Peroxisomal Diseases and Treatment Strategies

#### **Chapter 3**

## Apoptosis-Related Diseases and Peroxisomes

*Meimei Wang, Yakun Liu, Ni Chen, Juan Wang and Ye Zhao*

#### **Abstract**

Apoptosis is a highly regulated cell death program that can be mediated by death receptors in the plasma membrane, as well as the mitochondria and the endoplasmic reticulum. Apoptosis plays a key role in the pathogenesis of a variety of human diseases. Peroxisomes are membrane-bound organelles occurring in the cytoplasm of eukaryotic cells. Peroxisomes engage in a functional interplay with mitochondria. They cooperate with each other to maintain the balance of reactive oxygen species homeostasis in cells. Given the key role of mitochondria in the regulation of apoptosis, there could also be an important relationship between peroxisomes and the apoptotic process. Peroxisome dysfunction severely affects mitochondrial metabolism, cellular morphological stability, and biosynthesis, and thus contributes directly or indirectly to a number of apoptosis-related diseases. This chapter provides an overview of the concept, characteristics, inducing factors, and molecular mechanisms of apoptosis, as well as evidence for apoptosis in cancer, cardiovascular diseases, and neurodegenerative disorders, and discusses the important role of the peroxisome in the apoptosis-associated diseases.

**Keywords:** apoptosis, mitochondria, peroxisome, ROShomeostasis, Cancer, cardiovascular diseases, neurodegenerative diseases

#### **1. Introduction**

Death is the final fate of cells and organisms and is a normal biological phenomenon in the living world. Cell death plays a crucial role in the development of plants and animals in nature and in maintaining ecological balance [1]. For example, in the developing vertebrate nervous system, as many as half or more of the nerve cells usually die soon after they are formed. In a healthy adult human, billions of cells die every hour in the bone marrow and intestines. So much cell death seems very wasteful, especially when the vast majority of cells are perfectly healthy at the time of suicide.

In general, cell death can be divided into two types: programmed cell death (PCD) and accidental cell death (necrosis) [2]. The former is a controlled process of intracellular death program, also vividly referred to as cellular suicide. The latter is caused by external factors (i.e., injury, infection, etc.). The study of PCD (especially apoptosis) processes has led to a better understanding of the pathogenesis of certain diseases. The 2002 Nobel Prize in Physiology and Medicine was awarded to Britons Sydney Brenner, Jone E. Sulston, and H Robert Horvitz for their discovery of how genes

regulate organ growth and programmed cell suicide processes, using the nematode *Caenorhabditis elegans* (*C. elegans*) as an animal model.

A coordinated balance between cell proliferation and apoptosis is crucial for normal development and tissue homeostasis. Once this balance is permanently disrupted, normal cells may be transformed into mutant cells whose clonal survival and uncontrolled proliferation may lead to the development of tumors and various other diseases.

#### **2. Apoptosis**

#### **2.1 The concept, characteristics, and inducing factors**

#### *2.1.1 Key concepts*

Apoptosis is the process of cellular suicide by activating an intracellular death program or by the orderly breakdown of cells from within. The term was first introduced by Kerr J. F. R. in the 1970s and was not accepted by the general public until the 1990s.

Although apoptosis is only one form of Programmed cell death (PCD), it is by far the most common and well-understood form, and, confusingly, biologists often use the terms PCD and apoptosis interchangeably [3].

For a multicellular organism, a highly organized community, cell numbers are tightly regulated not only by controlling the rate of cell division but also by controlling the rate of cell death. Thus, apoptosis is important not only for tissue remodeling and elimination of transitional organs during the development of an organism, but also for the clearance of cellular senescence inactive metabolic organs, such as blood cells and epithelial cells in the digestive system, and cells with damaged or mutated DNA [4–6]. In a nutshell, apoptosis is an essential mechanism complementary to proliferation to ensure homeostasis in all tissues.

Unlike apoptosis, necrosis is a form of cell injury that leads to the premature death of cells in living tissues due to autolysis, usually caused by stronger external factors such as infection, toxins, or trauma, ultimately resulting in the unregulated of cellular components, always harmful and potentially fatal to the organism [7, 8]. Necrosis usually causes a local inflammatory response. The reason for this is that when nearby macrophages engulf these necrotic cells, they may release microorganisms that destroy the surrounding tissue causing collateral damage and inhibiting the healing process.

Typically, cell death due to necrosis does not follow the apoptotic signaling transduction pathway, but rather various receptors are activated, leading to loss of cell membrane integrity and uncontrolled release of cell death products into the extracellular space. In contrast, apoptosis is a naturally occurring programmed and targeted cause of cell death and usually provides beneficial effects to the organism. A brief comparison of them can be summarized as follows (**Figure 1**).

#### *2.1.2 Characteristics*

As mentioned above, necrosis is a form of traumatic cell death caused by acute cellular injury. In contrast, apoptosis is a process of active cellular suicide. Multicellular organisms eliminate mutated, damaged, or unwanted cells by this type of active suicide. Apoptosis plays an important role in tissue sculpting during embryonic development and in the maintenance of tissue homeostasis throughout life [6].

*Apoptosis-Related Diseases and Peroxisomes DOI: http://dx.doi.org/10.5772/intechopen.105052*

**Figure 1.** *Structural change of cells undergoing necrosis and apoptosis.*

The process has distinct morphological features, including cell rounding and contraction, blebbing and PS externalization of the plasma membrane, cytoplasmic vacuolization including endoplasmic reticulum expansion and cisternae swelling to form vesicles and vacuoles, nuclear condensation, border aggregation or fragmentation, chromatin compaction, pyknosis, and ultimately fragmentation between nucleosomes by endonucleases, resulting in regular DNA degradation and inhibition of protein translation, and ultimately to the eventual rupture of the cell into small spheres surrounded by membranes called apoptotic bodies, which contain "packed" cell contents with an electron cloud density similar to chromatin; and a sub-G1 curve preceding the G1 phase peak is observed in cytometric histogram [9]. Apoptotic bodies can be recognized and digested by phagocytosis of neighboring macrophages through the presence of phosphatidylserine (PS) on their surface [10]. In this way, the apoptotic cells can be rapidly removed by tissue phagocytes through phagocytosis, without releasing harmful substances that can initiate inflammation, which can cause a significant amount of tissue damage. Because apoptotic cells are always rapidly eaten and digested, dead cells are usually rarely seen, even when large numbers of cells die from apoptosis. This may be the reason why biologists once ignored the phenomenon of apoptosis and may still underestimate its extent.

Abnormal apoptosis contributes to many important diseases, including cancer, autoimmune diseases, diabetes, and neurodegenerative diseases. Various types of cellular stress, such as DNA damage or growth factor deprivation, can trigger apoptosis through intrinsic or extrinsic pathways.

#### *2.1.3 Inducing factors*

Apoptosis can be triggered by both internal stimuli, such as abnormalities in DNA, and external stimuli, such as certain cytokines from different pathways, respectively [11]; or it can be induced by physiological or pathological factors.

Specifically, physiological triggers can include the following two aspects [12]:

(1) Direct action of certain hormones and cytokines: for example, glucocorticoids

are typical signals of apoptosis in lymphocytes; thyroxine plays an important role in the apoptotic degeneration of tadpoles' tails; TNF can induce apoptosis in a variety of cells. (2) Indirect effects of certain hormones and cytokines: for example, testosterone deficiency caused by testicular dysplasia can lead to apoptosis of prostate epithelial cells. Inadequate secretion of adrenocorticotropic hormone by the pituitary gland can promote apoptosis of adrenocortical cells, etc.

While pathological triggers usually include the following two aspects: (1) It is generally believed that apoptosis can be induced by many factors that can cause damage to cells, such as stress, radiation, chemical toxins, viral infections, and chemotherapeutic drugs, and even malnutrition and excessive functional complexes can induce apoptosis. (2) Some factors such as various chemical carcinogens and certain viruses (e.g., EBV) inhibit apoptosis. Therefore, it is thought that the ability to induce cells may be related to the type, intensity, and duration of the harmful factors.

#### **2.2 Molecular mechanisms of apoptosis (Signaling pathways and related enzymes)**

#### *2.2.1 Apoptotic Signaling pathways*

The initiation of apoptosis is tightly regulated by different signaling pathways. The best-understood two are the intrinsic pathway (also known as the mitochondrial pathway) and the extrinsic pathway (also known as the death receptor pathway). The mitochondrial pathway is generally activated by intracellular signals and depends on proteins released from the intermembrane space between the mitochondrial bilayers. The death receptor pathway is activated by extracellular ligands, and the activated extracellular ligands bind to their specific death receptors on the cell surface, inducing the formation of death-inducing signaling complexes (DISC) [13, 14]. Here, we will discuss the extrinsic and intrinsic pathways separately. However, it should be noted that there is crosstalk between these pathways and that extracellular apoptotic signaling can also lead to activation of the intrinsic pathway.

#### *2.2.1.1 The extrinsic pathway of apoptosis*

The extrinsic death pathway triggers receptor-mediated apoptosis. Its major components include pro-apoptotic ligands, receptors that recognize/bind ligands, and adaptor proteins that bind to the cytoplasmic face of the receptor. In addition, the pathway recruits other molecules, including cysteine-specific proteases (caspases), the initiator of the death process, and the executors, to execute the apoptotic process [15]. For example, TNF is a common pro-apoptotic ligand and TNFR1 on the cell membrane is the receptor. When TNF binds to TNFR1, the activated receptor binds to two different cytoplasmic adaptor proteins (tumor necrosis factor-related death domain protein, TRADD, and fas-associating protein with death domain, FADD) and procaspase-8, forming a multi-protein complex on the inner surface of the plasma membrane, containing an 80 amino acid death structure domain through which a death-inducing signaling complex (DISC). The cytoplasmic structural domains of the TNF receptor, FADD, and TRADD interact through homologous regions called death structural domains present in each protein [16]. Procaspase-8 and FADD interact through homologous regions called death effector domains. Procaspase 8 in DISC is activated and active caspase 8 is released into the cytoplasm, where it cleaves and activates effector caspases (e.g., procaspase 3), triggering a caspase cascade that

#### *Apoptosis-Related Diseases and Peroxisomes DOI: http://dx.doi.org/10.5772/intechopen.105052*

further cleaves a number of death substrates, including BID and cytoskeletal proteins, if glued, leading to apoptosis (**Figure 2**). Notably, inhibitors of apoptosis (IAPs) can inactivate caspases by specifically binding to their active sites. Caspase activator (SMAC)/Diablo and its functional homologs in flies, including Grim, Reaper, and Hid, can in turn target binding and degrade IAPs [17].

In addition, it should be noted that the interaction between TNF and TNFR1 may also activate other signaling pathways and allow cell survival rather than self-destruction.

#### *2.2.1.2 The intrinsic pathway of apoptosis*

In general, internal stimuli such as irreparable genetic damage, hypoxia (lack of oxygen), very high concentrations of cytosolic Ca2+, viral infection, or severe oxidative stress (i.e., production of large amounts of damaging free radicals) and cytotoxic drug treatment trigger apoptosis via the intrinsic pathway.

The intrinsic death pathway, i.e., the mitochondrial-received apoptotic pathway, is a death receptor non-dependent apoptotic pathway [18]. This pathway is activated by the release of cytochrome C (Cyto C) from mitochondria in response to various stresses and developmental death cues. The process specifically involves multiple steps as follows: apoptotic signals (various types of cellular stress), lead to the insertion of pro-apoptotic members of the Bcl-2 family of proteins (e.g., Bax), into the outer mitochondrial membrane, forming pores that mediate the release of Cyto C from the mitochondrial intermembrane space into the cell membrane. Once in the cell membrane, Cyto C molecules bind to Apaf-1 (a homolog of mammalian CED4) and further recruit procaspase-9 to form a complex of multiple subunits called the apoptosome. Then procaspase-9 is activated to become active caspase-9. Then the caspase-9 molecule cleaves and activates the downstream executor caspase (Caspase-3, 6,7) to carry out the apoptotic process (**Figure 2**) [19].

**Figure 2.** *Schematic diagram of apoptotic signaling.*

Bcl-2, the mammalian homolog of Ced-9, prevents apoptosis by inhibiting the release of CytoC from mitochondria [20]. IAPs, second mitochondrial activators of caspases (Smac), endonuclease G (Endo G), and AIF also have important roles in the apoptotic process [21]. Notably, Endo G and AIF are specifically activated by apoptotic stimuli and are able to induce ribosomal breakage of DNA independently of caspases. Endo G is a mitochondria-specific nuclease that translocates to the nucleus and cleaves chromatin DNA during apoptosis. AIF is a flavin adenine dinucleotidecontaining, NADH-dependent oxidoreductase that resides in the mitochondrial intermembrane space, and its specific enzymatic activity remains unknown. In the presence of apoptosis, AIF undergoes proteolysis and translocates to the nucleus, where it triggers chromatin condensation and massive DNA degradation in a caspaseindependent manner.

