The role of oxidative stress in ovarian cancer pathogenesis

Ovarian cancer is the deadliest form of gynecological malignancy. The presence of non-specific symptoms, together with the identification of the disease in an advanced stage, are the primary determinants of an unfavorable prognosis, resulting in an overall five-year survival rate of 45%. Currently, the treatment of choice for ovarian cancer  is surgery aimed at completely removing the illness macroscopically, along with adjuvant chemotherapy. The significance of early diagnosis is evident in the several conducted prospective studies, which have not identified a successful method for early identification of this disease. Oxidative stress (OS) is a well-established contributor to carcinogenesis in several types of malignancies. It occurs when there is an imbalance between the production and clearance of reactive oxygen species. These molecules affect both pathological and physiological processes. Literature findings indicate that oxidative stress has a role in the development and pathophysiology of ovarian cancer by affecting the following four areas of cellular functioning: (i) genetic alterations, (ii) signaling pathways, (iii) transcription factors, and (iv) tumor microenvironment. Reactive oxygen species can induce mutations, which initiate the formation, proliferation, and maturation of tumor tissue. Also, prolonged elevation of the concentration of free radicals harmfully affects cells, causing the initiation of apoptotic pathways. Specifically, the presence of free radicals leads to the impairment of biomolecules and the creation of byproducts from this interaction. Consequently, these compounds may be found in various body structures and products such as tissue, plasma, urine, and other, where they can be detected. The study aims to enhance the readers’ understanding of the pathogenesis of ovarian cancer, which is characterized by its undetermined cause and diverse nature. Previous research has primarily focused on surgical or medicamentous treatment, therefore the present study aims to shed light on the role of oxidative stress in this process.

INTRODUCTION

Ovarian cancer is the deadliest gynecological malignancy. Worldwide, 230,000 women are diagnosed with ovarian cancer annually, while 150,000 women lose their lives as a result of this disease [1]. Regardless of the great efforts of the scientific and academic community to fully understand the underlying mechanisms of this disease, ovarian cancer is the seventh most frequently diagnosed cancer in the female population, while the five-year survival rate is currently only about 45% [2]. Late detection and non-specific complaints that occur in the advanced stages of the disease are the main cause of an unfavorable outcome.

Histologically, ovarian tumors can originate from all three germ layers. Epithelial ovarian cancer ranks first in terms of frequency, while non-epithelial ovarian tumors make up a significantly smaller percentage of this disease. Risk factors for ovarian cancer include benign gynecological conditions such as endometriosis, polycystic ovary syndrome, and pelvic inflammatory disease, as well as low parity, early menarche, and late menopause. The first-line therapy is primary debulking surgery, which aims to achieve zero residual disease in the abdomen. It is usually followed by systemic chemotherapy composed of alkylating agents and taxane-based preparations [3].

EPIDEMIOLOGY OF OVARIAN CANCER

Ovarian cancer belongs to the group of rare diseases with an incidence of 12 cases per 100,000 women [4]. Epithelial ovarian cancer makes up most of the diagnosed cases, as compared to germinal and gonadal cord tumors, with a frequency of about 90% in the population of women suffering from ovarian tumors. The characteristics of the usual profile of women suffering from ovarian cancer are the following: 50 years of age, nulliparity, the absence of a lactation period, and a long period of continuous ovulation. Most cases of ovarian cancer occur sporadically, while 10% - 15% of cases have a hereditary component [5]. Within the group of patients suffering from hereditary forms of the disease, 65% - 75% of cases are caused by BRCA 1 and BRCA 2 gene mutations, with a small percentage of hereditary forms associated with Lynch syndrome [6].

IMPORTANCE OF EARLY DIAGNOSIS

Given that the disease is most often diagnosed in an advanced stage, with already existing distant metastases on the surface of the intestine, the liver capsule, and within the lymph nodes, a lot of effort has been put into discovering effective screening methods for ovarian cancer. Unfortunately, the assumption that early detection can reduce mortality and morbidity was quickly refuted by a large cohort study conducted in the United Kingdom, wherein over 200,000 women were randomized into three groups. The first group was monitored by determining the serum level of carcinoembryonic antigen 125 (Ca-125); the second group was monitored with regular annual ultrasound examinations, while the third group was monitored passively. Throughout the study, a reduction in mortality by 15% and 11% was recorded for the groups of patients monitored with the Risk of Ovarian Cancer Algorithm (ROCA) and for patients designated for regular ultrasound follow-ups, as compared to the population of women who were not monitored with any specific screening [7]. However, these results were not statistically significant.

