked to dysregulation of the brain function during Alzheimer's disease (AD) and, therefore, is strongly associated with the brain function loss through the course of AD. The accumulation of Beta Amyloid triggers the induction of neuronal cytotoxic pathways, involving microglia induced activation of pro-inflammatory cytokines IL-β and TNF-α and formation of free radicals [25, 26].

TNF-α can promote tumorigenesis leading to prostate and other cancers, and initiate apoptotic cell death [27]. Inflammatory processes are correlated with the neuronal apoptosis found in neurodegenerative diseases. Whether apoptosis plays an overall beneficial or detrimental role in neuroinflammation is unclear and topic remains controversial.

PRP-1 has roles as a caspases-2 and -6 activator [28], immunocompetent cells (Т and В lymphocytes and macrophages) stimulator [8, 9], and pro-apoptotic caspases-3 and -9 inhibitor. Furthermore, it is a tumor necrosis factor alfa (TNFα) and interleukins (IL-1, IL-6) inductor in lymphocytes, astrocytes, macrophages, and fibroblasts.

PRP-1 exhibits regulatory effect on myelo- and lymphopoiesis [29–31] and neuroprotection, countering multiple toxic endogen agents [32, 33], as well as demonstrates strong neurotrophic effect on glial fibrillary acidic protein (GFAP) biosynthesis in astrocytes [34].

PRP-1's influence on staurosporine-promoted apoptosis of postnatal hippocampal cells as well as on doxorubicin-induced bone marrow mono/granulocyte apoptosis was investigated [35]. We characterized PRP-1's activity on the neuron survival rate (in a myelopoiesis context) by demonstrating the significant reduction of staurosporine-induced apoptosis of postnatal hippocampal cells from PRP-1 treatment. PRP-1's protective function against apoptosis was shown to be both dose- and time- dependent. Prolonged PRP-1 treatments showed more pronounced neuroprotection against staurosporine-induced apoptosis. A similar significant reduction was seen in bone marrow monocyte and granulocyte apoptosis by doxorubicin. The neuroprotective effect lasted for 2–4 hours and was no longer effective at 24 h when doxorubicin and PRP-1 were simultaneously added. In conclusion, the endogenous peptide PRP-1 has the primary functions of regulating myelopoiesis and neuroprotection.

Multiple experiments were carried out to understand the effect of PRP-1 on the proliferation and the colony formation of multipotent mesenchymal stromal cells (MMSCs). The dose response effect of PRP administration to rats was observed with the increased number of MMSCs in bone marrow and spleen. In ex vivo condition, the addition of PRP into the culture medium led to up to 2.5-fold increase by stimulation.

On the contrary, the proliferation was inhibited 1.5 to 2-fold in the cultures of giant-cell tumor (GCT) stromal cells, when the same PRP concentrations and cultivation periods were used. PRP-1 demonstrated also the opposite effects on the proliferation of the human bone marrow stromal cells obtained from normal humans, and the stromal cells isolated from human GCT [36].

### **6. Effect of PRP-1 on cell death in the cancer diseases**

Other results demonstrating antitumor activity of PRP1 followed, opening up new perspective of PRP-1 antitumorigenic activity. PRP-1 induced the decay of tumor cells L929 and decreased the mitotic activity of transformed mouse fibroblast cells [37–40]. The peptide caused shrinkage of tumor in sarcoma C45 after subcutaneous injection [39].

These experimental results served as predecessors of intensive studies on other musculoskeletal malignancies, particularly chondrosarcoma. Chondrosarcoma is the second most common bone malignancy, which primarily affects the cartilage cells of the femur (thighbone), arm, pelvis, knee, and spine and even larynx, head, and neck. Chondrosarcoma is a rare disease and it does not appear to respond to either chemotherapy or radiation.

Surgical resection is the only option for the treatment, although metastatic spread to lungs occurs, eventually leading to dismal prognosis as these tumors are highly aggressive. Therefore, the search for new therapies is extremely important and urgent.

To date, only a few drugs have been identified that have been successfully shown to have clinical efficacy through the inactivation of a specific oncogene phenotype [41]. Inactivation of a single oncogene can induce cancer cells to differentiate into cells with a normal phenotype, or to undergo cellular senescence and/or apoptosis [42]. Upon MYC inactivation [43], tumors variously undergo proliferative arrest, cellular differentiation, and apoptosis.

In our most early publications, we provided experimental data indicating that PRP-1 caused inactivation of cMyc oncogene in human chondrosarcoma cells, prompting us to further investigate this peptide antitumorigenic potential [44].