#### *2.2.1.3 ER-dependent apoptotic pathways*

Previously, it was thought that the only apoptotic pathways were the mitochondrial pathway and the death receptor signaling pathway. Now, an increasing number of studies have shown that the endoplasmic reticulum (ER) also senses and transmits apoptotic signals [22, 23]. The sustained action of various apoptosis-inducing factors may induce a complex unfolded protein response (UPR) by interfering with the correct protein folding process. The UPR response causes endoplasmic reticulum stress, leading to cellular apoptosis due to the accumulation of intracellular misfolded proteins. ER, in addition to being the site of protein folding, it is also the main intracellular Ca2+ reservoir. Disturbing intracellular Ca2+ homeostasis can also induce the typical ER stress response. Interestingly, the localization of Bcl-2 family proteins (including Bcl-1, Bax, Bak, *et al.*) in the ER affects the cellular Ca2+ homeostasis. Overexpression of Bcl-2 or deficiency of Bax and Bak decreases the Ca2+ concentration in the ER and protects cells from apoptotic stimuli that trigger cell death by inducing Ca2+ influx from the ER to the cell membrane (**Figure 2**).

It has been suggested that procaspase-12 is a proximal effector of apoptosis associated with the ER. Recent studies have found that although caspase-12 is processed and activated in ER stress-induced apoptosis in mouse cells, the enzyme is not absolutely necessary for this process. On the other hand, cells lacking caspase-8 or caspase-9 were highly resistant to ER stress-induced apoptosis. One of the mechanisms that could explain caspase-8 activation in the ER involves the recent discovery of an ER-resident potential apoptosis initiator, named neurotrophic receptor-like death domain protein (NRADD). This protein has a transmembrane and cytoplasmic region that is highly homologous to the death receptor. Induction of apoptosis by NRADD is dependent on caspase-8 activation but does not require the mitochondrial component of the death program.

In addition to propagating death-inducing stress signals, ER contributes to apoptosis initiated by cell surface death receptors and to pathways resulting from DNA damage. Modulation of ER calcium stores can sensitize mitochondria to direct pro-apoptotic stimuli and promote activation of cytoplasmic death pathways.

In short, the extrinsic (receptor-mediated), intrinsic (mitochondria-mediated), and endoplasmic reticulum stress-mediated apoptotic pathways ultimately converge by activating the same caspases, which cleave the same cellular targets. Apoptosisinducing factors can be involved in diseases by activating apoptotic pathways that affect the rate of apoptosis, and may predominantly involve the first two pathways or all three of these pathways.

#### *2.2.2 Apoptosis-related enzymes*

The mechanism by which apoptosis occurs is highly conserved in all animal cells. It is dependent on a family of proteins called caspases (c for cysteine and asp for aspartic acid). This family of proteins has many members and generally exists as inactive precursors (procaspases). Procaspases are generally activated by the catalytic cleavage of other (already active) caspases, forming an amplified network of protein cascades. The activation process of procaspases involves the formation of a heterodimer by cleavage and the combination of two dimers to form an active tetramer. During apoptosis, those responsible for initiation are known as initiator caspases; those responsible for cleavage of specific target proteins (e.g. nuclear lamina proteins, DNA degradation enzymes, cytoskeletal proteins, and cell–cell adhesion proteins) are the executor caspases.

Apoptotic mechanisms are present throughout the initial to final stages of animal development. Only the process requires a trigger to be activated for its occurrence. So, how is the first member of the caspase cascade reaction described above initiated? Initiator procaspases usually contain a caspase recruitment domain (CARD). This structural domain can assemble into an activation complex with an adaptor protein when the cell receives an apoptotic signal. The formation of this complex means that the promoter caspase will be activated by cleavage.

As mentioned above, there are numerous members of the caspases family, most of which are involved in apoptosis, but not all of them mediate apoptosis [24]. For example, the first discovered caspase, human interleukin-l-converting enzyme (ICE), was not associated with apoptosis but was responsible for mediating the inflammatory response. After the discovery of ICE, similar proteins to ICE were identified in *C. elegans* and were confirmed to be involved in the apoptotic process.

#### **2.3 Apoptosis and peroxisomes**

Peroxisomes, similar to the mitochondria, are a membranous subcellular organelle within eukaryotic cells. The peroxisome contains enzymes related to fatty acid and amino acid oxidation processes that produce hydrogen peroxide and also degrade hydrogen peroxide [25]. This gives the peroxisome its name and it plays an important role in maintaining intracellular oxidative metabolic homeostasis.

Because of the crucial role of the peroxisome, its dysfunction is associated with various pathological conditions, organ dysfunction, and aging [26–28]. For example, deficiency of Pex3, a peroxisomal membrane protein essential for membrane assembly, a member of the peroxisome (Pex) family, leads to complete loss of peroxisome function, while deficiency of Pex5, a peroxisome transporter, leads to Pex5 (a peroxisomal transporter) leads to the loss of peroxisomal matrix proteins. Mutations in this class of Pex genes may lead to human developmental abnormalities, such as human autosomal recessive disorders [29].

Peroxisomes play important roles in biosynthesis and signal transduction, which cannot be achieved without interaction with other organelles in the cell. In particular, peroxisomes interact functionally with mitochondria [30]. They cooperate with each other to perform biological functions such as production, fission, proliferation and degradation through vesicular transport, signaling, and membrane contact [31]. On the other hand, they can act synergistically to clear excess intracellular ROS, resist extracellular stresses through immune responses, and play an important role in the maintenance of lipid homeostasis through fatty acid β-oxidation [32–34]. In one

word, peroxisomes are essential for the maintenance of normal mitochondrial and even whole cell function. Some chemotherapeutic drugs have been found to trigger mitochondrial dysfunction, leading to apoptosis by overwhelming cells with ROS. For example, Vorinostat (Vor), an FDA-approved histone deacetylase inhibitor (HDACi) for lymphoma treatment, has been well documented to trigger mitochondrial-mediated apoptosis through ROS accumulation. Acute Vor treatment has been shown to induce the expression of peroxisome proteins, thereby increasing peroxisome proliferation in a lymphoma model system. In addition, the knockdown of peroxisomes by gene silencing of Pex3 enhances Vor-induced ROS-mediated apoptosis [35].

In short, peroxisome dysfunction severely affects mitochondrial metabolism, cellular morphological stability, and biosynthesis, directly or indirectly contributing to a number of apoptosis-related diseases such as cancer [36, 37], cardiovascular disease [38–40], and neurodegenerative disorders [41].

#### **2.4 Apoptosis-related diseases and peroxisomes**

Apoptosis is an important way for the organism to maintain the numerical homeostasis of the cell population. Excessive or insufficient apoptosis can lead to disease.

#### *2.4.1 Cancer*

Crosstalk between mitochondria and other organelles is important in tumorigenesis. Mitochondria and peroxisomes are important organelles for ROS production and scavenging. Under normal conditions, both maintain intracellular ROS homeostasis. Impaired peroxisome function inevitably leads to increased levels of ROS in mitochondria, which impairs mitochondria, exacerbates impaired ROS clearance, leads to low levels of apoptosis, and thus promotes tumorigenesis and progression [42–44].

ROS act as signaling molecules to regulate various physiological and pathological processes [45]. H2O2 is a member of the ROS family and plays an important role in the signaling of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). H2O2 prevents protein tyrosine phosphatase 1B (PTP1B) from dephosphorylating EGF, thereby facilitating EGF stimulation. In addition, activation of PDGF requires H2O2 to promote oxidation and inactivation of PDGF-receptor-associated phosphatases and SHP-2, thereby facilitating the signaling pathway [46, 47]. Excessive ROS production can lead to cellular genomic instability (including mutations in the mitochondrial genome) on the one hand. Notably, ROS can promote tumor cell proliferation under hypoxic conditions. The reason for this is that the transcription factors hypoxia-inducible factors (HIFs) are upregulated under hypoxic conditions, thus promoting the expression of oncogenes. Although some proteases such as prolyl hydroxylases (PHDs) can degrade HIFs, the increased release of ROS induced by hypoxia can prevent the action of PHDs on HIFs. In this case, HIFs can then promote tumor progression under hypoxic conditions.

Briefly, because disruption of the functional balance between mitochondria and peroxidases may lead to increased ROS production, the increased ROS may inhibit apoptosis-inducing genes (bcl2 and p53, etc.), resulting in non-apoptosis of cells that should be apoptotic. Alternatively, the apoptotic process may be inhibited due to a decrease in the activity of apoptosis-related enzymes (caspases, etc.), leading to malignant cell transformation and tissue malignant proliferation. Both of these aspects are considered to be one of the important mechanisms leading to tumorigenesis and infiltrative metastasis.

#### *2.4.2 Cardiovascular diseases (CVDs)*

Apoptosis is a form of death of terminally differentiated cardiomyocytes. Clinical data suggest that ROS generation, DNA damage, and other factors activate apoptosis, resulting in the loss of large numbers of cardiomyocytes in patients with advanced congestive heart failure, patients with myocardial infarction, and patients with diabetic cardiomyopathy. The evidence suggests that apoptosis may be an important pathogenetic mechanism in cardiovascular disease [38]. Apoptosis, in concert with necrosis, may also lead to foam cell death and thus to the formation of a necrotic core, which contributes to lesion instability and increases the risk of lesion rupture and thrombosis.

Lower levels of ROS production can lead to chronic remodeling of the heart, whereas high levels of ROS can directly lead to apoptosis in the cardiomyocytes [48]. It is therefore interesting that catalase overexpression inhibits cardiomyocyte apoptosis by protecting the cells from ROS [49]. Peroxisomal antioxidant enzymes and plasmalogens protect cardiomyocytes via the degradation and trapping of ROS and the maintenance of ROS homeostasis. Apoptosis of cardiac cells has been demonstrated in several cardiovascular diseases, including myocardial ischemia–reperfusion injury (I/R) and atherosclerosis [50–52]. Atherosclerosis, a major cause of heart failure and myocardial infarction, can likewise predispose to acute coronary heart disease. There is evidence that thrombosis and plaque rupture may be due to apoptosis of a large number of smooth muscle cells and macrophages in unstable atherosclerotic plaques [53, 54]. Rupture of atherosclerotic plaques with concomitant thrombus formation may lead to coronary artery occlusion, which affects the blood supply to the myocardium, resulting in myocardial infarction and leading to patient death. Reperfusion is an effective treatment for acute myocardial infarction, but it may cause reperfusion injury while restoring blood flow [55]. Studies in the last decade or so have shown that cardiac cell death occurring during reperfusion after myocardial infarction is mainly apoptosis, not cell necrosis, which breaks the long-held misconception [56–58]. Usually, what occurs during I/R is mostly cell apoptosis, whereas necrosis occurs more often after prolonged ischemia. In addition, apoptosis also plays an important role in myocardial remodeling after infarction. There is evidence that a large number of apoptotic cells can be detected in myocardium at the marginal zone of myocardial infarction [56]. Since the regenerative capacity of myocardium is limited, people show great interest in preventing apoptosis of myocardial cells during I/R.

There is also a connection between chronic heart failure and apoptosis [59]. It has been reported that patients with advanced heart failure have higher rates of cardiac myocyte apoptosis than normal subjects. Using transgenic mice with cardiac tissuespecific expression of caspase-8, it was found that apoptosis of cardiomyocytes, even at very low levels, can lead to fatal dilated cardiomyopathy as long as it occurs chronically [60]. In addition, the use of caspase inhibitors prevented left ventricular dilatation and improved ventricular function, suggesting that long-term apoptosis can lead to a significant reduction in cardiomyocyte numbers, which in turn gradually decreases cardiac contractile function. As a result, the remaining cardiomyocytes become overcompensated and contribute to cardiac hypertrophy, leading to the development of heart failure [61].

Regarding the major pathways involved in apoptotic signaling in the heart, the death receptor pathway, the mitochondrial, and ER-stress death pathways are all involved [62]. The cross-talk between death receptors and mitochondrial cell death pathways has been demonstrated in cardiomyocytes and the heart [63, 64]. For example, Date *et al*. found that overexpression of FasL of the death receptor pathway activated both caspase-8 and -9 in cardiac myocytes [65]. Cardiacrestricted overexpression of TNF-α promoted apoptosis, but when these mice overexpressing TNF-α were crossed with mice overexpressing Bcl-2 in the heart, both left ventricular remodeling and cardiac apoptosis in the progeny mice were be alleviated [66].

