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

**3. Oxidative stress and cancer**

, peroxynitrite; SOD, superoxide dismutase.

glutathione; GST, glutathione S-transferase; H<sup>2</sup>

86 Ovarian Cancer - From Pathogenesis to Treatment

iron (II); Fe<sup>3</sup>

ONOO<sup>−</sup>

+

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

, iron (III); GPX, glutathione peroxidase; GSH, glutathione; GSR, glutathione reductase; GSSG, reduced

−

, nitrite; NO<sup>3</sup>

, hydrogen peroxide; H<sup>4</sup>

HOCl, hypochlorous acid; iNOS, inducible nitric oxide synthase; L-Arg, L-arginine; MPO, myeloperoxidase; NAD(P)H,

, chloride ion; Fe<sup>2</sup>

•−, superoxide;

B, tetrohydrobiopterin; HO•, hydroxyl radical;

, nitrate; O<sup>2</sup>

−

+ ,

**Figure 1.** Summary of key oxidant and antioxidants in cancer [1]. Abbreviations are CAT, catalase; Cl−

O2

nicotinamide adenine dinucleotide phosphate; NO•+, nitrosonium cation; NO<sup>2</sup>

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]. Thus, cancer cells trigger several critical adaptations that are essential for their survival such as suppression of apoptosis, alteration of glucose metabolism, and stimulation of angiogenesis [10, 29]. Oxygen depletion, due to a hypoxic microenvironment, significantly stimulates mitochondria to produce high levels of ROS and RNS which is known to activate HIF-1α and consequently promote cell survival in such an environment [29]. The half-life of HIF-1α is extremely short as it is rapidly inactivated through hydroxylation reactions mediated by dioxygen, oxaloglutarate, and iron-dependent prolyl 4-hydroxylases, located in the nucleus and cytoplasm [40, 41]. Nitric oxide and other ROS, as well as H2 O2 efflux into the cytosol due to dismutation of O2 ●−, can inhibit prolyl 4-hydroxylases activity, leading to the stabilization of HIF-1α [29, 42]. More importantly, stabilization of HIF-1α, under hypoxic conditions, can be blocked when inhibiting ROS production in mitochondria that lack cytochrome c [29, 43].

apoptosis in EOC cells [54]. Moreover, the remarkably higher levels of iNOS/NO, produced by EOC cells, resulted in the generation of high levels of nitrate and nitrite, powerful protein nitration agents that are known to stimulate the initiation and progression of tumor cells [53].

(HO●), further increasing oxidative stress [22, 53]. Additionally, EOC cells are also characterized by enhanced expression of NAD(P)H oxidase, a potent oxidant enzyme that is known

●– in the cell. Such high levels of O2

which modifies proteins and DNA structure and function in cells [57].

**6. Biomarkers for the early detection of ovarian cancer**

high levels of NO generates peroxynitrite, another powerful nitrosylation and nitration agent,

Recently we have gathered compelling evidence demonstrating that talc, through alteration of the redox balance, can generate a similar pro-oxidant state in both normal ovarian epithelial and ovarian cancer cells. Talc and asbestos are both silicate minerals, and the carcinogenic effects of asbestos have been extensively studied and documented in the medical literature [58]. Asbestos fibers in the lung initiate an inflammatory and scarring process, and it has been proposed that ground talc, as a foreign body, might initiate a similar inflammatory response [58]. Although there is strong epidemiological evidence to suggest an association between talc use and ovarian cancer, the direct link and precise mechanisms have yet to be elucidated. We investigated the effect of talc on both oxidants and antioxidants in normal ovarian epithelial and ovarian cancer cell lines. There was a marked increase in mRNA levels of the pro-oxidant enzymes, iNOS and MPO in talc treated ovarian cancer cell lines and normal ovarian epithelial cells, all as compared to their control, as early as 24 hours. Additionally, there was a marked decrease in the mRNA levels of the antioxidant enzymes CAT, GPX, SOD3, but with a marked increase in GSR, and no change in GST, in talc treated ovarian cancer cell line and in normal ovarian epithelial cells, all compared to their control, as early as 24 hours (*data not published*). Thus, there is a direct effect of talc on the molecular levels of oxidant and antioxidants, elucidating a potential mechanism for the development of ovarian cancer in response to talc.