The cytostatic effect of PRP-1 in human chondrosarcoma JJ012 cell line was demonstrated as 80% inhibition of cell proliferation on PRP-1 treatment in comparison with the nontreated cells. Interestingly, PRP-1 did not have any effect on immortalized chondrocytes culture, which spoke to the fact that PRP-1 selectively targeted malignant sarcoma cells and not the benign cells. Caspase-3 activity was not affected and no apoptosis was detected, thus the inhibition was due to cytostatic and not cytotoxic effect [45].

The mammalian target of rapamycin (mTORC) is an intracellular serine/threonine protein kinase, which is linked to cell growth because of its important role in nutrient signaling processes. PRP-1 was revealed as mTORC1 inhibitor, as it was able to inhibit this kinase activity in statistically significant manner [46].

The fact that the concentration lower than 10 μg/ml peptide with cytostatic effect did not inhibit mTORC1 but inhibited its target cMyc prompted us to assume the possibility of PRP-1 binding to two different receptors, facilitating the antiproliferative effect.

The cytostatic, antiproliferative action of PRP-1 was also demonstrated in the triple negative breast carcinoma MDA MB 231 cell lines [47] and the cell cycle experiments pointed on obvious stall in S phase, delaying the progression to the next stage of cell cycle upon PRP-1 treatment.

### *Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

Through our study, we sought to ascertain the condition of JJ012 human malignant chondrosarcoma cells' expression of intercellular junction proteins, as well as determine the effect of the antitumorigenic cytokine PRP-1 on their expression. The experimental data suggested that tumor suppressor desmosomal proteins expression in JJ012 chondrosarcoma cells is restored and H3K9 demethylase activity comprised of a pool of JMJD1 and JMJD2 is inhibited by PRP-1, suppressing the tumorigenic potential of chondrosarcoma cells [48].

Identifying the PRP-1's receptor was very important to discern the mechanism behind its action. G protein coupled receptors (GPCR) and nuclear pathway receptor assays determined that PRP-1 receptors do not belong to nuclear or orphan receptor families, and neither were they G protein coupled. We have demonstrated in our study that the interacting partners of PRP-1 binding belong to the gel forming secreted mucin MUC5B, as well as to the innate immunity pattern recognition toll-like receptors TLR1/2 and TLR6. The experimental data indicated that the aforementioned receptors had tumor suppressive function in this cellular context [49].

When examined, the microRNA expression profiles specific to tumors showed that, throughout diverse cancers, there was widespread deregulation of these molecules. MicroRNAs have been reported to have the potential to function in disease diagnostics and therapy as novel biomarkers, as well as a novel class of tumor suppressor and oncogenes. Tumor suppressors, such as miR20a, miR125b, and miR192, were significantly upregulated by mTORC-1 inhibitor PRP-1 while onco-miRNAs, miR509-3p, miR589, miR490-3p, and miR550 were downregulated in the human chondrosarcoma JJ012 cell line [50]. The fact that PRP-1 manifests itself as a powerful epigenetic regulator was confirmed with the experiments demonstrating inhibition of BAFF Chromatin remodeling complexes [51].

Experimental results indicated that among the miRNA significantly downregulated by PRP-1 treatment was miRNA 302c. miRNA 302c is a part of the embryonic human stem cell stemness regulator cluster miR302367. miR302367 is expressed in embryonic stem cells, as well as in certain tumors, but its expression is not found in normal tissue or in adult HMSCs [52]. PRP-1 had a strong effect on chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type) viability by inhibiting their proliferation. However, PRP-1 did not have any cell proliferation inhibitory action on glioblastoma, because the miR-302-367 cluster in glioblastoma exhibits an opposite effect and its expression is enough to inhibit the stemness inducing properties. The antiproliferative activity of PRP-1 and its action on downregulation of miR302c has an observed correlation that explains the peptide's opposite effects on the downregulation of miR302c targets, the stemness markers Nanog, c-Myc, and polycomb protein Bmi-1 [52].

We concluded that the inhibition of H3K9 demethylase activity by PRP-1 leads to downregulation of miR302c and its targets, defining the antiproliferative role of PRP-1.

Effects of PRP-1 on a 3D chondrosarcoma tumor model in vitro (known as spheroids) and on the cancer stem cells (CSCs) that form the spheroids, was evaluated in another study [53]. Spheroid formation and colony formation assays of cell fractions (including CSCs) were used in comparing PRP-1 treated groups with the controls. The CSCs were assessed with a modified Annexin V/propidium iodide assay for early apoptosis and cell death. Western blotting confirmed mesenchymal marker expression, and the spheroid self-renewal assay demonstrated the presence of the self-renewing CSCs. The study's results determined that PRP-1 eliminates spheroid formation and independent CSC growth, indicating the PRP-1 potential to inhibit tumor formation in a murine model. Another indication of an advantageous decline in tumor stromal cells is the decrease in non-CSC bulk tumor cells. These findings lead us to conclude that PRP-1 inhibits CSC proliferation in 3D tumor models that mimic the behavior of in vivo chondrosarcoma.