As opposed to screening performed in the population of healthy women, a study conducted in America included 2,359 women with a positive family history of breast or ovarian cancer or the existence of BRCA 1 and BRCA 2 gene mutations. The primary aim of this study was to determine the positive predictive value and specificity of the tests for the prevention of ovarian cancer. The patients were followed up for nine years with the help of the ROCA test. Using this test, 18 out of a total of 19 cases of ovarian cancer were detected in the examined cohort, with the measured specificity being 90% in the general population, i.e., 92% in the high-risk patient population [8]. Currently, the only recommendation of the European Society of Gynaecological Oncology (ESGO) for preventing the development of tumors and for early detection of the disease is the application of prophylactic or risk-reducing removal of the fallopian tubes on both sides, in accordance with the hypothesis that epithelial ovarian cancer is transferred from the fimbrial part of the tubes to the surface of the ovary and then spreads further within the abdominal cavity.

PATHOGENESIS OF OVARIAN CANCER

The dualistic model of epithelial ovarian carcinogenesis, based on a molecular-genetic basis, as opposed to the usual morphological one, has been established for two decades [9]. Attempting to make a more definitive grouping, in an otherwise extremely heterogeneous group of tumors, has long been a major scientific challenge. Moreover, literature describes numerous attempts to classify ovarian tumors based on structural, pathohistological, molecular, genetic, and antigenic composition. This dualistic classification describes two tumor populations. The characteristics of Type I carcinoma are the following: unilateral occurrence, cystic nature, large size, and low-grade histology of the tumor. On the other hand, Type II carcinomas usually present at a late stage, they account for over 75% of cases, and are histologically characterized by a high grade as well as a severe clinical presentation of the disease [10].

In addition to the difference in the aggressiveness and invasiveness of the disease, one of the main characteristics of the principle behind the dualistic categorization of the tumors is based on the presence of precursor lesions. Namely, in Type I carcinoma, there is a sequential cascade, i.e., the development of borderline or atypical lesions, which are of similar etiopathogenesis as in cervical or colon cancer. In contrast, Type II carcinoma is characterized by the development of de novo tumor lesions, which are, according to the latest research, usually located on the fimbrial part of the fallopian tube, designated as intraepithelial carcinoma lesions [10].

GENETIC FACTORS

Mutations in the KRAS, BRAF, and ERBB2 genes result in the activation of the MAP kinase signaling pathway (mitogen-activated protein kinase) [11]. The MAP kinase pathway plays a key role in transmitting signals induced by growth factors, which consequently leads to neoplastic transformation. KRAS and ERBB2 mutations occur relatively early in oncogenesis, and they lead to the upregulation of the glucose transporter, with a consequent increase in the intensity of the glucose metabolism, which is necessary for malignant transformation [12].

By changing resection techniques and the techniques of the pathohistological examination of fallopian tube preparations in patients with the BRCA mutation, the presence of precursor lesions of high-grade serous epithelial ovarian cancer has been observed. In contrast to Type I cancers, tumors of high malignant potential are characterized by mutations in the TP53, BRCA, and PTEN oncogenes, with widespread structural genomic variations [13].

HORMONAL FACTORS

The ovaries are the primary reproductive organs wherein estrogen, progesterone, and testosterone production occurs [14]. Estrogens affect numerous physiological and pathophysiological processes. There are classical (Erα, Erβ) and non-classical (G protein-coupled estrogen receptor – GPER1) estrogen receptors [15]. Despite the common belief that cholesterol-derived hormones, due to their liposolubility, easily penetrate the cell membrane and have receptors in the cytoplasm, numerous studies have shown the presence of a G protein-coupled estrogen receptor on the surface of the cell membrane. After the binding of estradiol to the GPER1 receptor, a downstream signaling cascade is triggered that leads to the activation of PI3K/Akt and ERK/MAPK signaling pathways, involved in the process of oncogenesis [16]. Yan et al., in a 2005 study, showed that exposure to selective agonists of GPER1 receptors leads to the modulation and proliferation of cell lines through the production and activation of matrix metalloproteinase 9 (MMP-9) [17].