In recent years, ER stress pathway has been reported to be in cross-talk with both the death receptor pathway and the mitochondrial pathway [13, 67, 68]. One study found that application of TNF-α induced HL-1 myoblast cell lines that activated both caspase-3 and -12 [69]. Bcl-2, which targets ER, inhibited mitochondrial membrane depolarization in apoptotic cells and also inhibited cytochrome c release [70]. Caspase-8 cleaves BAP31, an ER-associated protein, and the cleaved fragment induces Ca2+ release from ER, into the mitochondria, and initiates apoptosis [71]. It has also been reported that Bik proteins can activate Bax/Bak in the ER membrane after localization to the mitochondria, initiating Ca2+ release [72].

Regardless of the causative factor, and regardless of which signal transduction pathway or pathways are involved, oxidative stress due to the interaction of peroxisomes and mitochondria plays a pivotal role in triggering apoptosis and thus contributing to the development of cardiovascular disease.

#### *2.4.3 Neurological disorders*

Apoptosis plays a key role in the normal development of the central nervous system and is involved in the pathogenesis of adult brain-related diseases, such as stroke [73] and neurodegenerative diseases [74].

There is growing evidence that the decline in peroxisome function with age may be associated with age-related neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) [75]. In the brains of patients with Alzheimer's disease and Parkinson's disease, plasmin levels are significantly reduced [76, 77], which suggests peroxisome dysfunction in neurodegenerative diseases. The lack of peroxisome activity in aged cells accumulates cellular ROS, which can compromise the integrity of organelles including mitochondria and the peroxisome itself. Subsequent defects in energy production mediated by peroxisomal fatty acid metabolism and mitochondrial oxidative phosphorylation may lead to metabolic failure in aged postmitotic cells, thereby inducing apoptosis associated with neurodegeneration.

Huntington's disease (HD), a prototypical neurodegenerative disorder, is caused by a mutation in the Huntingtin protein due to a repeat amplification of the CAG in the Huntington gene. Patients with this disease suffer from neuronal dysfunction due to massive apoptosis of nerve cells, which in turn manifests as mental cognitive and motor impairment, and even disability [74].

ROS can easily poison neurons due to their series of characteristics, such as rich in fatty acids, easy intracellular production of large amounts of hydroxyl radicals, weak antioxidant capacity, and low regenerative capacity. In addition, because of the high metabolic rates, neurons require a high energy supply from mitochondria, which are both the most important intracellular organelle for ROS production and also vulnerable to ROS attack. It has been shown that treatment of isolated cultured cerebellar granule neurons with hydrogen peroxide induces mitochondrial fission within 1 hour [78]. Furthermore, treatment of mice with nitric oxide in stroke leads to massive fission of neuronal mitochondria before the onset of neuronal loss [79]. In the presence of calcium, acute exposure to high levels of ROS can induce massive opening

#### *Apoptosis-Related Diseases and Peroxisomes DOI: http://dx.doi.org/10.5772/intechopen.105052*

of mitochondrial membrane transition pores and increased permeability, which in turn causes cell Apoptosis or necrosis occurs. ROS production in mitochondria forms a vicious cycle with oxidative stress and is toxic to cells. There is some evidence in transgenic mouse models of HD that showed that Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid with antioxidant properties, prevents the production of reactive oxygen species, mitigates mitochondrial insufficiency and apoptosis, in part, by inhibiting Bax translocation from cytosol to the mitochondria [80]. TUDCA prevented striatal degeneration and ameliorated locomotor and cognitive deficits in a 3-NP (3-nitropropionic acid) rat model of HD. Keene *et al*. [81] showed that systemically administered TUDCA significantly reduced striatal neuropathology, decreased striatal apoptosis, reduced the size of ubiquitinated neuronal intranuclear htt inclusions, and improved locomotor and sensorimotor deficits in the R6/2 transgenic HD mouse.

#### **3. Conclusions**

Apoptosis is a highly regulated cell death program that can be induced by a variety of physiological and pathological factors and has specific morphological and biochemical characteristics. The mechanism of its onset has not been completely elucidated to date, and it is now accepted that it is mediated by a number of pathways including the death receptor signaling pathway, the mitochondrial signaling pathway, and the endoplasmic reticulum signaling pathway. As an important way for the organism to maintain the numerical homeostasis of the cell population, apoptosis plays a key role in the pathogenesis of various human diseases. Peroxisomes and mitochondria are membrane-bound organelles in the cytoplasm of eukaryotic cells and are closely related to each other in their organelle synthesis and function. One of their important roles in cooperating with each other is to regulate the level and extent of apoptosis by maintaining the homeostasis of reactive oxygen species in the cell. Peroxisome dysfunction severely affects mitochondrial metabolism, cellular morphological stability, and biosynthesis, and therefore contributes directly or indirectly to a number of apoptosis-related diseases. Based on the available relevant findings, this chapter presents and summarizes the important potential role of peroxisomes in apoptosis-related diseases such as tumors, cardiovascular diseases, and neuropsychiatric disorders.

#### **Acknowledgements**

This work was supported in part by grants from National Natural Science Foundation of China (22176002), Anhui Provincial Natural Science Foundation (2008085 MB49), Natural Science Foundation of Anhui Provincial Department of Education (KJ2021A0215), Anhui Medical University Research Enhancement Program (2021xkjT004), and Open Project Fund of the Key Laboratory of the Ministry of Education for the Birth Population (JKZD20202).

#### **Conflict of interest**

The authors report no conflicts of interest.

### **Author details**

Meimei Wang1 \*, Yakun Liu1 , Ni Chen2 , Juan Wang3 and Ye Zhao2

1 Department of Pathophysiology, School of Basic Medical Science, Anhui Medical University, Hefei, Anhui, P.R. China

2 Department of Nuclear Medicine, School of Basic Medical Science, Anhui Medical University, Hefei, Anhui, P.R. China

3 Department of Public Health Inspection and Quarantine, School of Public Health, Anhui Medical University, Hefei, Anhui, P.R. China

\*Address all correspondence to: wangmm@ustc.edu.cn

© 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 4**

## Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights from Preclinical and Clinical Evaluations

*Nishat Fatima*

#### **Abstract**

Peroxisome function has long been associated with oxygen metabolism. High concentrations of hydrogen peroxide (H2O2−) producing oxidases are in the set of peroxisomes and their antioxidant enzymes, especially catalase. Reactive oxygen species (ROS) can certainly be considered as an intracellular multifunctional biological factor which are released and scevenged in peroxisomes. They are known to be involved in normal cellular functions such as signaling mediators, overproduction under oxidative stress conditions leading to adverse cellular effects, cell death, and various other pathological conditions. This review provides an insight into the relationship between peroxisomes and ROS, which are emerging as key players in the dynamic rotation of ROS metabolism and oxidative damage. Various conditions upset the balance between ROS production and removal in peroxisomes. The current review also targets the ROS-inhibiting enzymes and exemplifying the effects of antioxidants in pre-clinical and clinical evaluation of natural and herbal supplements.

**Keywords:** antioxidants, oxidative stress, free radicals, vitamin C, E

#### **1. Introduction**

Reactive oxygen species (ROS) can certainly be considered as an intracellular multifunctional biological factor. They are known to be involved in normal cellular functions such as signaling mediators, overproduction under oxidative stress conditions leading to adverse cellular effects and eventually cell death. Under normal conditions in every human cell, the release of pro-oxidants in the form of ROS (reactive oxigen species) and RNS (reactive nitrogen species) are scrutinized by antioxidant levels. The equilibrium maintained is shifted in favor of pro-oxidants resulting in oxidative stress, when exposed to adverse circumstances such as atmospheric pollutants, unfavorable physicochemical, environmental or pathologicl agents including cigarette smoking, toxic chemicals, ultra violet rays and radiation and also excess formation of advanced glycation end products (AGE), in diabetes [1, 2]. This has been associated in the origin of various (>100) human diseases. Peroxisome function has long been

associated with oxygen metabolism. High concentrations of H2O2 − producing oxidases are in the set of peroxisomes and their antioxidant enzymes, especially catalase. This review provides an insight into the relationship between ROS and peroxisomes, which have emerged as key players in the dynamic rotation of ROS metabolism and oxidative damage. The counterparts of ROS-producing enzymes such as antioxidants are also discussed; exemplifying their effects in pre-clinical and clinical evaluation of natural and herbal supplements [3].

Peroxisomes contain at least 50 different enzymes involved in a variety of biochemical pathways in different cell types. Peroxisomes were originally defined as organelles that perform oxidation reactions that result in the production of hydrogen peroxide. Because hydrogen peroxide is harmful to the cell, peroxisomes also contain the enzyme catalase, which breaks down hydrogen peroxide either by converting it to water or by oxidizing another organic compound. A variety of substrates are degraded by such oxidative reactions in peroxisomes, including uric acid, amino acids, and fatty acids. The oxidation of fatty acids is a particularly important example as it is a major source of metabolic energy. In animal cells, fatty acids are oxidized in both peroxisomes and mitochondria, but in yeast and plants, fatty acid oxidation is restricted to peroxisomes. Peroxisomes are one of the main sites in the cell where oxygen free radicals are both generated and scavenged. The balance between these two processes is believed to be of great importance for the proper functioning of cells and has been linked to aging and carcinogenesis. The peroxisome is a single, membranebound organelle present in virtually every eukaryotic cell and biosynthetic pathways, however, these pathways may differ between species. The significance of the peroxisome for the normal regulation of cellular activities can be explained by the fact that more than 20 human peroxisomal disorders dwell because of the absence of protein or due to loss of protein function [4].

Antioxidants act either by neutralizing free radicals or their consequences [5]. The natural cellular environment provides sufficient protective pathways against unfavorable effects of free radicals: glutathione reductase, glutathione peroxidase, superoxide dismutase (SOD), disulphide bonding, thiols and thioredoxin are buffering systems in every cell. The relation between free radicals and disease can be explained by the concept of "Oxidative stress" [3].

All biological molecules present in our body are at risk of being attacked by free radicals. Such damaged molecules can impair the cell functions and even lead to cell death eventually resulting in disease states. Antioxidants may prevent and improve different diseased states [1, 2]. Several investigators have demonstrated the positive effects of antioxidants like Vitamin C, E to name a few in both preclinical and clinical setup.

#### **2. Oxidative stress as a marker of endothelial dysfunction**

The vascular endothelium, which promotes the passage of macromolecules and circulating cells from blood to tissues, is an important target of oxidative stress, playing a pivotal role in the pathophysiology of several vascular diseases and disorders. It is has been also reported that exclusively, oxidative stress increases vascular endothelial permeability and promotes leukocyte adhesion, which is coupled with alterations in endothelial signal transduction and redox-regulated transcription factors [6]. The reactive oxygen species (ROS) which originate at the sites of inflammation and injury have been emerged as the major contributing factor in the pathogenesis of endothelial

#### *Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights… DOI: http://dx.doi.org/10.5772/intechopen.105827*

dysfunction. These reactive oxygen species at low concentrations can function as signaling molecules participating in the regulation of fundamental cell activities such as cell growth and cell adaptation responses; whereas at higher concentrations, ROS can cause cellular injury and death. Predominantly, under normal body functions, the vital enzymes such as NAD(P)H dependent oxidases and superoxide dismutases (SOD) conscientiously regulate release of superoxide along with maintenance of intracellular redox balance. On the other hand, if production of superoxide anion surpasses the scavenging capacity of endothelial cells, this active intermediate react with nitric oxide resulting in formation of peroxynitrites. These peroxynitrites are potent oxidants that cause structural and functional changes in various components of the cell. ROS are also well known to suppress NO and restrict the formation of peroxynitrite [7]. It is a cytotoxic oxidant that causes endotheial dysfunction through nitration of protein function. Peroxynitrite plays a key role in oxidation of LDL and as proatherogenic [8]. In addition, peroxynitrite leads to the degradation of the eNOS cofactor tetrahydrobiopterin (BH4), resulting in an uncoupling of eNOS [9]. An excess of oxidant also leads to a reduction of BH4 with an increase in BH2. When this occurs, the formation of the active dimer of eNOS with oxygenase activity and the production of NO is restricted. The reductase function of eNOS is activated and more ROS are produced, so NO synthase switches from its NO-producing oxygenase function to its ROS-producing reductase function, with consequent exaggeration of oxidant excess and deleterious effects on endothelial and vascular function of the vessel wall. ROS upregulate adhesion (VCAM-1 and ICAM-1) and chemotactic molecules (macrophage chemoattractant peptide-1 (MCP-1) [8]. Inflammation decreases the bioavailability of NO [8]. The main source of oxidative excess in the vasculature is NAD(P)H oxidase and xanthine oxidase [10], the mitochondria [11] and uncoupled NOS constitute as other sources.

