**11. Chemoresistance is associated with point mutations in key redox enzymes in EOC cells**

genes, and loss of functional p53 that augments NF-κB activation [13, 131]. We have previously shown that chemoresistant EOC cells manifested increased iNOS and nitrate/nitrite levels as well as a decrease in GSR expression as compared to sensitive EOC cells, suggesting a further enhancement of the redox state in chemoresistant cells [1, 45]. Additionally, CAT, GPX, and iNOS were shown to be significantly increased while, GSR, SOD, and the NAD(P)H oxidase subunit (p22phox) were decreased in chemoresistant EOC cells as compared to their sensitive counterparts [21]. These finding supports a key role for oxidative stress, not only in the development of the oncogenic phenotype, but also in the development of chemoresistance (**Figure 2**).

**Figure 2.** Summary of the role of oxidative stress in the development of sensitive and chemoresistant ovarian cancer [1].

**10. Common polymorphisms in redox enzymes are associated with** 

A single nucleotide polymorphism (SNP) occurs as a result of gene point mutations with an estimated frequency of at least one in every 1000 base pairs that are selectively maintained and distributed in populations throughout the human genome [132]. An association

**ovarian cancer**

94 Ovarian Cancer - From Pathogenesis to Treatment

To date, the acquisition of chemoresistance in ovarian cancer is not fully understood. The enhanced oxidant state reported in chemoresistant EOC cells may be linked to point mutations in key redox enzymes [21]. Chemoresistant EOC cells manifested increased levels of CAT, GPX, and iNOS and decreased levels of GSR, SOD, and NAD(P)H oxidase as compared to their sensitive counterparts [21]. Interestingly, chemoresistant EOC cells, and not their sensitive counterparts, 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].

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

acid [55, 150–153]. We, and others, have demonstrated that MPO utilizes NO, produced by

hydrolyzed forming nitrite as end-product [55, 56, 154, 155]. The ability of MPO to generate

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

[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

EOC cells through a mechanism that involved S-nitrosylation of caspase-3 by MPO.

, 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

, in combination with chloride to generate hypochlorous

New Insights into the Pathogenesis of Ovarian Cancer: Oxidative Stress

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

97

●– and other molecules, determines the net effect of NO on apoptosis

, a labile nitrosating species that is rapidly

S-nitrosothiol (SNO) formation *in vivo* are not well understood.

iNOS, as a one-electron substrate generating NO+

, O2

O2

Myeloperoxidase typically uses H2

NO+

combination with O2

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 signature mutations for oxidants and free radicals [21].

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 chemoresistance in nature [21].