ENVIRONMENTAL FACTORS

Substances present in the environment as the result of pollution, or those that make up various cosmetic or food additives, can affect hormone-dependent tissues due to their structural and functional similarity to natural estrogens. These substances are called xenoestrogens and can bind directly or indirectly to estrogen receptors. Numerous studies conducted on different cell lines describe the different effects of environmental factors on ovarian cancer cells. Genistein belongs to the group of isoflavonoids, which activate signaling pathways associated with ER receptors [18]. Compounds such as phytoestrogens lead to the altered modulation, activation, and expression of the Erβ receptor, which results in an altered response of the PI3K/AKT signaling pathway [19]. It has also been shown that the ubiquitous compound bisphenol A, which is released when plastic packaging is heated, induces the proliferation of ovarian cancer cell lines by regulating the Erα signaling pathway [20].

OXIDATIVE STRESS

A key characteristic of free radicals is the existence of unpaired electrons in molecules, atoms, and ions, which makes them highly reactive. The presence of an unpaired electron was proved by Fenton, as early as 1894, when he described a reaction wherein a small amount of violet light is released in the presence of iron ions [21]. Reactive oxygen species (superoxide anion, hydrogen peroxide, and the hydroxyl radical) influence both physiological and pathological processes. As early as 2007, it was shown that redox-dependent signaling pathways influence the activity of transcription factors and thereby alter the cellular response. It has been shown that when a suitable stimulus acts on the cell, a small amount of hydrogen peroxide is created, which then modulates the activity of certain signaling pathways, thus indicating that reactive oxygen species, in addition to their basic role, also play the role of secondary messengers in the cell [22]. Namely, free radicals and hydrogen peroxide can lead to the activation of tyrosine kinases and the subsequent stimulation of MAP (mitogen-activated protein kinase) kinases and phospholipase C. According to one theory, as a consequence of the activation and production of a larger quantity of free radicals inside the cell, activation of transcription factors occurs, which can be significant for the process of oncogenesis.

DEFINITION OF OXIDATIVE STRESS AND ITS EFFECT ON THE BODY

Oxidative stress is defined as a disruption of the balance between the production of free radicals and their elimination by protective mechanisms, i.e. antioxidants. This imbalance leads to the damage of biomolecules and cells, with a potentially harmful effect on the whole body [23]. Atoms, molecules, or ions with one or more unpaired electrons in their structure are very unstable and very reactive, which is why they can cause a chain reaction of new free radicals in the body. The instability of these compounds stems from the need for the unpaired electron or electrons to become paired, whereby electrons are “stolen” from other compounds, thus creating new radicals and damaging various cell structures.

According to their origin, free radicals in the body can be divided into two large groups. The first group includes reactive oxygen species formed on the mitochondrial respiratory chain, while the second group includes radicals originating from the metabolism of xenobiotics. During endogenous aerobic metabolic reactions, reactive oxygen species (ROS) are produced in the mitochondria, such as superoxide anion and hydroxyl radical, which are products of partial reduction of molecular oxygen [24].

Numerous positive effects of small concentrations of ROS enable the proper functioning of the organism. However, the scientific community is especially interested in the connection between oxidative stress and inflammation, which is one of the main mechanisms of many chronic diseases. Although oxidative stress was previously viewed exclusively as a pathological process, today the focus has shifted towards discovering an increasing number of physiological roles of free radicals.

Antioxidative mechanisms of protecting the organism from oxidative stress can be endogenous and exogenous. Endogenous systems of antioxidant protection can be enzymatic and non-enzymatic. The endogenous enzymatic system of antioxidant protection is produced by the cells of the organism and their role is to defend against the unwanted effects of the primary partial products of oxygen reduction (superoxide anion and hydrogen peroxide) and includes the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. The main endogenous non-enzymatic antioxidant is glutathione, an intracellular thiol compound that prevents oxidation of the thiol groups of enzymes and proteins.