#### **3. Significance of antioxidants**

Many investigators have studied the significance of antioxidants in relation to disease and showed that zinc is an essential trace element, being a cofactor for about 200 human enzymes, including cytosolic antioxidant Cu-Zn SOD, isoenzyme of SOD mainly present in cytosol. Selenium is also an essential trace element and a cofactor for glutathione peroxidase. There is a vast information which suggests that chronic administration of antioxidants may be beneficial in improving cardiovascular risk. Vitamin E and tocotrienols (such as those from palm oil) are efficient lipid soluble antioxidants that function as a chain breaker during lipid peroxidation in cell memebranes and various lipid particles including LDL [12].

Vitamin E is considered the standard antioxidant against which other compounds with antioxidant activity are compared, particularly in terms of its biological activity and clinical relevance. Daily dietary intake varies between 400 IU and 800 IU. Vitamin C is also another important water-soluble free radical scavenger. The recommended daily dose is 60 mg. Apart from these, carotenoids like beta-carotene, lycopene, lutein and other carotenoids act as important antioxidants and quench superoxide (O2.) and (ROO..) [12, 13].

The effects of short-term dietary supplementation of tomato juice (source of lycopene), vitamin E and vitamin C on susceptibility of LDL to oxidation and circulating levels of C-reactive protein (CRP) and cell adhesion molecules measured in patients with type 2 diabetes. In this study 57 patients with well controlled type 2 diabetes

melliitus were randomized to receive tomato juice (500 mg/day), Vitamin E (800 U/ day), Vitamin C (500 mg/day) or placebo treatment for 4 weeks. It was observed that lycopene and vitamin E were both associated with resistance of LDL to oxidation, but only Vitamin E showed a decrease in C-reactive protein. It was also found that levels of cell adhesion molecules and plasma glucose did not change significantly during the study. Thses investigators then suggested that these findings may be relevant to strategies aimed at reducing risk of myocardial infarction in patients with diabetes [14].

In another study, it was suggested that vitamin E (1,600 IU/day, 10 weeks) decreased the susceptibility of LDL to oxidation in comparison with placebo. Vitamin E had this effect in both bouyant and dense LDL subfractions. This protection occurred in an environment where glycemic indices did not change and protein glycation was unaffected. The hypothesis that endothelial function and LDL oxidation might be linked was advanced further by Pinkney et al. [15]. These investigators studied 46 patients with type 1 diabetes without nephropathy and compared the results to 39 controls using a 3-month, randomized, double-blind, placebo-controlled study of vitamin E, 500 IU/day. The results indicated that in the absence of changes in LDL oxidation, vitamin E intake enhanced flow mediated dilatation FMD in type I diabetics [16].

Another [17] study reported that intracellular Vitamin C levels are reduced in patients with type 1 diabetes, particularly those who are poorly controlled. Histologically, the microvascular lesions of scurvy bear a surprising resemblance to those seen in long-standing diabetes, making them an attractive therapeutic alternative. Many short-term studies have shown the beneficial effects of ascorbic acid on vascular function, particularly in smokers and after ingestion of high-fat meals. However, the effect of vitamin C is not chronically sustainable, at least in smokers. Based on these observations, hypothesized that the antioxidant vitamin C might enhance endothelium-dependent vasodilation in forearm resistance when tested [18]. These investigators studied 10 subjects with diabetes and 10 age-matched, nondiabetic control subjects. FBF was determined by venous occlusion plethysmography, and endothelium dependent vasodilatation. The results from this study indeed support the hypothesis that acute administration of vitamin C improves endothelial function associated with the diabetic state., however, no information on chronic effects can be found from this study. But still more research has to be taken up in exploring the possible use of these vitamins to prevent atherosclerosis and/or microvascular disease in patients with diabetes. A study showed for the first time effects of consumption of flavonoid rich dark chocolate on endothelial function, aortic stiffness, wave reflections and oxidant status in healthy adults [19].

According to world health organization, traditional medicines are widely used globally. Approximately 80% of the population of developing countries rely on traditional medicines for their primary health care needs [20–22]. These medicinal plants contain several phytochemicals such as Vitamins (A, C,E and K), carotenoids, terpenoids, flavonoids, polyphenols, alkaloids, tannins, saponins, enzymes and minerals etc. These phytochemicals possess antioxidant activities, which can be used in the treatment of multiple ailments [23]. Many herbs along with potent antioxidant activity also possess anti-inflammatory and cardioprotective properties and are used by patients with increased risk of cardiovascular morbidity and mortality. Thus it is necessary to through light on the beneficial effects of the herbs such as *Terminalia arjuna, Emblica officinalis, Withania somnifera, Boerhaavia diffusa* and *Ocimum sanctum etc.*

#### **4. Preclinical studies with** *Terminalia arjuna*

#### **4.1 Antioxidant and anticancer activities**

The effect of Terminalia arjuna aqueous extract on the antioxidant defense system in lymphoma-bearing AKR mice was examined. The antioxidant effects of T. arjuna were monitored through the activities of catalase, superoxide dismutase and glutathione S-transferase. These enzyme activities are low in lymphoma-bearing mice, indicating an impaired antioxidant defense system. Oral administration of different doses of aqueous extracts of T. arjuna caused a significant increase in the activities of antioxidant enzymes. Here, T. arjuna was found to downregulate anaerobic metabolism by inhibiting lactate dehydrogenase activity in lymphoma-bearing mice, which was increased in untreated cancerous mice. The results demonstrated the antioxidant effects of Terminalia arjuna aqueous extract, which may play a role in anticarcinogenic activity by reducing oxidative stress [24].

#### *4.1.1 Cardio protective activity*

Sumitra et al., demonstrated that Arjunolic acid, a new triterpene and a potent principle from the bark of *Terminalia arjuna* has been shown to produce significant cardiac protection in isoproterenol induced myocardial necrosis in rats and prevents decrease in the levels of super oxide dismutase, catalase and reduced glutathione. This study explains that Arjunolic acid at a dosage of 15 mg/kg body weight (Pre and post treatment) produces cardioprotective effect [25].

#### *4.1.2 Antiplatelet activity*

Some researchers have shown that oleanane-type triterpene glycosides designated as Termiarjunoside I and Termiarjunoside II isolated from stem bark of *Terminalia arjuna*, potently suppressed the release of nitric oxide and superoxide from isolated macrophages and also inhibited aggregation of platelets [26].

#### **5. Clinical studies with** *Terminalia arjuna*

#### **5.1 Antioxidant and Cardioprotective activity**

The antioxidant constituents in Terminalia arjuna are reported to reverse endothelial dysfunction in chronic smokers. The study was conducted with 18 healthy male smokers and an equal number of non-smokers of the same age. The baseline brachial artery reactivity test was done using high-frequency ultrasound according to the standard protocol under identical conditions to determine endothelium-dependent flow-mediated and endothelium-independent nitroglycerinmediated dilatation. The two groups were matched for age, body mass index, blood pressure, serum cholesterol, mean resting vessel diameter, and flow velocities after occlusion. Smokers then received Terminalia arjuna (500 mg every 8 hours) or a matched placebo randomly in a double-blind, cross-over design for two weeks each, followed by repeated brachial artery reactivity studies to determine various parameters, including flow-mediated dilatation after each period. However, flow-mediated dilatation showed a significant improvement from baseline after Terminalia arjuna

therapy. The study concluded that smokers have impaired endothelium-dependent but normal endothelium-independent vasodilation as determined by brachial artery reactivity studies. In addition, two weeks of Terminalia arjuna therapy resulted in significant regression of this endothelial abnormality in smokers [27].

The effect of *Terminalia arjuna* (500 mg 8 hourly) was evaluated in fifty-eight males with chronic stable angina (NYHA class II-III) and with isosorbide mononitrate (40 mg/daily) on treadmill exercise induced ischemia, or a matching placebo for one week each. A wash-out period of at least three days was observed between the groups in a randomized, double-blind, crossover design. The treadmill exercise test parameters improved significantly during therapy with both treatments compared to those with placebo [28].

#### *5.1.1 E. officinalis*

The fruits of *E. officinalis* (Amla) family: Euphorbiaceae, commonly known as Indian gooseberry is widely used in many of the indigenous medical preparations against a variety of disease conditions [29].

*E. officinalis* is considered as a rich source of a vitamin C, which plays an important role in scavenging free radicals. For many years the therapeutic potential of fruits of *E. officinalis* was attributed to their high content of ascorbic acid [30]. It was further determined through comprehensive, chromatographic, spectroscopic and crucial chemical analyzes that the antioxidant property is due to the low molecular weight hydrolyzable tannins of fresh fruit skin. These tannins, namely Emblicanin A, Emblicanin B, Pedunculagin and Punigluconin, have been found to provide protection against oxygen radical-induced hemolysis of rat peripheral erythrocytes [31]. Purification and fractionation process was conducted in another study and phytochemicals like gallic acid, methyl gallate and geranin were identfied [32].

#### **6. Preclinical studies with** *E. officinalis*

#### **6.1 Antioxidant activity**

Invitro and animal studies have shown that Amla has potent antioxidant activity against multiple test systems such as superoxide radicals, induction of lipid peroxide formation by the Fe+3/ADP ascorbate system, hydroxyl radical scavenging activity. It also caused systemic increase in antioxidant enzymes in laboratory animals [33].

#### **6.2 Hypolipidemic activity**

In a study conducted in rats showed that flavonoids from *E. officinalis* effectively reduced lipid levels in serum and tissues and had significant inhibitory effect on hepatic 3-hydroxy-3-methylglutaryl-CoenzymeA (HMG CoA) reductase activity [34].

Effect of amla on the lipid metabolism and protein expression involved in oxidative stress during the aging process were evaluated in laboratory rats. Sun Amla or ethyl acetate extract of amla, a polyphenol-rich fraction, on oral administration significantly increased the hepatic PPAR [α] protein level. Furthermore, the amla extracts reduced the expressions of hepatic NF-[kappa] B, inducible NO synthase (iNOS), and cyclo-oxygenase-2 (COX-2) protein levels which were increased with

*Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights… DOI: http://dx.doi.org/10.5772/intechopen.105827*

aging. The results suggested that amla may prevent age-related hyperlipidaemia through attenuating oxidative stress in the aging process [35].

E. officinalis (Amla), showed improvement in treatment of dyslipidemia and intima-media thickening and plaque formation in the aorta in hypercholesterolemic rabbits [36].

#### **7. Clinical studies with** *E. officinalis*

#### **7.1 Hypolipidemic and anti-inflammatory activity**

In a pilot clinical study the effect of *E. officinalis* extract (AMLAMAX ™) was evaluated on markers of systemic inflammation and dyslipidemia. Amlamax™ a purified, standardized, dried extract of amla containing about 35% galloellagic tannins along with other hydrolysable tannins showed reduction in total and LDL cholesterols, in blood CRP levels and enhancement of beneficial HDL cholesterol [37].

#### **7.2 Hypoglycaemic activity**

The hypoglycemic and lipid lowering effects of *E. officinalis* fruits were evaluated in normal and diabetic patients. The data showed a significant decrease (p < 0.05) in fasting and 2 hour post- prandial blood glucose levels along with total cholesterols (TC) and triglycerides (TG) in both normal and diabetic volunteers upon 21 days of treatment [38].

#### **7.3** *W. somnifera*

*W. somnifera* (ashwagandha, WS) Family: Solanacae is widely used in Ayurvedic medicine, the traditional medical system of India and is an important medicinal plant, which is used in to cure many diseases. Some researchers have demonstrated that *W. somnifera* possesses powerful antioxidants. Preclinical studies also suggested the herb to produce an increase in the levels of natural antioxidants- superoxide dismutase, catalase and glutathione peroxidase [39].

#### **8. Preclinical studies with** *W. somnifera*

#### **8.1 Anti-inflammatory and Antistress activities**

Anti-inflammatory properties have been investigated to validate its use in inflammatory arthritis and animal stress studies have been performed to investigate its use as an antistress agent [40, 41].

