Known as the silent killer, ovarian cancer (OC) has the lowest survival rate and the worst prognosis among all gynecologic malignancies in the US; and is the eighth most common cancer in women worldwide [1,2]. In 2022, the American Cancer Society estimates about 21,000 new cases of OC will be diagnosed, and approximately 14,000 women will die from this type of cancer. The overall 5-year survival rate is only 48% due to OC’s ambiguous symptoms and inadequate screening capabilities at the early stages of the disease. Due to late detection, about 60% of new cases are diagnosed when the disease has already progressed to the advanced stage [2]. OC is a heterogeneous disease with several subtypes that differ in their gene expression, tumor origin, pathway alterations, and pathogenesis. The majority of OC originates from three main cell types: epithelial cells (90%), stromal cells (7%), and germ cells (3%) [1,3,4]. In general, epithelial OC can be further divided into five histotypes: high-grade serous (HGSOC; 70%), endometrioid (ENOC; 10%), clear cell (CCOC; 10%), mucinous (MOC; 5%), and low-grade serous (LGSOC; less than 5%) OC [4]. In addition, another classification system was introduced a decade ago that divided OC into type I and II tumors. Type I tumors are low-grade neoplasms, including mucinous carcinomas, endometrioid carcinomas, malignant Brenner tumors, and clear cell carcinomas. Type I tumors are typically characterized by mutations in BRAF, KRAS, and PTEN with DNA instability. Type II tumors are high-grade serous carcinoma, carcinosarcoma, and undifferentiated carcinoma, which are frequently observed with mutations in p53, BRCA1/2, HER-2/HER-3 overexpression, and p16 inactivation [5,6,7,8]. Depending on the specific subtype and histopathology, OC treatment involves a combination of surgery and chemotherapy. For patients with advanced-stage tumors, debulking surgery is recommended; however, large tumors or residual tumors may show negative side effects leading to blockage of the perfusion area and the possibility of developing drug resistance [1,9]. Platinum-based chemotherapy is the standard line of treatment for OC, either in conjunction with or following surgery [10,11,12]. The combination of paclitaxel/carboplatin has been recognized as the standard postoperative chemotherapy for many years [13]. In recent years, PARP inhibitors have been incorporated into clinical treatment as a recommended maintenance drug [14]. However, due to the aggressive growth rates and the propensity of advanced tumors to evade treatment, there are critical limitations to the current lines of therapy. A better understanding of the molecular biology of OC is allowing more research efforts to establish new effective treatment options for advanced-stage tumors.

Reactive oxygen species (ROS) have remained a highly relevant topic over the last few decades due to their expansive effects on normal cellular function. Oxidative stress is generated through the accumulation of ROS, either through exogenous exposure or endogenous production. ROS are oxygen ions with unpaired electrons (singlet oxygen ¹O₂, superoxide O₂·⁻) or oxygen-containing molecules, such as hydroxyl radicals (OH·⁻), hydrogen peroxide (H₂O₂), nitric oxide (NO), and nitrogen dioxide (NO₂) [15]. Superoxide radicals are converted into H₂O₂ by the enzyme superoxide dismutase (SOD). However, superoxide can also react with nitric oxide to produce peroxynitrite (ONOO⁻), a strong oxidizer with damaging cellular effects [16]. The accumulation of H₂O₂ has detrimental effects on nuclear and mitochondrial DNA, which may lead to genetic instability to drive cancer progression with increased expression of oncogenes and decreased expression of tumor suppressors [17,18]. Several enzymes work in conjunction to convert H₂O₂ into the water, including catalase, glutathione peroxidases 1 and 4, and peroxiredoxins 3 and 5 [19,20,21,22]. Furthermore, H₂O₂ can also participate in the Fenton reaction, in which free iron Fe(II) reacts with H₂O₂, generating highly reactive hydroxyl radicals (·OH)(shown below). The production of hydroxyl radicals (·OH) by the Haber–Weiss reaction (shown below) further perpetuates the damaging effects of the accumulation of ROS.

Fenton Reaction:

Haber–Weiss Reaction:

Original studies implicated the mitochondria as primary endogenous sources of superoxide through the process of cellular respiration, a process dependent on the availability of O₂ [23,24,25]. Based on this view, the production of ROS was thought to be a harmful by-product of intracellular metabolism. Then a family of transmembrane enzymes known as NADPH oxidase (NOX) proteins was identified, whose primary function was the production of endogenous ROS. NOX2, the first NOX protein discovered, was the primary producer of endogenous ROS in leukocytes to generate an oxidative burst, an essential process for the neutralization of pathogens [26,27,28,29]. The characterization of a disease called chronic granulomatous disease (CGD) caused by a mutation in the phagocytic NOX gene provided insight into the emerging role of endogenous ROS production on cellular functionality [30,31]. Subsequent work demonstrated a pivotal role of NOX proteins in mammalian cell transformation through the production of superoxide radicals and H₂O₂ [32,33]. Our group demonstrated that the accumulation of ROS in OC cells was attributed to H₂O₂ increased levels induced by NOX4 [34], identifying an endogenous mechanism for the overproduction of ROS and alteration of intracellular signaling in OC tumor development.