Although the cytostatic effect of PRP-1 has been demonstrated in various tumors we studied, the potential of PRP-1-related apoptosis in other types of cancer has not been ruled out.

PRP-1 action is cellular and disease context dependent. Morpho-functional study on the effect of PRP-1 on a mouse Ehrlich ascites carcinoma (EAC) model was conducted [54]. The number of viable cells in the suspension was determined by the histological method of exclusion with trypan blue (diazo live dye). The percentage of dead and alive cells was calculated after 24 h of incubation in the control samples and in those treated with PRP-1 at 0.1 and 1 μg/ml concentrations. The effect of PRP-1 on the number of tumor cells incubated for 24 h and their viability led to a 44% reduction in the number of viable cells on day 11 post-inoculation, vs. the 22% inhibition of viable cells after PRP-1 treatment (0.1 μg/ml) on day 7 postinoculation (**Figures 12** and **13**).

### **Figure 12.**

*Effect of the hypothalamic PRP-1 on the growth and viability of mouse isolated EAC cells on the 7th day of tumor growth. By the histological method with Tr-Bl staining, viable EAC cells were revealed in the control samples before their culture (control). Few number of dead Tr-Bl-positive tumor cells were detected among the viable cells in the non-treated control samples 24 h after culture (control 24 h). An increased number of Tr-Bl-positive dead cells was evident 24 h after 0.1 and 1 μg/ml PRP-1 administration. The PRP-1 (0.1 and 1 μg/ml) inhibitory effect on the number of (B) total and (C) viable tumor cells treated for 24 h was statistically (\*\*\*P<0.001) significant, difference compared to the control at 24 h. Histological method with Tr-Bl staining.*

*Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

#### **Figure 13.**

*Effect of the hypothalamic PRP-1 on the growth and viability of mouse isolated EAC cells on the 11th day of tumor growth. (A) Histological method with Tr-Bl staining detected viable EAC cells before their culture (control). EAC control cells after 24 h of incubation were mainly viable, although several dead Tr-Bl-positive cells were present (control 24 h). In samples treated with 0.1 and 1 μg/ml PRP-1, an increased number of Tr-Bl-positive non-viable cells was detected; along with various viable cells (arrows), apoptotic cells with fragmented nuclei (double arrows), as well as various Tr-Bl-positive cells surrounded by apoptotic bodies (arrow heads) were observed. The PRP-1 (0.1 and 1 μg/ml) inhibitory effect on the number of (B) total and (C) viable tumor cells treated for 24 h was statistically (\*\*\*P<0.001) significant, difference compared to the control at 24 h. Histological method with TR-Bl staining.*

Based on the PRP-1-induced morphological features of EAC cells, the apoptotic nature of PRP-1 was confirmed histologically as manifested by cell shrinkage, membrane blebbing, chromosome condensation (pyknosis), and nuclear fragmentation (karyorrhexis) (**Figures 14** and **15**).

To verify this observation, a series of experiments were performed, which were focused on the determination of apoptosis in cultured tumor cells using an Annexin V-Cy3 apoptosis detection kit and fluorescence microscopy. The analysis of the apoptosis on the 7-day inoculated mice EAC-cultured cells exposed to 0.1 μg/ml PRP-1 for 24 h revealed a significant increase in the number of apoptotic cells, reaching 50.33%, compared to 8.33% in the control sample on day 7. Besides, early apoptotic cells, as well as late apoptotic cells, containing and surrounded by fragments of necrotic nuclei were also detected, in contrast to the numerous viable tumor cells detected in the untreated control samples.

In late apoptotic cells, the apoptotic bodies undergo secondary necrotic changes and turn to detritus, known as a secondary form of necrosis mainly *in vitro* experiments when phagocytosis does not occur due to the absence of macrophages [55] (**Figure 16**).

In addition, a series of experiments aimed at elucidating the possible participation of PRP-1 in antitumorigenic processes was carried out by detecting the immunohistochemical localization of PRP-1 in the control and experimental EAC cells (**Figures 17** and **18**).