#### **8.2 Hypoglycaemic and Hypolipidemic activities**

In a study flavonoids were isolated from the extracts of *W. somnifera* root and leaf and further hypoglycaemic and hypolipidemic effects were investigated in alloxaninduced diabetic rats. Eight weeks of treatment with *W. somnifera* and glibenclamide restored the changes in parameters to normal, indicating that it possesses hypoglycaemic and hypolipidemic activities [42].

#### **8.3 Anti-oxidant activity**

Researchers at Banaras Hindu University in Varanasi have discovered that some of the chemicals found in W. somnifera are powerful antioxidants. Studies conducted on rat brains showed that the herb increased the levels of superoxide dismutase, catalase, and glutathione peroxidase [39].

#### **8.4 Anti-carcinogenic activity**

The anti-carcinogenic property of Ashwagandga has been confirmed. Animal cell cultures has shown that the herb lowers [43] tumor size [44]. In another study, the herb was examined for its antitumor effects on urethane-induced lung tumors in adult male mice. After administration of ashwagandha for a period of seven months, the histological study of the lungs was similar to that observed in the lungs of control animals [45, 46].

#### **9. Clinical studies with** *W. somnifera*

#### **9.1 Hypoglycemic and hypolipidemic activity**

The hypoglycemic, hypocholesterolemic and diuretic effects of Ashwagandha has been studied in human clinical trials. A decrease in blood sugar levels comparable to that caused by the administration of a hypoglycaemic drug has been observed. Significant increases in urinary sodium, urine volume, and decreases in serum cholesterol, triglycerides, and low-density lipoproteins were also observed [46].

#### **9.2** *O. sanctum*

*O. sanctum* also known as Tulsi belonging to family: Labiatae and its extracts are used in ayurvedic remedies. The use of this herb has been reported in the Indian traditional medical system, and its modern uses receive widespread attention over the years. Various parts of the plant have been claimed to be valuable in a wide range of diseases. It has been observed that Tulsi exerts hypocholesterolemic, hypotriglyceridemic and hypophospholipidemic effects. Among the chemical constituents contained in essential oil of *O. sanctum* leaves eugenol, a phenolic compound, is considered to be an active ingredient contributing for its hypolipidemic and antioxidant action [47].

#### **9.3 Antioxidant and antineoplastic activity**

In a study at Bangladesh, the antioxidant activity of Tulsi leaves extract was evaluated invitro. *O. sanctum* extract showed significant free radical scavenging activity. In the same study, antineoplastic activity of *O. sanctum* was demonstrated against Ehrlich Ascites Carcinoma (EAC) in mice. Tulsi leaves extract was administered at a dose of 50mgKg−1 body weight intraperitoneally. Heamatological studies reveal that hemoglobin levels were reduced in EAC-treated mice, while near-normal recovery was observed in extract-treated animals. There was also a significant decrease in RBC count and an increase in WBC count in extract-treated mice compared to EAC-treated animals. From the results it was concluded that the extract has significant antioxidant and antineoplastic activity [48].

#### **9.4 Hypolipidemic activity**

In a study administration of fresh leaves of O. sanctum for four weeks resulted in significant changes in the lipid profile of normal albino rabbits. Significant reduction in serum total cholesterol, triglyceride, phospholipid and LDL cholesterol levels and an increase in stool HDL cholesterol and total sterol content were recorded [49].

Hypolipidemic activity of shade dried leaf powder of Tulsi along with the extracts and their fractions have shown *invitro* hypolipidemic and anti-peroxidative activity at very low concentrations in male albino rabbits. Aqueous extract feeding also provided significant protection of liver and aortic tissue from hypercholesterolemia-induced peroxidative damage [50].

#### **10. Protection against radiation induced lipid peroxidation**

A study was conducted to see if aqueous extract of *O. sanctum*, protects against radiation induced lipid peroxidation in liver and to determine the role, if any, of the inherent antioxidant system in producing radioprotection. Glutathione (GSH) and the antioxidant enzymes glutathione S-transferase (GST), reductase (GSRx), peroxidase (GSPx) and superoxide dismutase (SOD), as well as lipid peroxide (LPx) activity were estimated in the liver of adult swiss mice. The mice were injected intraperitoneally with 10 mg/kg of Tulsi for 5 consecutive days and exposed to 4.5 Gy of gamma radiation 30 min after the last injection. The aqueous extract itself increased GSH and enzymes significantly above normal levels, while irradiation significantly reduced all levels. The maximum drop was 30–60 min for GSH and related enzymes and 2 h for SOD. Pretreatment with the extract controlled the radiation-induced depletion of GSH and all enzymes and kept their levels within or above the control range. Irradiation significantly increased the lipid peroxidation rate, reaching a maximum value (about 3.5 times that of control) 2 hours after exposure. Aqueous extract pretreatment significantly reduced lipid peroxidation and accelerated recovery to normal levels [51].

#### **11. Clinical studies with** *O. sanctum*

In a study Tulsi leaves were tested on anthropometric measurements, diabetic symptoms and blood pressure in male patients with non-insulin dependent diabetes mellitus. Daily dosage of four capsules i.e. 2 g powder (Lunch and dinner) was given and supplementation was carryout for a period of 3 months. Significant percent reduction in the symptoms like polydypsia (35%), polyphagia (21%), and headache (27%), was observed in patients treated with Tulsi. It was concluded from the study that tulsi leaves are helpful in reducing subjects' diabetic symptoms and blood pressure. No significant improvement in subjects' anthropometric parameters was observed tulsi leaf powder supplementation [52].

#### **11.1** *Boerhaavia diffusa*

*Boerhaavia diffusa* is a medicinal plant widely used in Ayurvedic medicine. The plant was named in the honor of Hermann Boerhaave, a famous Dutch physician of the 18th century [53]. It is also known as Spreading Hogweed in English, belonging to family, Nyctaginaceae.

#### **12. Preclinical studies with** *Boerhaavia diffusa*

#### **12.1 Hypolipidemic activity**

The efficacy of antioxidant and hypolipidemic agents tocotrienols and *Boerhaavia diffusa* by analyzing all the parameters in plasma lipoprotein lipids, total lipids (TL), total cholesterol (TC), triglycerides (TG), VLDL-C, LDL-C, HDL-C and MDA in oxidized cholesterol feeded rats. In the same study invitro oxidizability of LDL, was also demonstrated. All the plasma lipid parameters and MDA levels were significantly increased in hyperlipidemic control rats. After 4 weeks of administration of tocotrienols and *Boerhaavia diffusa* significantly reduced the overall oxidative burden and effectively ameliorated the above altered parameters. Thus indicating a strong hypolipidemic/antiatherogenic and antioxidant effect [54].

#### **12.2 Anti-oxidant activity**

The root extracts of *Boerhaavia diffusa* were evaluated using different solvents for free radical scavenging activity (FRSA) at a dose of 1000 mg/Kg body weight, prior to irradiation with 8 Gy gamma radiation as compared to mice pre-treated with extract at the dose of 250 and 500 mg/Kg body weight prior to irradiation with same dose of radiation. The data obtained showed that hydroethanolic extract produced potent free radical scavenging activity in DPPH**.**, ABTS**.+** and NO**.** assays and was found to be beneficial in reducing symptoms of radiation sickness, changes in body weight and mortality were minimum in the experimental animals. The antioxidant effect of *B. diffusa* roots was attributed to the presence of certain phenolic constituents like quercetin, caeffic acid, kempferol and their derivatives [55].

#### **12.3 Hypoglycemic activity**

Another study focused on blood glucose concentration and hepatic enzymes in normal and alloxan induced diabetic rats after daily oral administration of aqueous solution of *Boerhaavia diffusa* L. leaf extract (200 mg/kg) for 4 weeks. Significant improvement was recorded in blood glucose and glycosylated hemoglobin A1C levels. The action of hepatic enzymes such as hexokinase was significantly increased. Similarly, glucose-6-phosphatase, fructose-1,6-bisphosphatase were significantly decreased by the administration of BLEt in normal and diabetic rats. The results of BLEt were more potent when compared with antidiabetic drug—glibenclamide (600 μg/kg) [56].

#### **12.4 Hepatoprotective activity**

The hepato-protective activity of *Boerhaavia diffusa* alcoholic extract of the whole plant administered orally, was evaluated against experimentally induced *Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights… DOI: http://dx.doi.org/10.5772/intechopen.105827*

hepatotoxicity using carbon tetrachloride in rats and mice. The extract also produced an increase in normal bile flow, indicating potent choleretic activity and no signs of toxicity were observed up to an oral dose of 2 g/kg.

#### **13. Clinical studies with** *Boerhaavia diffusa*

#### **13.1 Antioxidant and Hypolipidemic activity**

The effects of methanolic extract of Boerhaavia diffusa on oxidative stress in healthy and diabetes mellitus patients were studied. Results through this research demonstrated that diabetic patients experience increased oxidative stress when compared with normal subjects, significant increase in plasma, TG, TC, VLDL-C, LDL-C, and decrease in HDL-C. This may be due to markedly increased production of oxidant and significantly diminished antioxidant defense including a decline in total plasma antioxidant power. The study depicted that daily intake of *Boerhaavia diffusa* extract by diabetes mellitus patients significantly reduced TC, TG, LDL-C and increased HDL-C levels. The study concluded that extract of *Boerhaavia diffusa* may be useful in the prevention and treatment of the diabetes-induced hyperlipidemia and atherosclerosis. In addition, daily use of *Boerhaavia diffusa* can be efficacious and cost effective and good source of natural antioxidant [57].

#### **14. Discussion**

Over the past three decades, various experimental startegies have revealed the existence of cellular functions of peroxisomes related to reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the function of peroxisomes as key centers of the cellular signaling apparatus. Peroxisomes of different origins have been detected which strongly indicate the interest of them as a cellular source of various signaling molecules, including ROS. In this review, we have focused on the generation and regulation of ROS in peroxisomes and the different antioxidant systems in this cell organelle [58]. We also enlighten the supporting evidence for application of antioxidants in preclinical and clinical evaluation of herbal supplements used in the management of associated disease complications.

Uncontrolled ROS production leads to structural modification of cellular proteins and alteration of their functions, resulting in cellular dysfunction and dysregulation of important cellular processes [59, 60]. Enhanced levels of ROS cause lipid, protein, and DNA damage. Specifically ROS can distort the lipid membrane and increase the fluidity and permeability of the membrane. Impairment of protein includes site-specific amino acid modification, peptide chain fragmentation, aggregation of crosslinked reaction products, modification of electric charges, immobilization of enzymes, and sensitivity to proteolysis [61]. Eventually, ROS can damage DNA by oxidizing deoxyribose, strand breakage, removal of nucleotides, changes in bases, and crosslinking DNA protein [62–65].

Literature suggests that peroxisomes are powerful and metabolically active organelles and are a very vital source of reactive oxygen species (ROS), H2O2, O2 (.-) and · OH, which are the products of diverse metabolic pathways, such as, photorespiration, fatty acid β-oxidation, nucleic acid and polyamine catabolism, ureide metabolism, to name a few. ROS were originally associated with oxygen toxicity. However, these

reactive species also play a significant role in the signaling network that regulates essential processes in the cell. Peroxisomes have the ability to produce and scavenge H2O2 and O2(.-) rapidly, allowing to regulate dynamic alterations in ROS levels. The flexibility of these organelles, and based on varied developmental and environmental stimuli, render these organelles to perform a pivotal role in cellular signal transduction. The catalase and glycolate oxidase loss-of-function mutants have provided insights to study the consequences of modifications in endogenous H2O2 levels in peroxisomes. This has also facilitated the understanding of transcriptomic profile of genes regulated by peroxisomal ROS. It is now well established that peroxisomal ROS are involved in complicated signals which employ hormones, calcium, and redox homeostasis [66].

Antioxidants render an important role in these defense mechanisms. The antioxidat therapies target for maintenance of critical balance between oxidants and proxidants. In aerobic organisms, the steady release of free radicals needs to be equalized at the same degree of utilization of antioxidant. The naturally occuring enzymatic or non-enzymatic antioxidant systems prevent the formation of free radicals, and neutralize or repair the damage caused by them [62]. A wide range of endogenous and exogenous antioxidants are responsible, for providing protection against oxidative damage leading to development of chronic diseases [67]. The different types of antioxidant systems present both in plants [68] and the human body, contributes for controlling ROS homeostasis [69]. Release of natural ROS by the mitochondrial respiratory chain suggests that under certain conditions ROS can be metabolically beneficial but at the same time may also be harmful to cells [70–72].