Under normal cellular conditions, low levels of endogenous ROS activate several signaling pathways involved in cell proliferation. However, the accumulation of ROS causes extensive damage to DNA, RNA, proteins, and lipids, thus causing a significant hindrance to normal cellular functions and contributing to the development of multiple human pathologies [35,36,37,38]. The damage can induce cell death pathways or trigger the mutation of DNA, as commonly found in cancer [39,40]. In addition to the endogenous production of ROS and oxidative stress, external or environmental exposure to ROS can have detrimental effects on mammals [41]. For instance, many chemotherapeutic agents induce oxidative stress as a means of inducing cellular damage and cell death pathways [42]. However, as demonstrated by more recent findings, ROS play an important role in the progression and advancement of human diseases. The counterweight for endogenous ROS is the genetically programmed redox system. This includes groups of genes coding for antioxidant proteins such as superoxide dismutase (SOD), catalase, and the glutathione system, which neutralize the ROS produced in cells [43,44,45]. The failure to neutralize endogenous ROS leads to a build-up of harmful oxygen species and, consequently, oxidative stress. In normal cells, oxidative stress leads to deleterious cellular effects, such as protein, lipid, and DNA damage, organelle dysfunction, and cell cycle arrest [46]. Higher levels of oxidative stress cause the activation of cell death pathways such as apoptosis and necrosis [46], which may be mitigated in cancer cells by an increase in antioxidant production. The upregulation of nuclear factor erythroid 2-related factor 2 (NRF2), a master transcriptional regulator of antioxidant genes, contributes to the neutralization of endogenous ROS in OC cells [47,48,49], making NRF2 a viable target for chemotherapeutic treatment in certain cases of OC. In addition, the genetic mutation of cellular pathways that induce cell death mechanisms in response to increased oxidative stress allows cancer cells to evade the activation of cell death pathways [50], thus providing cancer cells the ability to continue continuous proliferation in the presence of adverse cellular conditions, such as oxidative stress.

There is an established link between an increase in ROS production and cancer development in humans [51]. As secondary cellular signaling molecules, ROS are involved in the activation of several signaling pathways involved in cell proliferation and growth. Consequently, these pathways are constitutively activated in cancer cells with increased ROS levels that contribute to tumorigenesis [51]. For example, endogenously derived ROS activate the ERK1/2 MAPK signaling pathway and the AKT signaling pathway in OC, both of which promote cell proliferation [52,53]. The increased ROS generation also contributes to a genetic mutation in cancer cells, further contributing to cell transformation [54,55]. As opposed to the traditional view of ROS generation in cancer as a harmful secondary by-product, the increasing knowledge of cancer cell metabolism and signal transduction is exposing ROS as a positive contributing factor in tumorigenesis and cancer development. The increased metabolic activity of cancer cells was originally thought to be responsible for the accumulation of ROS as a byproduct of increased glycolytic metabolism and mitochondrial respiration [56]. However, the discovery of the role of NOX proteins in endogenous ROS production revealed a more important role for ROS production in non-phagocytic cells, particularly in cancer [57,58,59]. The endogenous production of ROS by NOX1 was found to be responsible for increased viability and proliferation in colon cancer [60,61]. Similarly, the role of NOX2-mediated ROS production was discovered to be critical for cell viability and proliferation in breast, colorectal, myelomonocytic leukemia, gastric, and prostate cancers [62,63,64,65,66,67]. NOX4 overexpression contributed to an oncogenic proliferation in renal cell carcinoma, melanoma, glioblastoma, ovarian, prostate, and lung cancers [34,68,69,70,71,72]. In OC cell lines, there is a significant increase in ROS production, which contributes to tumorigenesis [34]. The increase in ROS is a result of NADPH oxidase activity and mitochondrial metabolism, as this increase is diminished by NADPH oxidase and mitochondrial complex I inhibitors [34]. Moreover, the increased levels of ROS result from the upregulation of the NADPH oxidase subunit NOX4, which serves as the main contributor to ROS production in OC cells to promote tumor growth and angiogenesis [34]. Furthermore, the activation of NOX4 is positively correlated with TGF-β1 and NF-κB activity, which is suppressed by their inhibitors [34]. This system demonstrates that endogenous NOX4-derived ROS are a driving force in OC development. Moreover, NOX4 is a potential target for the therapeutic resistance of OC which is dependent on ROS production for an increase in oncogenic signaling.