The discovery of MPO expression in ovarian EOC cells and tissues was surprising, as it is only expressed by cells of myeloid origin. Intriguingly, the combination of serum MPO and free iron was reported to potentially serve as biomarkers for early detection of ovarian cancer [22]. A robust detection method based on molecular profiles for ovarian cancer has not yet been developed because the disease exhibits a wide range of morphological, clinical and genetic variations during its progression. The search for non-invasive, cost-effective ovarian cancer biomarker tests has been ongoing for many years. Immunizations of mice with ovarian cancer cells has led to hybridoma validation by ELISA, while flow cytometry analysis permitted the discovery of cancer antigen (CA)-125 and mesothelin [59]. Furthermore, the screening of an array of 21,500 unknown ovarian cDNAs hybridized with labeled first-strand cDNA from ten ovarian tumors and six normal tissues led to the discovery of human epididymis protein 4 (HE4) [60]. Most interestingly, HE4 is overexpressed in 93% of serous and 100% of endometrioid

O2

●– are elevated, MPO was reported to serve as

http://dx.doi.org/10.5772/intechopen.73860

89

●– combined with significantly

and generated highly reactive hydroxyl radical

New Insights into the Pathogenesis of Ovarian Cancer: Oxidative Stress

Under oxidative stress, where both NO and O2

a source of free iron which reacts with H2

to be the major source of O2

Pro-oxidant enzymes such as myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and NAD(P)H oxidase have been associated with initiation, progression, survival, and increased risk in cancers such as breast, ovarian, lung, prostate, bladder, colorectal and malignant melanoma [21, 44]. Moreover, the expression of those key pro-oxidant enzymes was found to change based on the histological type and grade of the tumor [21, 45, 46]. Likewise, antioxidants have also been associated with initiation, progression, survival, and increased risk in cancers such as lung, head and neck, and prostate cancer [47–50]. The expression of GSR and GPX, key antioxidant enzymes, has also been reported to be altered in various types of cancer [21]. The activity and expression of SOD, a powerful antioxidant enzyme, has been reported to be decreased in colorectal carcinomas, pancreatic, lung, gastric, ovarian, and breast cancers [21, 45, 46]. Likewise, the expression and activity of CAT, a key antioxidant enzyme, was reported to be decreased in breast, bladder, and lung cancers but increased in brain cancer [21, 45, 46]. Antioxidant enzymes play a critical role in maintaining the redox balance in the presence of microenvironment stress, and thus, alteration of this balance may provide a unique and complex microenvironment for cancer cell survival.