OXIDATIVE STRESS AS A RISK FACTOR FOR OVARIAN CANCER

The role of oxidative stress in the pathogenesis of ovarian cancer is dualistic in nature. Reactive oxygen species can lead to mutations that cause tumor initiation, growth, and progression. Long-term increased concentration of free radicals has a cytotoxic effect, which leads to the activation of apoptosis pathways [25]. Due to the high degree of activity and proliferation, tumor cells have to find ways to deal with the high concentration of reactive molecules, i.e., to avoid the unwanted effects of reactive oxygen species that would lead to the initiation of apoptosis or ferroptosis [26].

Oxidative stress participates in the etiopathogenesis of ovarian cancer through four aspects: (i) development of a genetic alteration, (ii) modification of signaling pathways, (iii) activity of transcription factors, (iv) tumor microenvironment [27]. DNA strand damage and genetic instability that occur as the result of oxidative stress play a key role in the onset and development of ovarian cancer. The most common product of oxidative DNA damage is 8-hydroxy-2-deoxyguanosine (8-OHdG), which is formed by the oxidation of guanine [28]. Namely, the interaction of the hydroxyl radical with the nucleobases of the DNA chain leads to damage that causes the formation of promutagenic lesions, which contribute to the process of carcinogenesis, confirmed in patients with bladder, kidney, and other tumors [29],[30]. In addition, Valavanidis et al. showed the presence of 8-OHdG in urine as a possible marker of oxidative DNA damage in patients with ovarian cancer [31].

EFFECT OF OXIDATIVE STRESS ON PROLIFERATION, INVASION, AND METASTASIS OF OVARIAN CARCINOMA

Mitochondrial DNA (mtDNA) is particularly important in the process of oxidative stress. Furthermore, research has shown that the presence of free electrons originating from the respiratory chain (electron transport chain), as well as the absence of the usual DNA chain repair mechanisms, makes mtDNA particularly sensitive to oxidative stress. Ni et al. showed that highgrade serous ovarian cancer with mtDNA mutations has a higher frequency of platinum resistance as well as a higher incidence of disease relapse, as compared to the group of patients without somatic mtDNA mutations [32]. By analyzing tumor tissue sections and examining somatic mutations resulting from oxidative damage, insight into the exposure of tumor cells to oxidative stress can be obtained. The tumor microenvironment is a space where tumor cells interact with host cells, i.e., cells of the tissue stroma, including fibroblasts, endothelial cells, and various cells originating from the immune system. Oxidative stress acts on this microenvironment through tumor-associated neutrophils and macrophages, T regulatory cells, as well as many other components of the extracellular matrix [27],[33].

Ascites, i.e. the presence of free fluid in the peritoneal cavity, is a key factor in the tumor microenvironment in ovarian cancer. Via the liquid, the disease characteristically spreads throughout the abdomen, counterclockwise. Namely, Pakula et al. have shown that malignant ascites originating from a serous ovarian tumor leads to oxidative stress in peritoneal mesothelial cells, through the induction of cytochrome c oxidase and NADPH dehydrogenase, resulting in the aging and inactivation of these cells. The loss of these cells leads to the development of adhesions, as well as to the proliferation and general migration of ovarian cancer cells in the abdomen [34].

OXIDATIVE STRESS AND TREATMENT IMPLICATIONS

As noted earlier, the role of oxidative stress in ovarian cancer is dualistic in its nature. On the one hand, oxidative stress, due to its promutagenic potential and the possibility of oxidative damage to macromolecules, is linked to the process of carcinogenesis, which indicates that an increase in antioxidant capacity and a decrease in oxidative stress can affect the prevention of ovarian cancer. On the other hand, the treatment approach in patients suffering from ovarian cancer is aimed primarily at increased production of free radicals and increased oxidative stress, for the purpose of inducing apoptosis in tumor cells and preventing the spread and progression of the disease in the abdomen.

The use of chemotherapy drugs whose action is based on changing the redox balance of tumor cells is not a new concept [27]. Medications belonging to this group of drugs are known to increase the intensity of oxidative stress and inflammation and lead to the induction of apoptosis in tumor cells. The treatment of choice for ovarian cancer is the combined use of platinum preparations and taxanes. Platinum is known to induce the production of reactive oxygen species, while taxanes have a lesser effect on the production of these compounds [35].

