**2. Oxidative stress**

has long been considered a heterogeneous disease with respect to histopathology, molecular biology, and clinical outcome. It comprises at least five distinct histological subtypes, the most common and well-studied being high-grade serous ovarian cancer (HGSOC) [3]. The majority of advanced-stage tumors are of epithelial cell origin and can arise from serous, mucinous, or endometrioid cells on the surface epithelium of the ovary or the fallopian tube [2]. The most obvious clinical implication of tumor heterogeneity is that molecular-targeted therapy, while

Because early-stage ovarian cancer presents with nonspecific symptoms, most often diagnosis is not made until after the malignancy has spread beyond the ovaries [4]. Mortality rates for this type of malignancy are high because of a lack of a sensitive and specific early-stage screening method [4]. Surgical cytoreduction followed by platinum/taxane chemotherapy results in complete clinical response in 50–80% of patients with stage III and IV disease, but most will relapse within 18 months and ultimately develop chemoresistant disease [2]. Resistance to chemotherapy can either be intrinsic, occurring at the onset of treatment, or acquired, when the disease recurs despite an initially successful response [5–7]. Attempts to overcome drug resistance are central to both clinical and basic molecular research in cancer chemotherapy [5, 8]. Cancer cells are known to be under intrinsic oxidative stress, resulting in increased DNA mutations or damage, genome instability, and cellular proliferation [9–13]. The persistent generation of cellular reactive oxygen species (ROS) is a consequence of many factors including exposure to carcinogens, infection,

inflammation, environmental toxicants, nutrients, and mitochondrial respiration [14–17].

Nevertheless, many cases of ovarian cancer continue to be described as *de novo*.

sarcomas are also classified as type II tumors.

The origin and causes of ovarian tumors remains under debate. Injury to surface epithelial ovarian cells due to repeated ovulation is thought to induce tumorigenesis in these cells and is known as the "incessant ovulation hypothesis." Additionally, hormonal stimulation of the surface epithelium of the ovary has been described to initiate tumorigenesis in surface epithelial cells and is known as the "gonadotropin hypothesis." Moreover, the fallopian tube, and not the ovary, has been suggested to be the origin for most epithelial ovarian cancer [2, 18, 19].

Histopathologic, clinical and molecular genetic profiles were successfully utilized to clearly discriminate between type I and type II ovarian tumors [19]. Accordingly, type I ovarian tumors develop from benign precursor lesions that implant on the ovary and include clear cell, endometrioid, low-grade serous carcinomas, mucinous carcinomas and malignant Brenner tumors [19]. Type II ovarian tumors develop from intraepithelial carcinomas of the fallopian tube and can then spread to involve both the ovary as well as other sites, such as high-grade serous carcinomas which comprise morphologic and molecular subtypes [19]. Additionally, high-grade endometrioid, poorly differentiated ovarian cancers, and carcino-

Attempts to identify specific genes in ovarian tumors to help in early detection of the disease and serve as targets for improved therapy had failed to identify reproducible prognostic indicators [2, 20–22]. Several oncogenic mutations and pathways have been identified in ovarian cancer. Specific inherited mutations in the *BRCA1* and *BRCA2* genes that produce tumor suppressor proteins, are known to be associated with a 15% increased risk of ovarian cancer overall [2]. Ovarian cancers associated with *BRCA1* and *BRCA2* mutations are much more common in

being effective at one tumor site, may not be as effective at all of them [3].

84 Ovarian Cancer - From Pathogenesis to Treatment

Homeostasis, the balance between the production and elimination of oxidants, is maintained by mechanisms involving oxidants and antioxidant enzymes and molecules. If this balance is altered, it leads to an enhanced state of oxidative stress that alters key biomolecules and cells of living organism [13]. Oxidant molecules are divided into two main groups; oxygen-derived or nitrogen-containing molecules. Oxygen-derived molecules, also known as reactive oxygen species (ROS), includes free radicals such as hydroxyl (HO●), superoxide (O2 ●−), peroxyl (RO2 ●), and alkoxyl (RO●), as well as oxidizing agents such as hydrogen peroxide (H2 O2 ), hypochlorous acid (HOCl), ozone (O3 ), and singlet oxygen (1 O2 ) that can be converted to radicals [13, 27]. Nitrogen containing oxidants, also known as reactive nitrogen species (RNS), are derived from nitric oxide (NO) that is produced in the mitochondria in response to hypoxia [13]. Exposure to inflammation, infection, carcinogens, and toxicants are major sources of ROS and RNS, *in vivo* [13, 16, 27, 28]. Additionally, RNS and ROS can be produced by various enzymes including cytochrome P450, lipoxygenase, cyclooxygenase, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase complex, xanthine oxidase (XO), and peroxisomes (**Figure 1**) [13, 28, 29].