#### **5. Ovarian cancer cells manifest a persistent pro-oxidant state**

Recent evidence suggests that oxidative stress is a critical factor in the initiation and development of several cancers, including ovarian cancer [40, 51]. Consistently, it has been reported that ovarian cancer patients manifested significantly decreased levels of antioxidants and higher levels of oxidants [10, 22, 40, 51–53]. An enhanced redox state, resulting from increased expression of key pro-oxidant enzymes and decreased expression of antioxidant enzymes, has been extensively described in epithelial ovarian cancer (EOC) [52–54]. We have previously reported that MPO, a hemoprotein present solely in myloid cells that acts as a powerful oxidant, and iNOS, a key pro-oxidant enzyme, are highly expressed and co-localized to the same cell in EOC cells [53]. These two enzymes, MPO and iNOS, work together to inhibit apoptosis, a hallmark of ovarian cancer cells. Nitric oxide, produced by iNOS, is used by MPO as a one-electron substrate to generate nitrosonium cation (NO+ ), a labile nitrosating species, resulting in a significant increase in S-nitrosylation of caspase-3, which inhibits apoptosis [53, 55, 56]. Indeed, attenuating oxidative stress by inhibiting MPO or iNOS significantly induced apoptosis in EOC cells [54]. Moreover, the remarkably higher levels of iNOS/NO, produced by EOC cells, resulted in the generation of high levels of nitrate and nitrite, powerful protein nitration agents that are known to stimulate the initiation and progression of tumor cells [53]. Under oxidative stress, where both NO and O2 ●– are elevated, MPO was reported to serve as a source of free iron which reacts with H2 O2 and generated highly reactive hydroxyl radical (HO●), further increasing oxidative stress [22, 53]. Additionally, EOC cells are also characterized by enhanced expression of NAD(P)H oxidase, a potent oxidant enzyme that is known to be the major source of O2 ●– in the cell. Such high levels of O2 ●– combined with significantly high levels of NO generates peroxynitrite, another powerful nitrosylation and nitration agent, which modifies proteins and DNA structure and function in cells [57].

Thus, cancer cells trigger several critical adaptations that are essential for their survival such as suppression of apoptosis, alteration of glucose metabolism, and stimulation of angiogenesis [10, 29]. Oxygen depletion, due to a hypoxic microenvironment, significantly stimulates mitochondria to produce high levels of ROS and RNS which is known to activate HIF-1α and consequently promote cell survival in such an environment [29]. The half-life of HIF-1α is extremely short as it is rapidly inactivated through hydroxylation reactions mediated by dioxygen, oxaloglutarate, and iron-dependent prolyl 4-hydroxylases, located in the nucleus

of HIF-1α [29, 42]. More importantly, stabilization of HIF-1α, under hypoxic conditions, can be blocked when inhibiting ROS production in mitochondria that lack cytochrome c [29, 43]. Pro-oxidant enzymes such as myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and NAD(P)H oxidase have been associated with initiation, progression, survival, and increased risk in cancers such as breast, ovarian, lung, prostate, bladder, colorectal and malignant melanoma [21, 44]. Moreover, the expression of those key pro-oxidant enzymes was found to change based on the histological type and grade of the tumor [21, 45, 46]. Likewise, antioxidants have also been associated with initiation, progression, survival, and increased risk in cancers such as lung, head and neck, and prostate cancer [47–50]. The expression of GSR and GPX, key antioxidant enzymes, has also been reported to be altered in various types of cancer [21]. The activity and expression of SOD, a powerful antioxidant enzyme, has been reported to be decreased in colorectal carcinomas, pancreatic, lung, gastric, ovarian, and breast cancers [21, 45, 46]. Likewise, the expression and activity of CAT, a key antioxidant enzyme, was reported to be decreased in breast, bladder, and lung cancers but increased in brain cancer [21, 45, 46]. Antioxidant enzymes play a critical role in maintaining the redox balance in the presence of microenvironment stress, and thus, alteration of this balance may

O2

●−, can inhibit prolyl 4-hydroxylases activity, leading to the stabilization

efflux into the cytosol due

), a labile nitrosating species,

and cytoplasm [40, 41]. Nitric oxide and other ROS, as well as H2

provide a unique and complex microenvironment for cancer cell survival.

**5. Ovarian cancer cells manifest a persistent pro-oxidant state**

as a one-electron substrate to generate nitrosonium cation (NO+

Recent evidence suggests that oxidative stress is a critical factor in the initiation and development of several cancers, including ovarian cancer [40, 51]. Consistently, it has been reported that ovarian cancer patients manifested significantly decreased levels of antioxidants and higher levels of oxidants [10, 22, 40, 51–53]. An enhanced redox state, resulting from increased expression of key pro-oxidant enzymes and decreased expression of antioxidant enzymes, has been extensively described in epithelial ovarian cancer (EOC) [52–54]. We have previously reported that MPO, a hemoprotein present solely in myloid cells that acts as a powerful oxidant, and iNOS, a key pro-oxidant enzyme, are highly expressed and co-localized to the same cell in EOC cells [53]. These two enzymes, MPO and iNOS, work together to inhibit apoptosis, a hallmark of ovarian cancer cells. Nitric oxide, produced by iNOS, is used by MPO