### **Figure 14.**

*Histological evaluation of the hypothalamic PRP-1 effect on mouse-isolated EAC cells on day 7 of tumor growth. Morphological changes of tumor cells (A) 24 and (B) 72 h after culture. (A) In control samples, numerous EAC cells linked with each other were detected at 24 h, whereas a decreased number of cells was observed with both doses of PRP-1. In the experimental samples, PRP-1-induced morphological changes were similar for both the two time-points of culture. Apoptotic membrane blebbing and apoptotic bodies (arrowheads), smaller and roundshaped cells with eosinophilic cytoplasm and condensed nuclei (pyknosis), and loss of reticular extensions and contacts with adjacent cells were observed. (B) Necrotic EAC cells containing no nuclei (karyolysis) or cells with lost membrane integrity (arrows) were mainly presented in the control samples after 72 h of culture, whereas few necrotic cells with lost plasma membrane integrity and released cell death products (arrows) were detected in the samples treated with PRP-1 for 72 h. Statistical data regarding the PRP-1 (0.1 and 1 μg/ml) effect on the apoptosis and necrosis in tumor cells treated for (C) 24 h and (D) 72 h were presented according to the H&E exclusion test in comparison with the findings in the untreated control cells. Data are presented as the mean ± standard deviation (n = 3), and represent* ≥*3 independent experiments. \*\*P<0.01; \*\*\*P<0.001, significant difference compared to (C) the control at 24 h and (D) the control at 72 h. Histological method with H&E staining.*

In the mice of control non-cultured EAC cells, PRP-1-IR was not detected on the seventh day of tumor growth. No intracellular PRP-1-IR was detected in the untreated control EAC cells cultured for 24 h however the presence of PRP-1-Ir cell membrane (21%) in the shape of narrow ring was registered. In the samples with 0.1 μg/ml PRP-1 treatment, strong PRP-1-IR was observed in the cytoplasm (46.5%) and nucleoli (10%).

However, the number of EAC cells with cytoplasmic PRP-1-IR of inoculated for 11 days mice and cultured for 72 h in the presence of PRP-1, constituted 73.4%, in contrast to 32.6% of the control cells with cytoplasmic PRP-1-IR. Notably, dense PRP-1-IR was noticed in 25% of apoptotic cells with membrane blebbing, in contrast to 4% of the untreated control cells.

Fluorescent nuclear staining of DAPI nevertheless proved statistically insignificant nuclear localization of PRP-1 in both control and PRP-1-treated samples after 72 h of incubation (4% of the total number of tumor cells).

*Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

### **Figure 15.**

*Histological evaluation of the hypothalamic PRP-1 effect on mouse-isolated EAC cells on the 11th day of tumor growth by H&E staining. Morphological changes of tumor cells at (A) 24 and (B) 72 h after culture. In the untreated control samples, tumor cells (arrows) with typical morphology were observed after both culture timepoints. In comparison to cells in the control group, cells exposed to 0.1 and 1 μg/ml PRP-1 were smaller in size and exhibited a round shape. EAC cells with the apoptotic bodies (arrowheads) were observed having no contact to adjacent cells. Statistical data regarding the PRP-1 (0.1 and 1 μg/ml) effect on the apoptosis and necrosis in tumor cells treated for (C) 24 h and (D) 72 h was presented according to the H&E exclusion test in comparison with the findings in untreated control cells. Data are presented as the mean ± standard deviation (n=3), and represent* ≥*3 independent experiments. \*\*P<0.01; \*\*\*P<0.001, significant difference compared to (C) the control at 24 h and (D) the control at 72 h. Histological method with H&E staining.*

#### **Figure 16.**

*Analysis of apoptosis/necrosis in cultured mouse EAC cells exposed to the hypothalamic PRP-1 on the 7th (A, B) and 11th days (C, D) of tumor growth according to fluorescence detection with Annexin V-cyanine 3. (A, C) viable EAC cultured cells (green) were detected 24 h after growing in the control untreated samples. In contrast to control samples, on the 7th day of tumor growth (B) an increased number of early apoptotic cells (orange) was revealed 24 h after treatment with 0.1 μg/ml PRP-1. Fragments of necrotic nuclei (red) were clearly detected in late apoptotic cells. (D) on the 11th day of tumor growth after treatment with 0.1 μg/ml PRP 1, the plasma membrane and certain weakly stained intracellular components could be indicative of early-stage apoptosis. Fluorescent method with Annexin V-Cy3 staining.*