To maintain the redox balance, ROS and RNS are neutralized by various important enzyme systems including superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione (GSH), thioredoxin coupled with thioredoxin reductase, glutaredoxin, glutathione peroxidase (GPX), and glutathione reductase (GSR) (**Figure 1**) [27]. Superoxide dismutase is known to convert O2 ●– to H2 O2 , which is then converted to water by CAT. Glutathione S-transferase is involved in detoxification of carcinogens and xenobiotics by catalyzing their conjugation to GSH that will aid in expulsion from the cell (**Figure 1**) [27]. Indeed, the GSHto-oxidized-GSH (GSH/GSSG) ratio is a good indicator of cellular redox buffering capacity [30, 31]. Under enhanced oxidative stress, the GSH/GSSG complex is known to stimulate the activity of the GS-X-MRP1 efflux pump, which removes toxins from cells. This mechanism has been investigated in the development of resistance to chemotherapeutic drugs [30, 31].

(Keap1), that binds Nrf2 in the cytoplasm and prevents its translocation into the nucleus, where it binds to promoters of antioxidant enzymes [13, 33]. Additionally, oxidative stress is known to activate certain signaling pathways, specifically, the MAPK/AP-1 and NF-κB pathways, which are critical for the initiation and maintenance of the oncogenic phenotype [34]. More importantly, ROS and RNS are known to induce genetic mutations that alter gene expression as well as induce DNA damage, and thus have been implicated in the etiology of several diseases, including cancer [2, 13, 35]. Damage to DNA by ROS and RNS is now accepted as a major cause of cancer, and has been demonstrated in the initiation and progression of several cancers including breast, hepatocellular carcinoma, and prostate cancer [34]. Oxidative stress is known to modify all the four DNA bases by base pair substitutions rather than base deletions and insertions. Modification of GC base pairs usually results in mutations, whereas, modification of AT base pairs does not [36]. Modification of guanine in cellular DNA, causing G to T transversions, is commonly induced by ROS and RNS [34]. If not repaired, the transversion of G to T in the DNA of oncogenes or tumor suppressor genes can lead to initiation and progression of cancer. Oxidation of DNA bases, such as thymidine glycol, 5-hydroxymethyl-2′-deoxyuridine,

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and 8-OHdG are now accepted markers of cellular DNA damage by free radicals [35].

MMP-10, and MMP-13 is enhanced by free radicals, specifically H<sup>2</sup>

**4. Cancer cells are under intrinsic oxidative stress**

Oxidants and free radicals are known to enhance cell migration contributing to the enhancement of tumor invasion and metastasis, main causes of death in cancer patients [2, 13]. Reactive oxygen species, through the activation of NF-κB, regulate the expression of intercellular adhesion protein-1 (ICAM-1), a cell surface protein in various cell types [13]. In response to oxidative stress, the interleukin 8 (IL-8)-induced enhanced expression of ICAM-1 on neutrophils enhances the migration of neutrophils across the endothelium, which is key in tumor metastasis [13]. Another important player that controls cell migration and consequently, tumor invasion, is the upregulation of specific matrix metalloproteinases (MMPs), essential enzymes in the degradation of most components of the basement membrane and extracellular matrix, such as type IV collagen [13, 37]. The expression of MMPs, such as MMP-2, MMP-3, MMP-9,

activation of Ras, ERK1/2, p38, and JNK, or the inactivation of phosphatases [13, 37]. Indeed, the major source of cellular ROS, the NAD(P)H oxidase family of enzymes, has been linked to the promotion of survival and growth of tumor cells in pancreatic and lung cancers [2, 13].

Oxidants and free radicals are also known to enhance angiogenesis, a key process for the survival of solid tumors [13]. Angiogenesis involves the upregulation of vascular endothelial growth factor (VEGF) or the downregulation of thrombospondin-1 (TSP-1), an angiogenesis suppressor in response to oxidative stress [13]. This process is controlled by several oncogenes and tumor-suppressor genes such as Ras, c-Myc, c-Jun, mutated p53, human epidermal growth factor receptor-2, and steroid receptor coactivators [38, 39]. Additionally, oxidants and free radicals are known to

Cancer cells are continuously exposed to high levels of intrinsic oxidative stress due to increased aerobic glycolysis (Warburg effect), a known process in cancer cell metabolism [10, 40].

stabilize HIF-1α protein and induce the production of angiogenic factors by tumor cells.

O2

and NO, through the

**Figure 1.** Summary of key oxidant and antioxidants in cancer [1]. Abbreviations are CAT, catalase; Cl− , chloride ion; Fe<sup>2</sup> + , iron (II); Fe<sup>3</sup> + , iron (III); GPX, glutathione peroxidase; GSH, glutathione; GSR, glutathione reductase; GSSG, reduced glutathione; GST, glutathione S-transferase; H<sup>2</sup> O2 , hydrogen peroxide; H<sup>4</sup> B, tetrohydrobiopterin; HO•, hydroxyl radical; HOCl, hypochlorous acid; iNOS, inducible nitric oxide synthase; L-Arg, L-arginine; MPO, myeloperoxidase; NAD(P)H, nicotinamide adenine dinucleotide phosphate; NO•+, nitrosonium cation; NO<sup>2</sup> − , nitrite; NO<sup>3</sup> − , nitrate; O<sup>2</sup> •−, superoxide; ONOO<sup>−</sup> , peroxynitrite; SOD, superoxide dismutase.