resulting in a significant increase in S-nitrosylation of caspase-3, which inhibits apoptosis [53, 55, 56]. Indeed, attenuating oxidative stress by inhibiting MPO or iNOS significantly induced

to dismutation of O2

88 Ovarian Cancer - From Pathogenesis to Treatment

Recently we have gathered compelling evidence demonstrating that talc, through alteration of the redox balance, can generate a similar pro-oxidant state in both normal ovarian epithelial and ovarian cancer cells. Talc and asbestos are both silicate minerals, and the carcinogenic effects of asbestos have been extensively studied and documented in the medical literature [58]. Asbestos fibers in the lung initiate an inflammatory and scarring process, and it has been proposed that ground talc, as a foreign body, might initiate a similar inflammatory response [58]. Although there is strong epidemiological evidence to suggest an association between talc use and ovarian cancer, the direct link and precise mechanisms have yet to be elucidated. We investigated the effect of talc on both oxidants and antioxidants in normal ovarian epithelial and ovarian cancer cell lines. There was a marked increase in mRNA levels of the pro-oxidant enzymes, iNOS and MPO in talc treated ovarian cancer cell lines and normal ovarian epithelial cells, all as compared to their control, as early as 24 hours. Additionally, there was a marked decrease in the mRNA levels of the antioxidant enzymes CAT, GPX, SOD3, but with a marked increase in GSR, and no change in GST, in talc treated ovarian cancer cell line and in normal ovarian epithelial cells, all compared to their control, as early as 24 hours (*data not published*). Thus, there is a direct effect of talc on the molecular levels of oxidant and antioxidants, elucidating a potential mechanism for the development of ovarian cancer in response to talc.

## **6. Biomarkers for the early detection of ovarian cancer**

The discovery of MPO expression in ovarian EOC cells and tissues was surprising, as it is only expressed by cells of myeloid origin. Intriguingly, the combination of serum MPO and free iron was reported to potentially serve as biomarkers for early detection of ovarian cancer [22]. A robust detection method based on molecular profiles for ovarian cancer has not yet been developed because the disease exhibits a wide range of morphological, clinical and genetic variations during its progression. The search for non-invasive, cost-effective ovarian cancer biomarker tests has been ongoing for many years. Immunizations of mice with ovarian cancer cells has led to hybridoma validation by ELISA, while flow cytometry analysis permitted the discovery of cancer antigen (CA)-125 and mesothelin [59]. Furthermore, the screening of an array of 21,500 unknown ovarian cDNAs hybridized with labeled first-strand cDNA from ten ovarian tumors and six normal tissues led to the discovery of human epididymis protein 4 (HE4) [60]. Most interestingly, HE4 is overexpressed in 93% of serous and 100% of endometrioid EOCs, and in 50% of clear cell carcinomas, but not in mucinous ovarian carcinomas [61]. Thus, HE4 was identified as one of the most useful biomarkers for ovarian cancer, although it lacked tissue-specificity [60, 62–64]. Secreted HE4 high levels were also detected in the serum of ovarian cancer patients [65]. Additionally, combining CA-125 and HE4 is a more accurate predictor of malignancy than either alone [66–68].

serum iron levels, but may also induce oxidative stress, which can promote lipid peroxidation, DNA strand breaks, and modification or degradation of biomolecules [78–80]. Iron reacts with

reported conflicting results regarding the use of iron levels alone for diagnosis [86–89].

as biomarkers for early detection and prognosis of ovarian cancer.

mouse fibroblasts and the DU-145 prostate epithelial cells [98].