Drugs known as PARP (poly‐ADP‐ribose polymerase) inhibitors affect the ability of the tumor cell to repair damage to its DNA strand. The use of this group of drugs is currently limited only to patients with BRCA mutations. In addition to their basic mechanism of inhibiting DNA strand repair enzymes, these drugs also lead to the up-regulation of NADPH oxidase 1 and 4, leading to antitumor effects by increasing oxidative stress in cancer cells [36].

It is known that there are many preparations on the market with more or less proven antioxidant effects. Numerous natural plant extracts are used to increase antioxidant capacity. It has been shown that Ganoderma lucidum leads to the induction of antioxidant enzymes superoxide dismutase, catalase, and glutathione S-transferase P1 via the NRF-2 signaling pathway, preventing the promutagenic effects of oxidative stress [27].

Ovarian cancer still represents a great challenge in all respects – prevention, diagnosis, and therapy. Great efforts are being made to discover new approaches to the treatment of this serious disease. Namely, research was conducted regarding the role of nanoparticles as a therapeutic modality in ovarian cancer, primarily due to their small dimensions and uniform distribution. Bai et al. described the association between the application of zinc oxide nanoparticles and the process of apoptosis and autophagy in an ovarian cancer cell line. The size of the particles was 20 nm on average, which enabled efficient penetration and a significant degree of cytotoxicity through a dramatic decrease in the amount of intracellular glutathione with a consequent decrease in antioxidant potential, which resulted in a significant increase in the sensitivity of these cells to oxidative stress [37].

It is possible to assume that there is a decisive moment in the pathogenesis of ovarian cancer, after which the mechanisms of protection against tumor promotion break down. Oxidative stress is considered to play a very significant role in various mechanisms that contribute to that moment, including the occurrence of DNA mutations, hypomethylation of certain DNA regions, somatic mutations of mtDNA, as well as changes in the activity of a large number of redox-dependent signaling pathways.

BIOMARKERS OF OXIDATIVE STRESS IN OVARIAN CANCER

Biomarkers are certain characteristics that are measured as indicators of physiological and pathological processes or responses to exposure or a procedure, including response to treatment. Biomarkers may include molecular, histological, radiographic, or physiological indicators, but do not include an assessment of the patient’s subjective symptoms. Most of the biomarkers associated with oxidative stress meet the basic criterion of being measurable. The results of numerous studies have linked oxidative stress to a large number of human diseases. However, for certain compounds to be classified as clinically relevant biomarkers, they must meet certain criteria such as: (i) prognostic value, (ii) clear correlation with disease progression, and (iii) specificity, i.e. diagnostic value for a specific disease [38].

Zheng et al. conducted research on the development of a scoring system for a risk model based on the expression level of genes associated with oxidative stress. Namely, the data obtained from the database on sequenced single-cell RNA were used to form 12 clusters, which were then evaluated by regression analysis. Among the initial 151 genes, the analysis detected nine prognostic gene markers, based on which risk scoring models were constructed. It was shown that the value of the score within this risk model predicted the outcome of the application of immunotherapy drugs, as well as that it could represent an independent prognostic factor for ovarian cancer [39].

Recently, the analysis of genes associated with oxidative stress has attracted considerable attention from the scientific community. More and more resources are being invested in the analysis and evaluation of large databases, in an attempt to obtain answers to the many questions regarding ovarian cancer. One such study was conducted by Liu et al., who analyzed the Omnibus gene database and identified 34 genes associated with the prognosis of ovarian cancer and oxidative stress. To assess the risk, a score was generated wherein 14 genes were included, showing that patients with a low score could have a better response to therapy, as compared to the group of patients who had a high score [40].

It is known that under the influence of free radicals, oxidative damage to biomolecules occurs and indicators of oxidative damage develop, which can result in the appearance of these compounds in tissue, plasma, urine, and other body fluids, where they can be identified and measured.

Lipid peroxidation is a process wherein the hydroxyl radical reacts with lipids, resulting in a lipid radical and water. Lipid peroxidation products are detected in blood plasma and used as oxidative stress biomarkers. Most commonly, the concentration of malondialdehyde, a marker of oxidative damage to polyunsaturated fatty acids, is measured.

