**12. Further increasing pro-oxidant enzymes: potential survival mechanism**

Apoptosis is a tightly regulated molecular process that removes excess or unwanted cells from organisms. Resistance to apoptosis is a key feature of cancer cells and is involved in the pathogenesis of cancer. We have previously reported that EOC cells have significantly increased levels of NO, which correlated with increased expression in iNOS [54]. We have also reported that EOC cells manifested lower apoptosis, which was markedly induced by inhibiting iNOS by L-NAME, indicating a strong link between apoptosis and NO/iNOS pathways in these cells [54]. Caspase-3 is known to play a critical role in controlling apoptosis, by participating in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins, resulting in disassembly of the cell [141–144]. Caspase-3 was found to be S-nitrosylated on the catalytic-site cysteine in unstimulated human lymphocyte cell lines and denitrosylated upon activation of the Fas apoptotic pathway [145]. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Caspase-3 S-nitrosylation/denitrosylation is known to serve as an on/off switch regulating caspase activity during apoptosis in endothelial cells, lymphocytes and trophoblasts [146–149]. The mechanisms underlying S-nitrosothiol (SNO) formation *in vivo* are not well understood.

manifested specific point mutations that corresponded to known functional SNPs, in key redox enzymes including *SOD2* (rs4880), *NOS2* (rs2297518), and *CYBA* (rs4673) [1]. However, altered enzymatic activity for CAT and GSR observed in chemoresistant EOC cells did not correspond to the specific SNP of interest in those enzymes, indicating involvement of other possible functional SNPs for those enzymes [21]. Coincidently, chemotherapy treatment induced point mutations that happen to correspond to known functional SNPs in key oxidant enzymes subsequently led to the acquisition of chemoresistance by EOC cells. Indeed, the induction of specific point mutations in *SOD2* or *GPX1* in sensitive EOC cells resulted in a decrease in the sensitivity to chemotherapy of these cells [21]. In fact, the addition of SOD to sensitive EOC cells during

chemotherapy treatment synergistically increased the efficacy to chemotherapy [21].

signature mutations for oxidants and free radicals [21].

chemoresistance in nature [21].

96 Ovarian Cancer - From Pathogenesis to Treatment

**mechanism**

Alternatively, the observed nucleotide switch in response to chemotherapy in EOC cells may be the result of nucleotide substitution, a process that includes transitions, replacement of one purine by the other or that of one pyrimidine by the other, or transversions, replacement of a purine by a pyrimidine or vice versa [21]. Indeed, hydroxyl radicals are known to react with DNA causing the formation of many pyrimidine and purine-derived lesions [21]. The oxidative damage to 8-Oxo-2′-deoxyguanosine, a major product of DNA oxidation, induces genetic alterations in oncogenes and tumor suppressor genes has been involved in tumor initiation and progression [21]. A GC to TA transversion has been reported in the *ras* oncogene and the *p53* tumor suppressor gene in several cancers. However, the GC to TA transversion is not unique to hydroxy-2′-deoxyguanosine, as CC to TT substitutions have been identified as

Moreover, the observed nucleotide switch in response to chemotherapy in EOC cells can be due to the fact that acquisition of chemoresistance generates an entirely different population of cells with a distinct genotype. Hence, chemotherapy kills the bulk of the tumor cells leaving a subtype of cancer cells with ability for repair and renewal, known as cancer stem cells (CSCs) [21]. Indeed, cancer stem cells have been isolated from various types of cancer including leukemia, breast, brain, pancreatic, prostate, ovarian and colon [21]. Interestingly, CSC populations were present in cultures of SKOV-3 EOC cells and have been shown to be

Apoptosis is a tightly regulated molecular process that removes excess or unwanted cells from organisms. Resistance to apoptosis is a key feature of cancer cells and is involved in the pathogenesis of cancer. We have previously reported that EOC cells have significantly increased levels of NO, which correlated with increased expression in iNOS [54]. We have also reported that EOC cells manifested lower apoptosis, which was markedly induced by inhibiting iNOS by L-NAME, indicating a strong link between apoptosis and NO/iNOS pathways in these cells [54]. Caspase-3 is known to play a critical role in controlling apoptosis, by participating in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of

**12. Further increasing pro-oxidant enzymes: potential survival** 

Myeloperoxidase typically uses H2 O2 , in combination with chloride to generate hypochlorous acid [55, 150–153]. We, and others, have demonstrated that MPO utilizes NO, produced by iNOS, as a one-electron substrate generating NO+ , a labile nitrosating species that is rapidly hydrolyzed forming nitrite as end-product [55, 56, 154, 155]. The ability of MPO to generate NO+ , from NO, led us to believe that not only does MPO play a role in S-nitrosylation of caspase-3 in EOC cells, but also highlights a possible cross-talk between iNOS and MPO. Indeed, we observed that MPO is responsible for the S-nitrosylation of caspase-3, which led to the inhibition of caspase-3 in EOC cells. Silencing MPO gene expression induced apoptosis in EOC cells through a mechanism that involved S-nitrosylation of caspase-3 by MPO.