The plant kingdom has served the mankind since ancient time and has provided remedies for various disease conditions. Over the period of time as the knowledge of plant derived medicines got advanced, it opened new avenues in improving the health and quality of life. Since many centuries, herbal drugs have been used both as food supplements and for medicinal requirements. When we mention about herbal medicine, it constitutes all parts of the plant like seeds, roots, bark, leaves, flowers and fruits from trees [73]. Most of the plant derived products and herbs act as potent scavengers of ROS or possess antioxidant activity. The phytoconstituents present in these herbs have been evaluated in numerous studies and are proved to rapidly stimulate the natural antioxidant enzyme systems such as catalase, superoxide dismutase, reduced glutathione etc., which protect the cells from oxidative damage and from progression of chronic diseases [74].

#### **15. Conclusion**

The conclusion of the present review is that peroxisomes are most common type of single layered membrane organelles identified in different types of eukaryotic cells. The origin of peroxisomes are through growth and division of cell and are independent organelles. These are also recognized as one of the most important and strong multifunctional organelles. The peroxisomes are able to facilitate the dynamic rotation of ROS generation and removal, fatty acid oxidation, β-oxidation of long-chain fatty acids, decomposition of purines, and glycerol, ether lipid and bile acid biosynthesis [75]. The metabolic processes of peroxisomes those which take place together with mitochondrial involvement are fatty acid β-oxidation and amino acid metabolism, but whereas the oxidation of different substrates is promoted by oxidases that consume oxygen. Several investigators in their work indicate the ability

#### *Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights… DOI: http://dx.doi.org/10.5772/intechopen.105827*

of peroxisomes to utilize 20% of the total oxygen consumption and can release up to 35% of the cellular hydrogen peroxide due to which are known to be major contributors of oxidative metabolism and in conserving oxidation balance [76]. Evidences from research show that the regulation of cell proliferation, apoptosis and carbohydrate metabolism is governed by hydrogen peroxide that act as a vital signaling molecule. However, at increased levels hydrogen peroxide is toxic and requires a regular check for its concentration. The other vital function of peroxisomes includes the action of antioxidant enzyme systems like the CAT, SOD, PRDX1, and PRDX5 [77–80]. CAT being the most significant enzyme and other antioxidant enzymes which metabolize the peroxidase hydrogen produced as a byproduct of peroxidases. Additionally super oxide dismutase 1 (SOD1) is regarded as a perfect peroxisomal protein, and a potent antioxidant enzyme that quenches the superoxide and accelerates the modification of oxygen to superoxide anion (O2− ) [81].

Plant-based bioactive molecules have received a lot of recognition since the past few decades. Several studies have demonstrated their therapeutic significance in the management of disease conditions and for prevention as well. The complete phytochemical profile of whole plants, plant extracts or even the isolated constituents are well explained in the literature, which can be utilized for planning treatment strategies for various diseases including diabetes mellitus, cardiovascular disease and neurodegenerative disorders. Extensive randomized trials are warranted to collect data for establishing the medical interest or probable hazards of antioxidant supplementation.

There is enormous substantiation that oxidative stress has been implicated in normal physiological processes and environmental interactions that occur in a cell. Several mechanisms are involved in antioxidant defense systems that render protection against oxidative damage. Literature suggests that in many conditions these processes seem to be tangled. ROS profusely disrupts the antioxidant balance, causing oxidative stress and results in constant alterations in the cellular material, which includes carbohydrate, protein and lipid substances [70, 82–84]. It is likely to presume that oxidative stress can be a cause of tissue damage and finally arresting the natural cellular-signaling processes. However, a thorough understanding of the biochemical events occurring at a cellular level to influence oxidative damage is mandatory to direct ensuing progress. Peroxisomes serve as very important sites for detoxification of ROS. But however peroxisomes itself release these radicals. With the advent of fluorescence methods and having vast knowledge of peroxisomal functions, we expect to read more about the role of this organelle in the near future that can be useful in the treatment of related disorders.

#### **Acknowledgements**

I thank Al Hawash Private University, Homs, Arab Republic of Syria, for providing the necessary support in completion of this chapter.

*The Metabolic Role of Peroxisome in Health and Disease*

#### **Author details**

Nishat Fatima Al Hawash Private University, Al-Mouzeina, Syria

\*Address all correspondence to: nishat\_fatima50004@yahoo.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.

*Association of Peroxisomes, Reactive Oxygen Species (ROS) and Antioxidants: Insights… DOI: http://dx.doi.org/10.5772/intechopen.105827*

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#### **Chapter 5**

## Peroxisomal Modulation as Therapeutic Alternative for Tackling Multiple Cancers

*Shazia Usmani, Shadma Wahab, Abdul Hafeez, Shabana Khatoon and Syed Misbahul Hasan*

#### **Abstract**

Peroxisomes are indispensably involved as a central player in the metabolism of reactive oxygen species, bile acids, ether phospholipids, very-long-chain, and branched-chain fatty acids. The three subtypes of PPARs are PPAR-alpha, PPARdelta, and PPAR-gamma which have been found to be instrumental in the control of cancer metabolism cascades. Any disproportionate expression of PPAR can lead to the progression of cell growth and survival in diverse types of cancers. It can be exploited both as an agonist or antagonist for utilization as a potential therapeutic alternative for the treatment of cancer. Therefore, the multifunctional PPAR modulators have substantial promise in various types of cancer therapies. Many recent studies led to the observations that a variety of phytochemicals, including phenolics, have been implicated in anticancer effects. Plant phenolics seem to have both palliative and treatment opportunities in combating cancer which requires deep insight into the proposed mechanisms. Henceforth, this chapter highlights the role of peroxisomal subtypes as an activator or suppressor followed by its modulation through bioactive obtained from a variety of crude drugs. A discussion on various challenges restricting proper utilization has also been incorporated.

**Keywords:** peroxisome, metabolism, PPARs, herbal, cancer

#### **1. Introduction**

Peroxisomes are small membrane-bound organelles with simple structures but contain enzymes that display a wide range of metabolic activities. About 50 peroxisomal enzymes have been identified where major [1, 2] pathways for metabolism involve α- and β-oxidation of fatty acids, biosynthesis of ether lipids, polyamines, D-amino acids, glyoxylate, and purines. The synthesis and assembly of peroxisomal proteins occur on free ribosomes which are then imported into these tiny organelles as completed polypeptide chains. The disorders related to peroxisomal functions can be attributed to a disturbance in the formation of the organelles or might be related to defects in either a particular peroxisomal enzyme or a related transporter [3, 4]. The metabolic disorders promote the accumulation of substrates that are usually degraded by specific peroxisomal enzymes. A variety of clinical symptoms has proven to be very severe leading to an early death.

#### **2. Metabolic implications of peroxisomes, a druggable target**

Peroxisome-related homeostatic balance is an indispensable mechanism of health where the removal of worn-out and defective peroxisomes occurs through autophagy. Association with mitochondria is reflected when commotion of peroxisomal function results in disruption of mitochondrial function. The impaired peroxisomal function has been found to be instrumental in special conditions of neurodegenerative disorders and diabetes, while dysregulation in peroxisomal function can result in cancer [5, 6]. There has been increasing evidence linking peroxisomal misregulation to the eruption of several diseases which potentiate an elevated possibility of targeting peroxisomal involvement in disease prevention or treatment.

Peroxisomes are amazingly active organelles, which have an important role in lipid and hydrogen peroxide metabolism making them elemental for human health [7]. Despite great advances in identification of essential components and related molecular mechanisms, an understanding of the process by which peroxisomes are incorporated into metabolic pathways is of elementary importance. The interaction of peroxisomes with other subcellular compartments, metabolic co-operations, peroxisome–peroxisome interactions, and the interaction of peroxisomes with microtubules needs to be addressed to utilize this information directly to combat the process of disease development.

Peroxisomes are consigned to clearing up the reactive oxygen chemical debris cast off by other organelles, where their functions extend far beyond hydrogen peroxide metabolism [8]. Peroxisomes are closely associated with mitochondria, and their ability to carry out fatty acid oxidation and lipid synthesis may be highly implicated in generating cellular signals required for normal physiology. The biology of peroxisomes and their relevance to human disorders, including cancer, obesity-related diabetes, and degenerative neurologic disease cannot be undermined [9].

Peroxisomes are multifarious where they invariably modulate the metabolism of reactive oxygen species and primary homeostatic mechanisms, such as oxidation of fatty acid, synthesis of bile acid, and transport of cholesterol. Henceforth, it is implicative that peroxisomal homeostasis is an important regulator of health, and disruption of peroxisomal function can lead to mitochondrial dysfunction, reflecting the intimate link between the two organelles [10].

The impaired peroxisomal function leads to neurodegenerative disorders and diabetes, but dysregulation may have far-reaching effects, such as the development of cancer [11]. The peroxisomal function is also transformed with aging owing to deviations in the expression and/or localization of peroxisomal matrix proteins.

The homeostatic mechanisms of peroxisomes are undermined by the existence of distressing genetic disorders attributed to impaired peroxisomal function. However, with amplified evidence connecting peroxisomal dysfunction to the pathogenesis of these acquired diseases, it can be utilized as a druggable target in disease prevention or treatment [12].

The immune system evasion is one of the mainstays of cancer, and peroxisomes have an indispensable role in the regulation of cellular immune responses. *Peroxisomal Modulation as Therapeutic Alternative for Tackling Multiple Cancers DOI: http://dx.doi.org/10.5772/intechopen.104873*

Investigations of individual peroxisome proteins and metabolites provide for their pro-tumorigenic functions [13]. It is, therefore, important to highlight new advances in our understanding of biogenesis, enzymatic functions, and autophagic degradation of peroxisomes, which shall avail enough evidence to link such activities to tumor development. Such findings shall add to the possibility of exploitation of peroxisomerelated processes for efficient battling against cancer.

#### **3. Peroxisome proliferator-activated receptors (PPARs)**

With the above, emerging evidence, exploring the possible sites of activation of peroxisomal receptors could be intriguing with respect to the benefit and risk ratio.

In this context, it was deduced that activation of peroxisome proliferator-activated receptors (PPARs) can be considered an efficient strategy for the treatment of metabolic dysregulation [14]. An ample of new moieties having the prospects to stimulate peroxisome proliferation have been discovered in the recent past.

The receptor which was cloned from a mouse liver, and titled a peroxisome proliferator-activated receptor (PPAR) could regulate the expression of sizable genes involved in the regulation of glucose and lipid metabolism [15].

Besides, the ligands which activate PPARs lead to the promotion of co-activators and inhibition of co-repressors remodeling the chromatin and initiating transcription [16].

#### **4. Metabolic regulation by PPARs and their repercussions**

The peroxisome proliferator-activated receptors (PPARs) are a set of nuclear receptors namely PPAR gamma, PPAR alpha, and PPAR delta, encrypted by diverse genes. PPARs are ligand-regulated transcription factors regulating gene expression by binding to specific response elements (PPREs) within promoters. PPARs bind as heterodimers with a retinoid X receptor and, upon binding agonist, interact with cofactors such that the rate of transcription initiation is increased [17].

The PPARs are major regulators of lipid metabolism where fatty acids and eicosanoids have been recognized as common ligands. Synthetic PPAR ligands, such as fibrates and thiazolidinediones, have been effectively used in the treatment of dysregulation of lipids and glucose metabolism.

The discovery of these ligands led to the disclosure of many impending functions for the PPARs in pathological metabolic situations, such as demyelination, atherosclerosis, and cancer [18].

#### **4.1 Peroxisome proliferator-activated receptor-alpha (PPAR-α)**

It has been recognized as the nuclear receptor for a class of rodent hepato-carcinogens leading to the proliferation of peroxisomes. PPAR-α is a transcription factor that happens to be the major regulator of lipid metabolism in the liver [19].

It is primarily activated via ligand binding where fatty acids, such as arachidonic acid and their metabolites from the ligand groups. Another category consists of synthetic ligands, such fibrate drugs referred to as peroxisome proliferators [20].

#### **4.2 Peroxisome proliferator-activated receptor beta or delta (PPAR-β or PPAR-δ)**

PPAR- δ is a nuclear hormone receptor that manages diverse biological processes involved in the progression of several chronic ailments, viz. obesity, atherosclerosis, and cancer [21].

PPAR-δ act as an integrated unit for transcription regulation and nuclear receptor signaling. It stimulates the transcription of a wide variety of target genes by binding to specific DNA elements.

Many fatty acids and their derivatives induce PPAR δ viz. arachidonic acid and its metabolites [22].