### **Figure 17.**

*Immunohistochemical localization of the hypothalamic PRP-1 in cultured mouse EAC cells on the 7th day of tumor growth. (A) All microimages demonstrated no PRP-1-IR in EAC cells before culture (control). PRP-1-IR in tumor cells (B) at 24 h and (C) 72 h after culture. (B) No intracellular PRP-1-IR was detected in control EAC cells after 24 h of culture, whereas the plasma membrane exhibited weak PRP-1-IR in the form of a narrow ring (arrows). In experimental samples, the sub-membrane cytoplasm with dense PRP-1-IR was detected in the tumor cells (arrows) exposed to 0.1 μg/ml PRP-1. (C) After 72 h of culture, nuclear localization of PRP-1 was detected in certain control (arrows) and PRP-1 treated (not shown) tumor cells. PRP-1-Ir cytoplasm was released from necrotic control cells with lost membrane integrity (double arrows). The strong PRP-1-Ir cytoplasm was revealed both in control (not demonstrated) and exposed to PRP-1 EAC cells. Notably, the apoptotic cells with the apoptotic bodies (arrowheads) also demonstrated strong PRP-1-Ir cytoplasm. ABC immunohistochemical method.*

### **Figure 18.**

*Immunohistochemical localization of the hypothalamic PRP-1 in cultured mouse EAC cells on the 11th day of tumor growth according to the ABC immunohisto-chemical method. (A) EAC control cells before culture, where PRP-1 was localized in the cell membrane, cytoplasm (arrows) and nucleoli (double arrows) of certain tumor cells. PRP-1-IR in the tumor cells (B) 24 h and (C) 72 h after their culture. (B) In the untreated control samples at 24 h, weak PRP-1-IR was mainly observed in the perinuclear zone of cell cytoplasm. In the experimental samples exposed to 0.1 μg/ml PRP-1 for 24 h, PRP-1-IR was observed in the cell nucleoli (double arrows). (C) At 72 h after EAC cell culture, dense cytoplasmatic IR for PRP-1 was detected in tumor cells both in the control and PRP-1 treated samples. Morphological changes of the cells undergoing death-related processes (apoptosis and necrosis) were clearly observed, including release of PRP-1-Ir intracellular contents from necrotic cells into the extracellular space, which was detected predominantly in the control samples (arrows), while PRP-1-Ir plasma blebs and apoptotic bodies (arrowheads) were revealed mainly in the experimental samples. ABC immunohistochemical method.*

*Diverse Effects of Hypothalamic Proline-Rich Peptide (PRP-1) on Cell Death… DOI: http://dx.doi.org/10.5772/intechopen.108632*

Thus, the findings provide evidence that the effect of PRP-1 is cellular context dependent in EAC cells, with PRP-1 acting as a cytotoxic agent by inducing programmed cell death type I apoptosis. With regard to the detection of PRP-1-IR in the nucleus and cytoplasm of apoptotic EAC cells cultured for 72 h in both control (untreated) and experimental (PRP-1 treated) samples, the possible biosynthesis of the endogenous PRP-1 in the studied cancer cells should be taken into account.

### **7. PRP-1 as a circulating biomarker**

Today, much attention is paid to the discovery of circulating biomarkers in the blood serum of patients with different disorders.

Previously, we succeeded in the detection and quantification of PRP-1 in rat blood serum by an enzyme linked immunosorbent assay (ELISA) developed for PRP-1 [56]. The minimum detectable concentration of PRP-1 in the intact rat blood serum has been shown to be approximately 1.78 ng/ml. Furthermore, the effect of the exogenous PRP-1 on the endogenous PRP-1 concentration was identified in the blood after 5 h and 2 days of its administration.

The significant increase of PRP-1 concentration observed in the blood samples in 5 h after the PRP-1 intraperitoneal injection was decreased in the 2-day post-injection period to approximately the control level.

Based on the recent data [57] pointing to PRP-1 being a new natural substrate for the multifunctional dipeptidyl protease (DPP-IV) that hydrolyses the peptide bonds formed by the proline residues, the decrease in the peptide concentration could be explained by the proteolytic processing of PRP-1 by DPP-IV.

The results serve as a basis for suggesting the involvement of different factors (neuropeptides, enzymes, neurotransmitters, etc.) in the mechanism of the PRP-1 action, and justify the need for additional studies for demonstrating the potential role of PRP-1 in the stress-induced disorders obtained on the animal models and in the pathogenesis of various human diseases.

### **Author details**

Silva Abrahamyan1 \* and Karina Galoian2

1 NAS RA Laboratory of Histochemistry and Functional Morphology, H.Buniatian Institute of Biochemistry, Yerevan, Armenia

2 Department of Orthopedics, University of Miami, Miller School of Medicine, Miami, Florida, USA

\*Address all correspondence to: silva.abrahamyan@gmail.com

© 2022 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.

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