Molecular alterations that lead to apoptosis can be inhibited by S-nitrosylation of apoptotic proteins such as caspases. Thus, S-nitrosylation conveys a key influence of NO on apoptosis signaling and may act as a key regulator for apoptosis in cancer cells. It has been known that the effects of NO on apoptosis are not only stimulatory but may also be inhibitory. These paradoxical effects of NO on apoptosis seem to be influenced by several factors. It has been suggested that biological conditions, such as the redox state, concentration, exposure time and the combination with O2 , O2 ●– and other molecules, determines the net effect of NO on apoptosis [156]. Also, NO is implicated in both apoptotic and necrotic cell death depending on the NO chemistry and the cellular biological redox state [57, 156]. As described earlier, we have previously demonstrated that the EOC cell lines, SKOV-3 and MDAH-2774, manifested lower apoptosis and had significantly higher levels of NO due to the presence of elevated levels of iNOS [54, 157]. We have also reported significant levels of MPO expression, which was found to be co-localized with iNOS, in both EOC cell lines SKOV-3 and MDAH-2774 [53]. We have demonstrated that 65% of the invasive epithelial ovarian carcinoma specimens tested expressed MPO in the neoplastic cells. The co-localization of MPO and iNOS has been demonstrated by immunohistochemical studies in cytokine-treated human neutrophils and primary granules of activated leukocytes [158]. Both plasma levels and tissue expression of MPO in gynecologic malignancies were previously evaluated and it was found that gynecologic cancer patients had higher plasma MPO compared to control subjects [159]. Using immunostaining, it was also demonstrated that MPO expression was higher in cancer tissues compared to control [159].

We have now characterized chemoresistant EOC cells to manifest an even further increase in pro-oxidant enzymes including MPO, and NO, a surrogate for iNOS activity in conjunction with a further increase in the S-nitrosylation of caspase-3 (*data not published*) and a concurrent decrease in the level of apoptosis [21]. Thus, we hypothesized that the decrease in apoptosis observed in chemoresistant EOC cells is a consequence of a further increase in the degree of S-nitrosylation of caspase-3. Since resistance to apoptosis is a hallmark of tumor growth, identifying mechanisms of this resistance such as S-nitrosylation may be a key in cancer progression and the development of chemoresistance. S-nitrosylation is reversible and seemingly a specific post-translational modification that regulates the activity of several signaling proteins. S-nitrosylation of the catalytic site cysteine in caspases serves as an on/off switch regulating caspase activity during apoptosis in endothelial cells, lymphocytes, and trophoblasts [147–149]. Targeting MPO may be a potential therapeutic intervention to reverse the resistance to apoptosis in sensitive and chemoresistant EOC cells.

state through alteration of the redox balance, which is further enhanced in their chemoresistant counterparts, as summarized in **Figure 2**. Forcing ovarian cancer cells to undergo oxidative phosphorylation rather than glycolysis has been shown to be beneficial for eliminating cells via apoptosis (**Figure 2**). Collectively, there is convincing evidence that indicated a causal relationship between the acquisition of chemoresistance and chemotherapy-induced genetic mutations in key redox enzymes, leading to a further enhanced oxidative stress in chemoresistant EOC cells. This concept was further confirmed by the observation that induction of point mutations in sensitive EOC cells increased their resistance to chemotherapy. Also, a combination of antioxidants with chemotherapy significantly sensitized cells to chemotherapy. Identification of targets for chemoresistance with either biomarker and/or screening