#### **4.3 Peroxisome proliferator-activated receptor gamma (PPAR-γ)**

PPAR-γ or the glitazone reverse insulin resistance receptor, is a type II nuclear receptor that is encoded by the PPAR-γ gene in humans [23, 24]. The protein encoded by this gene is PPAR-γ, which regulates the differentiation of adipose cells [25]

When the activity of PPAR-γ is regulated via phosphorylation through the MEK/ERK pathway, it results in decreasing transcriptional activity of PPAR-γ. The result is a loss of insulin sensitivity due to diabetic gene modifications. Owing to the above reasons, PPAR-γ has been implicated in the pathology of numerous diseases, including obesity, diabetes, atherosclerosis, and cancer [26].

PPAR-γ controls fatty acid storage and metabolism of glucose. The genes activated by PPAR-γ stimulate lipid uptake and adipogenesis by fat cells. The agonists have been reported to be used in the treatment of hyperlipidemia and hyperglycemia [27].

PPAR-γ decreases the inflammatory response of many cardiovascular cells. PPAR-γ activates the paraoxonase-1 gene, resulting in an increase of paraoxonase 1 in the liver, which reduces the incidence of atherosclerosis [28].

The prevalence of metabolic syndromes is growing in the adult and pediatric groups which include majorly atherogenic dyslipidemia raised blood pressure, and pre-eminent plasma glucose [29].

Peroxisome proliferator-activated receptors (PPARs) may come up as potential therapeutic targets for the treatment or prevention of metabolic syndromes. Further, there is substantial evidence that its agonists are, therefore, used in the treatment of metabolic syndrome and cardiovascular diseases [30].

Activation of peroxisome proliferators-activated receptor (PPAR) is invariably indulged in varied mechanisms related to lipid profile.

One of the researches in this area confirmed the role of herbs in the stimulation of PPARα. Among the tested plant extracts, about nine had shown moderate PPARα transactivation [31]. The bioactive, piperine, and capsaicin revealed substantial transactivational activities followed by a moderate activity in chalcones. It was concluded that a diet rich in natural products viz. herbs, act as PPARα agonists improving the lipid profile.

#### **5. Proposed mechanisms of PPARs in tumor suppression**

#### **5.1 Distressing metabolism**

PPAR ligands disturb the survival of cancer cells in such a way that the metabolism enters into complete devastation. Owing to the potential of PPAR ligands, they are been considered a potential source of anticancer agents, with minimal toxicities [32].

*Peroxisomal Modulation as Therapeutic Alternative for Tackling Multiple Cancers DOI: http://dx.doi.org/10.5772/intechopen.104873*

PPAR activation disrupts the metabolism of cancer cells mainly by blocking the synthesis of fatty acids and promoting fatty acid oxidation. Owing to nutrient depletion, in the tumor microenvironment, PPAR coordinates with AMP-dependent protein kinase in repressing oncogenic Akt activity, inhibiting cell proliferation, and inducing glycolysis-dependent cancer cells into "metabolic failure" [33].

There is substantial evidence for the antiproliferative role, and prevention of metastatic indulged by PPAR ligands, which prompts a detailed compilation on the possible potential of PPAR in tumor suppression [34].

#### **5.2 PPAR subdues cell proliferation by overpowering inflammation**

Suppression of inflammation is another mode contributing to anticancer effects. PPAR takeover the inflammation and activation of uncoupling proteins, which wanes the mitochondrial ROS generation and resultant cell proliferation. PPAR ligands can be considered as a low-toxic and well-tolerated therapeutic moiety to combat cancer [35].

The peroxisome proliferator-activated receptor γ ligands exhibited anticancer activity *in vitro*, against diverse neoplastic cells *whereas* animal studies also reflected that they are *in vivo* anticancer effects and chemopreventive proficiency. The effect may be attributed to slowing down the growth and induction of partial differentiation of several cancer cells, such as lipo-sarcoma, and cancers, such as colon, prostate, and breast cancers [36].

At the molecular level, these can decrease the levels of cyclin D1 and E, nuclear factor κB, and inflammatory cytokines. Some relevant data support the fact that PPAR γ might act as a gene for tumor suppression. On the other hand, several captivating pieces of evidence, suggest that under certain specific settings, PPAR γ ligands can lead to cancer [37].

Yet, the bulk of studies still reflects the fact that PPAR γ ligands bear antiproliferative potential against numerous transformed cells and may be applied in adjuvant treatments strategies for several common tumors [37–39].

As per research by Morinishi et al., activation of PPAR-*α* seems to be involved in the control of colorectal carcinomas, where nuclear expression of PPAR-*α* may be established as an indispensable therapeutic target for the respective treatment of the disease. It was deduced that the nuclear expression of PPAR-*α* was significantly higher in subtly differentiated adenocarcinoma than in mucinous adenocarcinoma [40].

Colorectal cancer poses one major threat due to excessive dietary fat posing as a major threat. As it is involved in the regulation of lipid and carbohydrate metabolism, it needs to be studied extensively in this case [41]. Despite the fact, that researchers have scrutinized the expression and clinical repercussions of PPARs in colorectal cancer, the exact mechanism needs to be further explored.

Diverse studies have been undertaken, focusing on the assumed link between the polymorphisms and mutations of the PPAR *γ* gene with the incidence of cancer [42, 43].

Ikezoe et al. [44] analyzed 397 clinical samples and cell lines, including colon, breast, and lung cancers for mutations of the PPAR *γ* gene. They indicated the absence of PPAR *γ* gene mutations in the tested cell lines ascertaining PPAR *γ* mutations may occur in cancers but very rarely.

There has been substantial experimental data supporting that synthetic PPAR *γ* ligands induce apoptosis in several types of cancer cells [45, 46]. Albeit, the majority of the evidence has documented that PPAR *γ* agonists inhibit growth in cancer

cells but the mechanism of the growth inhibition by PPAR *γ* agonists is not well understood and complicated.

#### **5.3 Differential behavior of peroxisome**

Specific tumors behave variably in terms of peroxisomal activity. It has been observed, thus, that the enzymatic activities of peroxisomal metabolism decline in the breast [47], colon [48], and hepatocellular carcinomas [49]. Similar observations were recorded in renal cell carcinoma [50]. In a related finding, von Hippel-Lindau (VHL)-deficient clear cell renal carcinoma displayed reduced peroxisomal activity. In contrast to this, some reports reveal that peroxisomal metabolic activities lead to enhancing the growth of tumors [51]. Few cancer cells count on peroxisomal lipid metabolism for energy and support the survival of cancer cells in the tumor microenvironment [52]. This controversial behavior of peroxisome indicates the fact that under a certain specific environment, it promotes or diminishes cancer growth, which may be attributed to the type of tumor. In this regard, it is implicative to further investigate the inducing factors that decide the fate of the metabolism of peroxisomes, closely related to its cancer proliferation effects.

Many studies have been undertaken to study the potential of the combinatorial approach where PPAR agonists can be used for the treatment of resistant cancers [53].

In research by Kaur et al., the probable effect of selective agonism by PPAR gamma receptors was studied for radiation therapy in non-small-cell lung carcinoma [54]. The agonist used was Rosiglitazone. A reasonably significant increase in the intensities of radiation-induced apoptosis was detected in H1299 cells attributed to enhanced PPARG expression. Consequently, it was deduced that PPAR gamma agonism stimulates the radio-sensitizing effect.

Another investigation was undertaken on the expression of PPAR gamma in human normal cervix and cervical carcinoma tissues. The effect of PPAR gamma ligands on the sustenance of cervical cancer cells was also an aim. It was observed that the PPAR gamma protein expression, was lessened in cervical carcinoma in comparison to normal cervical tissues [55].

Similar results were revealed using the effect of Ciglitizone on cell proliferation, which reflected noteworthy growth inhibition on human cervical cancer cell lines, C-33-A and C-4II. It further added to the substantial evidence for the role of PPAR in multiple human cervical cancer tissues and cell lines where a downregulation is encountered [56]. Several *in vitro* studies validated those high levels of free fatty acids induce the proliferation, migration, and invasion of prostate cancer cells (PC3 and 22RV1). Therefore, to test the fact, an assessment was done for serum lipid levels in patients suffering from prostate cancer in comparison to normal individuals. It was concluded that high levels of free fatty acids promote cancer by upregulation of expression in PPAR γ [57].

The fact potentiated was that obesity is undoubtedly an important risk factor, resulting in upregulation of PPAR gamma, consequently leading to incidence and progression of PCa [58]. In an interesting work, the expression of PPAR γ was studied in epithelial cells in the colon. There was a differential expression of PPAR in different segments of the colon. Specifically, in the cell lines, Caco-2, and HT-29 human adenocarcinoma cells, PPAR γ expression was amplified upon differentiation. A significant role was observed as reflected in the amplified expression of PPAR γ was observed in the colon (**Table 1**) [73, 74].

*Peroxisomal Modulation as Therapeutic Alternative for Tackling Multiple Cancers DOI: http://dx.doi.org/10.5772/intechopen.104873*


#### **Table 1.**

*Role of bioactive in the modulation of PPARs for treatment of various cancers.*

#### **6. Challenges in anticancer strategies of peroxisomes**

To exploit the peroxisomal metabolism for anticancer approaches, several constraints need to be addressed in the first place [75]. Foremost, it should be assessed for its possible side effects on non-malignant cells as overhauling the metabolism might result in serious side effects. Next, the concern might be the differential behavior of the diverse cancer cells and their lineage [76].

The tumor heterogeneity is anyhow allied with differential metabolic activities. In this context, the selection of the study group may be very crucial [77]. It may also affect the prerequisite of peroxisomal functions in a specific subset of tumors [78].

#### **7. Discussion**

PPARs, commonly known as modulators of genetic expression, exhibit variant tissue expression depending upon differential microenvironment and have thus attracted a lot of attention whether singly or in a combination strategy [25, 79].

This process can then activate the transcription of various genes involved in diverse physiological and pathophysiological processes that play main roles in the pathogenesis of several chronic diseases, such as atherosclerosis [80], diabetes [81], liver disease [82], cardiovascular diseases [83], and cancer, involving inflammatory effects and their corresponding clinical implications [84].

#### **8. Conclusions**

Though therapeutic approaches to target peroxisome metabolism in cancer have been on a rise and pursued very closely by keen researchers, not many modulators have been assessed completely. Henceforth, well-defined *in vivo* models have to be investigated for the potential of peroxisome inactivation to suppress cancer progression. The differential behavior of peroxisomes in different microenvironments will help to facilitate the development of a higher number of effective drugs for the modulation of peroxisomal functions [85].

Peroxisome metabolism has been invariably linked to the functions of organelles, viz. endoplasmic reticulum and mitochondria [86, 87]. The disruption of the association between the organelles and peroxisomes refurbishes the cancer cell metabolism. Further, it can be ascertained, that probable peroxisome targeting with drugs that inhibit the related organelles may lead to amplified anticancer mechanics [75].

It can be useful where targeting peroxisomes might enhance the targeting of other metabolic pathways in cancer. It, however, remains unveiled whether the alteration of peroxisome metabolism is a consequent event due to alterations in metabolism due to cellular changes during cancer or bears a prime position in the development [88].

However, it can never be disregarded that ample research has substantiated the potential of peroxisomes as absolute cancer targets while further exploration role of peroxisome metabolism in the genesis of tumors might prove to be a curtain-raiser. A rational approach to drug design can be attained by the revelation of the regulatory machinery and transcriptional focus of the PPARs. Focused research in this direction may provide a perfect perception of the development of metabolic diseases including cancer.

*Peroxisomal Modulation as Therapeutic Alternative for Tackling Multiple Cancers DOI: http://dx.doi.org/10.5772/intechopen.104873*

#### **Acknowledgements**

The authors are thankful to the Chancellor, Integral University, Lucknow-226026, Uttar Pradesh, India for his sustained encouragement, meticulous supervision, and valuable suggestions at all stages of completion of this chapter. The authors are thankful to the Faculty of Pharmacy, Integral University for providing all the necessary facilities related to the present work. The authors are also thankful for the research cell, Integral University.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Shazia Usmani1 \*, Shadma Wahab2 , Abdul Hafeez1 , Shabana Khatoon1 and Syed Misbahul Hasan1

1 Faculty of Pharmacy, Integral University, Lucknow, India

2 Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia

\*Address all correspondence to: shazia@iul.ac.in

© 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 6**

## Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise with Obestatin on Lipid Profile of DIO-C57BL/6 Mice

*Beekanahalli G. Mallikarjuna and Uma V. Manjappara*

#### **Abstract**

Obestatin and its fragment analog Nt8U were shown to upregulate glycerolipid metabolism and PPARg signaling and decrease fat accumulation in Swiss albino mice. It was further investigated if these peptides could decrease lipid accumulation under obese conditions. We chose to work on Diet-Induced-Obese (DIO) C57BL/6 mice to study the same. Both obestatin and Nt8U decreased lipid accumulation in DIO-C57BL/6 mice. PPARg was not upregulated in comparison to 60% high-fat diet (HFD) fed control mice, implying there was already enhanced PPARg expression due to HFD consumption. We also wanted to investigate if upregulation of PPARg signaling was a secondary effect of enhanced glycerolipid metabolism. To investigate the same, we administered obestatin pairwise with 2 agonists and 2 antagonists of PPARg. The results revealed obestatin is not a mere agonist of PPARg but can also decrease lipid accumulation brought about by rosiglitazone, a well-studied agonist of PPARg. The antagonists also show a further decrease in lipid accumulation, probably due to inhibition of PPARg activity brought about by HFD and the additive decrease brought about by obestatin in DIO-C57BL/6 mice. This chapter will be structured to briefly introduce obestatin, Nt8U, their effect on gene expression in the adipose tissue, and the effect of PPARg agonists and antagonists on their ability to reduce fat accumulation.