#### **3. Oxidative stress and cancer**

Oxidative stress has been implicated in the etiology of several diseases, including cancer. Alteration of the cellular redox balance modulates the initiation, promotion, and progression of tumor cells [13, 27]. The continuous generation of oxidants and free radicals affects key cellular mechanisms that control the balance of cell proliferation and apoptosis, which play a major role in the initiation and development of several cancers. Depending on the concentration of ROS and RNS in the cellular environment, oxidants can initiate and promote the oncogenic phenotype or induce apoptosis, and thus act as antitumor agents [32]. Several transcription factors that modulate the expression of genes critical to the development and metastasis of cancer cells are known to be controlled by oxidative stress. This includes hypoxia inducible factor (HIF)-1α, nuclear factor (NF)-κB, peroxisome proliferator-activated receptor (PPAR)-γ, activator protein (AP)-1, β-catenin/Wnt, and Nuclear factor erythroid 2-related factor 2 (Nrf2) [13]. The transcription factor regulator Nrf2 is known to control the expression of some key antioxidant enzymes that are needed to scavenge oxidants and free radicals [13, 33]. The activation of Nrf2 involves the suppressor protein, Kelch-like ECH-associated protein 1 (Keap1), that binds Nrf2 in the cytoplasm and prevents its translocation into the nucleus, where it binds to promoters of antioxidant enzymes [13, 33]. Additionally, oxidative stress is known to activate certain signaling pathways, specifically, the MAPK/AP-1 and NF-κB pathways, which are critical for the initiation and maintenance of the oncogenic phenotype [34].

More importantly, ROS and RNS are known to induce genetic mutations that alter gene expression as well as induce DNA damage, and thus have been implicated in the etiology of several diseases, including cancer [2, 13, 35]. Damage to DNA by ROS and RNS is now accepted as a major cause of cancer, and has been demonstrated in the initiation and progression of several cancers including breast, hepatocellular carcinoma, and prostate cancer [34]. Oxidative stress is known to modify all the four DNA bases by base pair substitutions rather than base deletions and insertions. Modification of GC base pairs usually results in mutations, whereas, modification of AT base pairs does not [36]. Modification of guanine in cellular DNA, causing G to T transversions, is commonly induced by ROS and RNS [34]. If not repaired, the transversion of G to T in the DNA of oncogenes or tumor suppressor genes can lead to initiation and progression of cancer. Oxidation of DNA bases, such as thymidine glycol, 5-hydroxymethyl-2′-deoxyuridine, and 8-OHdG are now accepted markers of cellular DNA damage by free radicals [35].

Oxidants and free radicals are known to enhance cell migration contributing to the enhancement of tumor invasion and metastasis, main causes of death in cancer patients [2, 13]. Reactive oxygen species, through the activation of NF-κB, regulate the expression of intercellular adhesion protein-1 (ICAM-1), a cell surface protein in various cell types [13]. In response to oxidative stress, the interleukin 8 (IL-8)-induced enhanced expression of ICAM-1 on neutrophils enhances the migration of neutrophils across the endothelium, which is key in tumor metastasis [13]. Another important player that controls cell migration and consequently, tumor invasion, is the upregulation of specific matrix metalloproteinases (MMPs), essential enzymes in the degradation of most components of the basement membrane and extracellular matrix, such as type IV collagen [13, 37]. The expression of MMPs, such as MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 is enhanced by free radicals, specifically H<sup>2</sup> O2 and NO, through the activation of Ras, ERK1/2, p38, and JNK, or the inactivation of phosphatases [13, 37]. Indeed, the major source of cellular ROS, the NAD(P)H oxidase family of enzymes, has been linked to the promotion of survival and growth of tumor cells in pancreatic and lung cancers [2, 13].

Oxidants and free radicals are also known to enhance angiogenesis, a key process for the survival of solid tumors [13]. Angiogenesis involves the upregulation of vascular endothelial growth factor (VEGF) or the downregulation of thrombospondin-1 (TSP-1), an angiogenesis suppressor in response to oxidative stress [13]. This process is controlled by several oncogenes and tumor-suppressor genes such as Ras, c-Myc, c-Jun, mutated p53, human epidermal growth factor receptor-2, and steroid receptor coactivators [38, 39]. Additionally, oxidants and free radicals are known to stabilize HIF-1α protein and induce the production of angiogenic factors by tumor cells.