**7. Modulation of oxidative stress**

from increased O2

Thus, the combination of serum MPO and iron levels should yield a higher power of specificity and sensitivity that should distinguish women with early-stage ovarian cancer from other disorders, specifically inflammation [22]. Additionally, combining serum MPO and iron levels with the best currently existing biomarkers through the creation of a logistic regression model may increase the overall predictive values. Collectively, there is a role for serum MPO and free iron in the pathophysiology of ovarian cancer, which thereby qualifies them to serve

Several studies have reported the beneficial effects of modulating the redox status of cancer cells, however few studies have been reported for ovarian cancer [90–92]. Inhibition of prooxidant enzymes, such as NAD(P)H oxidase, has been shown to significantly induce apoptosis of cancer cells [93, 94]. We investigated whether NAD(P)H oxidase-mediated generation of intracellular reactive ROS lead to anti-apoptotic activity and thus a growth advantage to EOC cells. Diphenyleneiodonium (DPI) has been used to inhibit ROS production mediated by NAD(P)H oxidase in various cell types [95–97]. Our results showed that NAD(P)H oxidase is over-expressed in EOC tissues and cells as compared to normal ovarian tissues and cells [52]. Indeed, high levels of NAD(P)H oxidase are known to promote tumorigenesis of NIH3T3

Inhibition of NAD(P)H oxidase has also been reported to decrease the generation of O2

as well as other oxidants [93, 94]. Cancer cells are known to manifest enhanced intrinsic oxidative stress and metabolic activity that lead to mitochondrial failure [99, 100]. Indeed, it was previously reported that ovarian tumors are characterized by increased ROS levels as evident

[101]. The NAD(P)H oxidase redox signaling is controlled by mitochondria, and thus loss of

**●–** generated from NAD(P)H oxidase as well as mitochondrial malfunction

●–, H2 O2 ,

 and catalyzes the generation of highly reactive hydroxyl radicals, which in turn further increases free iron concentrations by the Fenton and Haber–Weiss reaction [81]. Several studies from our laboratories have provided a mechanistic link between oxidative stress, MPO, higher levels of HOCl and higher free iron that could explain the observed accumulation of free iron in epithelial ovarian cancers tissues [53, 82–85]. Utilizing serum iron levels alone as a biomarker is also not sufficient for early detection of ovarian cancer due to many uncontrolled variables, i.e. dietary intake, supplements, effects of other iron-generating enzymes or factors, and more importantly they are not as specific as MPO levels. Specifically, in iron deficiency anemic patients, their free iron levels may become a confounding factor in its utilization for early detection of ovarian cancer. Thus, anemia should be ruled out to eliminate any overlap that would lead to misdiagnosis. The incorporation of iron deficiency anemic patients in a logistic regression model will help determine its overlap with early-stage ovarian cancer. Additionally, currently available clinical studies focused on either biochemical or more recently, genetic markers of iron overload have

New Insights into the Pathogenesis of Ovarian Cancer: Oxidative Stress

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91

H2 O2

Multi-marker panels have the potential for high positive predictive values (PPVs), but careful validation with appropriate sample cohorts is mandatory and complex algorithms may be difficult to implement for routine clinical use [59]. Panels of biomarkers have been extensively investigated to improve sensitivity and specificity and have included some of the most promising reported markers such as CA72–4, M-CSF, OVX1, LPA, prostacin, osteopontin, inhibin and kallikrein [69–71]. However, most of these tests frequently require certain equipments and complex computational algorithms that may not be available in a standard immunoassay laboratory, [32]. Among postmenopausal women in the U.S., only 1 in 2500 women are reported with ovarian cancer. Due to this low prevalence of the disease, a screening method that yield a 75% sensitivity and 99.6% specificity to achieve a PPV value of 10% to be effective for the detection of all stages of ovarian cancer [72]. To date, there is no single biomarker available that met these requirements.