New Insights into the Pathogenesis of Ovarian Cancer: Oxidative Stress

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

99

Portions of this chapter contain material that was previously published and is used with permission from Elsevier, IOS Press, and the authors. Reprinted from *Gynecologic Oncology*, 145(3), Saed GM, Diamond MP, Fletcher NM, Updates of the role of oxidative stress in the pathogenesis of ovarian cancer, 2017 Jun;145(3):595-602, with permission from Elsevier, 2017, License number 4091940523932; Reprinted from *Gynecologic Oncology*, 116(2), Saed GM, Ali-Fehmi R, Jiang ZL, Fletcher NM, Diamond MP, Abu-Soud HM, Munkarah AR, Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer, 2010 Feb;116(2):276- 81, with permission from Elsevier, 2017, License 4091940340178; Reprinted from *Free Radical Biology and Medicine*, 102, Fletcher NM, Belotte J, Saed MG, Memaj I, Diamond MP, Morris RT, Saed GM, Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer, 2017 Jan;102:122-132, with permission from Elsevier, 2017, License 4091940462337; Reprinted from *Gynecologic Oncology,* 122(2), Jiang Z, Fletcher NM, Ali-Fehmi R, Diamond MP, Abu-Soud HM, Munkarah AR, Saed GM, Modulation of redox signaling promotes apoptosis in epithelial ovarian cancer cells, 2011 Aug;122(2):418-23, with permission from Elsevier, 2017, License 4091940941920; Fletcher NM1, Jiang Z, Ali-Fehmi R, Levin NK, Belotte J, Tainsky MA, Diamond MP, Abu-Soud HM, Saed GM. Myeloperoxidase and free iron levels: potential biomarkers for early detection and prognosis of ovarian cancer. Reprinted from Cancer Biomark. 2011-2012;10(6):267-75 with permission from IOS Press. The final publication is available at IOS Press through http://dx.doi.org/10.3233/CBM-2012-0255.

and Nicole M. Fletcher1

potential will have a significant impact for the treatment of this disease.

**Acknowledgements**

**Author details**

Ghassan M. Saed1

\*, Robert T. Morris2

\*Address all correspondence to: gsaed@med.wayne.edu

1 Wayne State University, Detroit, MI, USA

2 Karmanos Cancer Institute, Detroit, MI, USA

#### **13. Ovarian cancer immunotherapy and oxidative stress**

It is well established that tumorigenic cells generate high levels of ROS to activate proximal signaling pathways that promote proliferation, survival and metabolic adaptation while also maintaining a high level of antioxidant activity to prevent buildup of ROS to levels that could induce cell death [160]. Moreover, there is evidence that ROS can act as secondary messengers in immune cells, which can lead to hyperactivation of inflammatory responses resulting in tissue damage and pathology [160]. Ovarian cancer is considered an ideal tumorogenic cancer because ovarian cancer cells have no negative impact on immune cells [161].

Effective immunotherapy for ovarian cancer is currently the focus of several investigations and clinical trials. Current immunotherapies for cancer treatment include therapeutic vaccines, cytokines, immune modulators, immune checkpoint inhibitors, and adoptive T cell transfer [162]. The discovery of a monoclonal antibodies (such as bevacizumab) directed against VEGF have been shown to improve progression free survival compared to cytotoxic chemotherapy alone was a major outcome of these clinical trials [163]. Other monoclonal antibodies currently approved for other cancers such as trastuzumab for breast cancer or cetuximab for colon cancer exhibited limited activity in ovarian cancer [163]. Several clinical trials are ongoing for the utilization of immune checkpoint blockade in ovarian cancer immune therapy [164]. Most recently tested were the programmed death (PD)-1 inhibitors, pembrolizumab and nivolumab, which showed a consistent response rate of 10–20% in phase 2 studies and then failed to improve outcomes in confirmatory trials [164]. Ultimately, larger phase 3 studies are needed to validate these findings for checkpoint inhibitors, particularly with regard to the duration of response seen with these agents. Additionally, the direct intraperitoneal delivery of interleukin (IL)-12, a potent immunostimulatory agent, exhibited some potential therapeutic efficacy in ovarian cancer [165]. Recently, targeting folate receptor alpha, which is found to be expressed in ovarian cancer, has shown promising therapeutic value. The targeting of the folate receptor was achieved by either a blocking monoclonal antibody (farletuzumab) or antibody conjugates of folate analogs, such as vintafolide [166].

#### **14. Summary and conclusion**

Oxidative stress has been implicated in the pathogenesis of several malignancies including ovarian cancer. Epithelial ovarian cancer is characterized to manifest a persistent pro-oxidant state through alteration of the redox balance, which is further enhanced in their chemoresistant counterparts, as summarized in **Figure 2**. Forcing ovarian cancer cells to undergo oxidative phosphorylation rather than glycolysis has been shown to be beneficial for eliminating cells via apoptosis (**Figure 2**). Collectively, there is convincing evidence that indicated a causal relationship between the acquisition of chemoresistance and chemotherapy-induced genetic mutations in key redox enzymes, leading to a further enhanced oxidative stress in chemoresistant EOC cells. This concept was further confirmed by the observation that induction of point mutations in sensitive EOC cells increased their resistance to chemotherapy. Also, a combination of antioxidants with chemotherapy significantly sensitized cells to chemotherapy. Identification of targets for chemoresistance with either biomarker and/or screening potential will have a significant impact for the treatment of this disease.