**Keywords:** DIO-C57BL/6, obestatin, rosiglitazone, GW9662, T0070907

#### **1. Introduction**

Obestatin is a regulatory peptide discovered in 2005 by Zhang et al. It was predicted to be a protease cleavage product of preproghrelin. The 23-residue peptide was isolated and characterized for its activity. It was shown to decrease food intake and body weight in rodents. They also continued to show GPR39 an orphan G-protein coupled receptor could be the cognate receptor for obestatin [1]. These claims have been debated upon

since [2]. More recent research shows GLP-1 receptor could be mediating its activity [3]. In our laboratory, we synthesized 3 overlapping fragments of obestatin and showed the N-terminal 13 residues mimicked the parent peptide obestatin the best [4]. Subsequently, we synthesized two analogs of the N-terminal peptide with alphaaminoisobutyric acid (Aib, denoted as U) at position 8 replacing a glycine (Nt8U) and cyclohexyl amino acid (Cha) at position 5 replacing phenylalanine (Nt5Cha). Experiments in Swiss albino mice showed Nt8U to be as active as obestatin in its ability to reduce fat accumulation [5]. Obestatin and its fragment analog Nt8U were shown to upregulate glycerolipid metabolism and PPARg signaling and decrease fat accumulation in Swiss albino mice [6]. It was further investigated if these peptides could decrease lipid accumulation under obese conditions. We chose to work on Diet-Induced-Obese (DIO) C57Bl/6 mice to study the same. Both Obestatin and Nt8U decreased lipid accumulation in DIO-C57BL/6 mice. PPARg was not upregulated in comparison to 60% high-fat diet (HFD) fed control mice, implying there was already enhanced PPARg expression due to HFD consumption [7, 8].

We also wanted to investigate if upregulation of PPARg signaling was a secondary effect of enhanced glycerolipid metabolism. To investigate the same, we administered obestatin pairwise with 2 agonists and 2 antagonists of PPARg. The results revealed obestatin is not a mere agonist of PPARg but can also decrease lipid accumulation brought about by rosiglitazone, a well-studied agonist of PPARg [9]. The antagonists also show a further decrease in lipid accumulation, probably due to inhibition of PPARg activity brought about by HFD and the additive decrease brought about by obestatin in DIO-C57BL/6 mice [5]. The effect of antagonists of PPARg in DIO-C57BL/6 mice will be discussed in this chapter concerning DIO and Rosiglitazone (PPARg agonist) + obestatin administered DIO-C57BL/6 mice.

#### **2. Effect of antagonists of PPARg, GW9662, and T0070907 on DIO-C57BL/6 mice individually and along with obestatin**

GW9662 is a potent and selective PPARγ antagonist with an IC50 of 3.3 nM. It has 10 and 600 fold less selectivity towards PPARα and PPARδ respectively. Mass spectrometric analysis revealed Cys285 was covalently modified by GW9662 [10]. T0070907 was identified as a potent and selective PPAR antagonist. It had an apparent binding affinity of 1 nM. It covalently modifies cysteine 313 in helix 3 of human PPAR2. **Figure 1** shows the structure of the antagonists [11].

**Figure 1.** *Structure of the PPARg antagonists GW9662 and T0070907.*

*Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*

#### **2.1 Induction of obesity and mice experiments**

Obesity was induced by administering a 60% calorie by high-fat diet (HFD) for a period of 24 weeks to four-week-old male C57BL/6 mice. The gain in weight over the induction period is shown in **Figure 2a** as a comparison with normal diet-fed male C57BL/6 mice. The HFD fed mice gained an average of 25 g whereas, the normal chow-fed mice gained 5 g of weight over the same period. Subsequently, the mice were grouped into six groups as follows:

Group 1: HFD Control.

Group 2: Obestatin Control.

Group 3: GW9662 Control.

Group 4: T0070907 Control.

Group 5: Obestatin + GW9662 treatment.

Group 6: Obestatin + T0070907 treatment.

Obestatin was synthesized, purified, and characterized in our laboratory as described previously [4]. GW9662 and T0070907 were purchased from Sigma Aldrich. As per the previous optimization done in our laboratory obestatin was administered at 160 nmol /kg/BW [12]. From the available literature, it was decided GW9662 and T0070907 should be administered at 1 mg/kg/BW to the respective groups [10, 11]. All samples were dissolved in 20% DMSO in 0.9% saline and the same solvent was used as control. All the administrations were intraperitoneal. The mice were administered the respective compound for 8 days after induction of obesity. Food intake was monitored for 5 h on an hourly basis after administration of saline or the respective compounds and gain in body weight was recorded every day. **Figure 3** shows the decrease in food intake upon administration of obestatin followed by further decreased food intake by the T0070907 group, Obestatin + T0070907 group, GW9662 group, and Obestatin + GW9662 group, respectively. **Figure 2b** shows the gain in body weight during 8 days. It can be seen that all the treated groups showed a negative gain in body weight even upon HFD administration. Only the HFD control group showed a steady gain in body weight. After 8 days of experimentation, the mice fasted for 6 hours and blood were

#### **Figure 2.**

*(a) Obesity induction over a period of 24 weeks in comparison to Normal diet fed mice. (b) Change in body weight upon treatment with the respective compounds.*

**Figure 3.** *Food intake for 5 h after administration of the respective compounds. The mice were administered the respective compounds and fed HFD after 15 min.*

drawn through the retro-orbital plexus and the mice were sacrificed according to established protocols. All adipose depots and vital organs were stored in formalin for histopathology studies and part of them were frozen in liquid nitrogen for RNA extraction and profiling.

#### **2.2 Effect of the treatments on the different fat pad**

Epididymal, perirenal, retroperitoneal, inguinal, BAT, gluteal, axillary, and cervical fat pads were collected from each group and weighed and normalized to the respective bodyweight to access the effect of the treatments on fat accumulation in each adipose depot. As not all adipose depots are metabolically the same, a decrease in fat accumulation in certain depots can be more beneficial. **Table 1** summarizes the overall effect of the treatment on the fat depots. GW9662 increased epididymal adipose tissue weight in comparison to the HFD and obestatin treated groups and decreased % epididymal fat upon coadministration with obestatin. It significantly decreased % Inguinal fat weight upon coadministration with obestatin by 26.4%. It also decreased total subcutaneous fat by 11.3% but increased % visceral fat by 15% and thereby increased total fat % in the adipose depots by 11.1%. On the other hand, T0070907 increased % epididymal fat by 24% and % visceral fat by 10%. It decreased perirenal fat by 54% and % subcutaneous fat by 10%. Upon coadministration with obestatin, T0070907 decreased % inguinal fat by 27% and % total subcutaneous fat by 15%. And maintaining total fat content is equal to that of the HFD control.

#### **2.3 Plasma biochemical analysis and lipid parameters**

SGOT, SGPT, alkaline phosphatase (ALP), fasting blood glucose, creatinine, urea, triglyceride, total cholesterol (TC), and high-density lipoprotein cholesterol


*Effect of obestatin (160 nmol /kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/*

≧*8). Data are expressed as the mean ± SEM* 

*BW, obestatin + T0070907 (160 nmol/kg/BW + 1 mg/kg/BW) treatment on % fat pad weight. Data are expressed as the mean ± SEM (N*

*(N*

≧*8). P < 0.05 was considered as statistically significant value.*

*Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*

(HDL-C) were estimated using commercially available kits. Phospholipid estimation was carried out by a colorimetric method [13]. Plasma Leptin and adiponectin concentrations were tested as per the instructions of the manufacturer of commercially available ELISA kits.

**Table 2** summarizes the plasma lipid parameters after the 8 days of the experiment. All the treated groups showed a significant decrease in plasma triglyceride. Obestatin + GW9662 and obestatin + T0070907 showed a maximum decrease of 39%. T0070907 and obestatin + T0070907 showed a maximum decrease in plasma total cholesterol by 10% whereas, GW9662 showed a significant decrease in total cholesterol by 4% only upon coadministration with obestatin. Obestatin increased phospholipids by 32%, the coadministered groups show a weak enhancement indicating there is a combined effect and not that of the individual components. Plasma-free fatty acids are decreased in all treated groups. A maximum decrease of 23% is seen in the obestatin + GW9662 treated group, followed by obestatin + T0070907 by 17%, GW9662 by 14%, T0070907, and obestatin by 10%.

**Table 3** summarizes the plasma biochemical parameters. Plasma glucose, protein, urea, and creatinine are in the normal range, did not show any significant changes. Marker enzymes SGOT, SGPT, and ALP are in the normal range for all the groups. Adipokine leptin that signals long-term fat reserves to the brain is significantly decreased most by obestatin + GW9662 group by 45% followed by obestatin + T0070907 by 37%, GW9662 by 33%, and T0070907 by 21% indicating a decrease in fat content. There were no significant changes in adiponectin levels.

#### **2.4 Adipose tissue lipid parameters**

Liver tissue, inguinal, and epididymal fat lipids were extracted by Folch's method of lipid extraction [14]. Tissue TAG, TC, and phospholipid were estimated by a colorimetric method [13, 15, 16]. **Table 4** summarizes the tissue lipid parameters. In epididymal adipose tissue, obestatin + T0070907 decreased triglyceride by 20%, followed by obestatin + GW9662 by 17% and GW9662 by 10%. Phospholipids were increased by about 80% in all the groups. No significant decrease in total cholesterol was observed. In inguinal adipose tissue, a significant decrease by 23% in total cholesterol was observed in both obestatin + GW9662 and obestatin + T0070907 groups followed by T0070907 at 18%. No significant changes were observed in triglyceride or phospholipids levels. In the liver tissue, a significant increase of 44% was seen only with respect to triglyceride in the obestatin treated group compared to that of the HFD control.

#### **2.5 mRNA profiling of epididymal and inguinal adipose tissue of lipid metabolism-related genes by quantitative real time-PCR (qPCR)**

Total RNA was isolated from the adipocyte cells using TRIzol reagent from Sigma, USA. The quantity and quality of the isolated RNA were assessed using a microspectrophotometer (Eppendorf). Samples having a ratio of A260/280 > 1.8 were used for cDNA synthesis by kit method (Thermo Scientific, Ltd.). Real-time PCR assays were performed using SYBR Green (BioRad CFX96 Touch Real-Time PCR Detection System) and primer sequences of the respective genes are given in SI **Table 1**. Fold change in gene expression was tabulated by normalizing the values of threshold cycle (CT) of the target gene with the CT value of housekeeping gene GAPDH. Briefly, the fold changes were calculated using the 2−ΔΔCt calculation


*Effect of obestatin (160 nmol /kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol /kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol /kg/BW + 1 mg/kg/BW) treatment on fasting plasma lipid parameters such as triglycerides, total cholesterol, HDL-cholesterol, and phospholipids level.* 

≧*8). P < 0.05 was considered as statistically significant value.*

*Data are expressed as the mean ± SEM (N*

### *Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*



*@Significant when compared toT0070907.*

*Effect of obestatin (160 nmol/kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol/kg/BW + 1 mg/kg/BW) treatment on fasting plasma biochemical parameters. Data are expressed as the mean ± SEM (N* ≧*8). P < 0.05 was considered as statistically significant value.*


*Effect of obestatin (160 nmol /kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol/kg/BW + 1 mg/kg/BW) treatment on lipid parameters such as triglycerides, total cholesterol, phospholipid content in epididymal fat and liver tissue. Data are expressed as the mean ± SEM (N*≧*8). P < 0.05 was considered as statistically significant value.*