The established role of MPO in oxidative stress and inflammation has been a leading factor in the study of MPO as a possible marker of plaque instability and a useful clinical tool in the evaluation of patients with coronary heart disease [73]. Recent genetic studies implicated MPO in the development of lung cancer by demonstrating a striking correlation between the relative risk for development of the disease and the incidence of functionally distinct MPO polymorphisms [74]. Myeloperoxidase levels reported for various inflammatory disorders are coincidentally lower than those levels found in all stages of ovarian cancer. A previous study reported normal serum MPO and iron levels as 62 ± 11 ng/ml and 96 ± 9 μg/dl, respectively [75]. However, there was a significant increase in serum MPO and iron levels to 95 ± 20 ng/ml and 159 ± 20 μg/dl, respectively, in asthmatic individuals [75]. Although there was an increase in this reported serum iron, these levels still fell within the normal range (50 to 170 μg/dl) [22, 75]. Other studies have showed that an elevated MPO levels, reaching up to 350 ng/ml, in serum plasma, was indicative of a higher risk for cardiovascular events in patients hospitalized for chest pain [76, 77]. A recent study showed a significant correlation between MPO levels and the stage of ovarian cancer, as is the linear trend for MPO with increasing stage [22]. Similarly, there was a significant difference in the level of free iron in serum and tissues obtained from stage I as compared to combined stages II, III, and IV ovarian cancer. There was an overlap between stage I ovarian cancer and inflammation (endometriosis) serum MPO levels, however serum free iron levels were significantly higher in stage I ovarian cancer as compared to inflammation. There was no significant change in free iron levels between the healthy control group, benign gynecologic conditions group, and inflammation group [22].

Due to the overlap of MPO levels in early-stage ovarian cancer and inflammatory conditions, there is a potential for a false positive with MPO alone in patients with cardiovascular, inflammation, and/or asthmatic disorders. It has been reported that MPO heme destruction and iron release is mediated by high levels of both HOCl (a product of MPO) and oxidative stress (i.e. cancer) [22]. The free iron generated by hemoprotein destruction not only contributes to elevation of serum iron levels, but may also induce oxidative stress, which can promote lipid peroxidation, DNA strand breaks, and modification or degradation of biomolecules [78–80]. Iron reacts with H2 O2 and catalyzes the generation of highly reactive hydroxyl radicals, which in turn further increases free iron concentrations by the Fenton and Haber–Weiss reaction [81]. Several studies from our laboratories have provided a mechanistic link between oxidative stress, MPO, higher levels of HOCl and higher free iron that could explain the observed accumulation of free iron in epithelial ovarian cancers tissues [53, 82–85]. Utilizing serum iron levels alone as a biomarker is also not sufficient for early detection of ovarian cancer due to many uncontrolled variables, i.e. dietary intake, supplements, effects of other iron-generating enzymes or factors, and more importantly they are not as specific as MPO levels. Specifically, in iron deficiency anemic patients, their free iron levels may become a confounding factor in its utilization for early detection of ovarian cancer. Thus, anemia should be ruled out to eliminate any overlap that would lead to misdiagnosis. The incorporation of iron deficiency anemic patients in a logistic regression model will help determine its overlap with early-stage ovarian cancer. Additionally, currently available clinical studies focused on either biochemical or more recently, genetic markers of iron overload have reported conflicting results regarding the use of iron levels alone for diagnosis [86–89].

Thus, the combination of serum MPO and iron levels should yield a higher power of specificity and sensitivity that should distinguish women with early-stage ovarian cancer from other disorders, specifically inflammation [22]. Additionally, combining serum MPO and iron levels with the best currently existing biomarkers through the creation of a logistic regression model may increase the overall predictive values. Collectively, there is a role for serum MPO and free iron in the pathophysiology of ovarian cancer, which thereby qualifies them to serve as biomarkers for early detection and prognosis of ovarian cancer.