Oxidized low-density lipoproteins (LDL) have also been identified in literature as biomarkers of oxidative stress, ever since the initial hypothesis about the importance of oxidative stress in the development of atherosclerosis was put forward. Namely, the oxidized form of LDL (oxLDL), as a potential biomarker for cardiovascular diseases, has been analyzed in numerous studies [41]. It has been shown that oxLDL most likely cannot represent a specific measure of the degree of oxidative stress, due to the limitations of the assays used in determining the concentration of this marker [42].

The degree of damage to DNA strands due to the presence of free radicals is related to the efficiency of DNA repair mechanisms. In conditions when the concentration of free radicals is low, there is a complete repair of DNA structures, which enables the proper functioning of the cell. However, in a state of redox imbalance, various lesions of the DNA chain occur, ranging from single-strand or double-strand breaks to the so-called oxidatively generated clustered DNA strand lesions [43]. As previously mentioned, one of the basic biomarkers of oxidative DNA strand damage is 8-hydroxy-2-deoxyguanosine (8-OHdG). Jelić et al. investigated the existence of a link between oxidative stress and cervical cancer. In a study including 153 female patients, they investigated the activity of antioxidant enzymes, as well as the concentration of 8-OHdG. It was shown that patients in the advanced stage of the disease had a statistically significantly higher activity of catalase, superoxide dismutase, and glutathione-S-transferase, as well as higher levels of 8-OHdG in the urine [44].

In Finland, in 2015, a group of authors conducted a retrospective study wherein serum 8-OHdG levels were analyzed in patients with ovarian cancer. The study was conducted on 112 subjects and showed that patients with higher 8-OHdG levels had significantly shorter overall survival, as well as a shorter disease-free period with a higher frequency of platinum resistance, as well as suboptimal surgical treatment [45]. However, a group of authors from Japan analyzed the level of 8-OHdG and heme oxygenase-1 activity, as well as total antioxidant capacity in a group of patients with benign endometriosis and endometriosis associated with ovarian cancer. This study showed that the values of 8-OHdG were statistically significantly lower in the group of patients with ovarian cancer, as compared to the group with benign endometriosis, which indicates contradictions regarding the role of 8-OHdG in the carcinogenesis of ovarian cancer [46].

Most of the protein modification processes caused by oxidative stress are based on carbonylation, glycation, and the production of advanced glycosylation products (AGEs). Oxidative deamination of lysine and glutamic acid can result in the formation of protein carbonyl groups. Since carbonyl groups can be formed through different mechanisms, their concentration is usually higher, as compared with other biomarkers of oxidative stress [47].

Although there are no direct data in literature on the prognostic significance of protein oxidation products in ovarian cancer, there are research results for other cancers. Namely, Gil et al. reported significantly higher levels of protein carbonyl groups in patients with hepatocellular carcinoma, as compared to the group of patients with hepatitis B [48]. Also, in 2004, a group of researchers analyzed the relationship between androgen levels in patients with polycystic ovary syndrome (PCOS) and the levels of AGEs. It was shown that androgen levels were significantly higher in the group of female patients with elevated AGEs [49]. In addition, Mažibrada et al. examined the levels of high-sensitivity C-reactive protein and fibrinogen in a group of normoinsulinemic, non-obese adolescent girls with PCOS. Research has shown that inflammatory markers can be useful in the follow-up of adolescent girls of normal body weight with PCOS, for the purpose of the timely prevention of unfavorable changes in body weight and the lipid profile, which may be in correlation with the results of previously mentioned studies [50].

CONCLUSION

Given the distinct degree of heterogeneity of ovarian cancer, both from a genetic and molecular perspective, as well as from the point of view of clinical presentation and response to treatment of these tumors, there is still a lot of controversy regarding pathogenesis. Among other things, oxidative stress can initiate a cascade of events based on DNA strand damage, changes in the tumor microenvironment, as well as on redox-dependent signaling pathways, and intracellular second messengers, which can result in malignant alteration of ovarian tissue. The currently available methods of ovarian cancer prevention are based on regular follow-up of women who are at increased risk of developing this disease, with possible prophylactic surgical treatment. All these facts are an incentive for future research into the role of oxidative stress in ovarian cancer, through further individual clarification of the numerous components of this complex disease.