Reactive oxygen species (ROS), the partially reduced metabolites of oxygen that possess strong oxidizing capabilities, are deleterious to cells at high concentrations but at low concentrations, they serve complex signaling functions. Reactive oxygen species formed as byproducts of normal cell metabolism are needed for maintaining homeostasis and cellular signaling. Apart from cellular metabolism they are generated by specific plasma membrane oxidases in response to growth factors and cytokines and serve as secondary messengers in specific signaling pathways and play a role in regulating gene expression [1]. Cells have a defense system to maintain ROS at physiologically normal levels, i.e., enzymes called antioxidants, responsible for transforming free radicals into stable, less damaging molecules, the impairment of which may lead to a state of oxidative stress [2]. These oxygen scavenging pathways include conversion of O₂⁻ to H₂O₂ by superoxide dismutase (SOD), the action of catalase on H₂O₂ to produce H₂O and O₂, decomposition of H₂O₂ and LOOH by Glutathione peroxidase, and the reduction of H₂O₂ by Thioredoxin reduction cycle to produce H₂O and also the exogenous detoxification of glutathione transferase [3]. Cancer cells are highly metabolically active and hypoxic cells, and due to massive growth and insufficient vascular irrigation tend to produce increased ROS, which damages DNA by diffusing through the mitochondrial membrane while also acting as signal-transducing messengers in many redox-sensitive molecular pathways involved in cell survival, therapeutic resistance, and progression [3]. Oxidative stress plays a major role in cancer hallmarks like angiogenesis, invasiveness, stemness, and metastatic ability, and hence, reducing oxidative stress with powerful antioxidants has been used as an important strategy for cancer prevention. Additionally, cancer cells develop mechanisms of keeping the increased oxidative stress in check. Therefore, some cancer therapeutic strategies also work by disrupting this check and making the cancer cells susceptible to apoptosis.

2. Source of Reactive Oxygen Species in Cancer Cells

2.1. Mitochondrial ROS

Mitochondria is one of the most prominent sources of reactive oxygen species within a cell which contribute to oxidative stress [4]. The electron transport chain located on the inner mitochondrial membrane generates the majority of mitochondrial ROS during the process of oxidative phosphorylation (OXPHOS). Leakage of electrons at complex I and complex III from ETC leads to a partial reduction of oxygen to form superoxide which undergoes spontaneous dismutation to hydrogen peroxide, both of which are collectively considered as mitochondrial ROS [5]. Endogenous modulators such as NO and Ca²⁺ have been observed to regulate the production of mtROS by regulating the metabolic states of mitochondria. The mitochondrial Ca²⁺ levels increase the rate of electron flow in the ETC and thus decrease mtROS generation [6]. However mitochondrial Ca²⁺ overload increases mtROS production [7]. STAT3, a transcription factor that regulates gene expression in response to cytokines interleukin (IL)-6 and IL-10, also modulates the activity of the ETC ^[8][9][8,9]. Hence a decrease in expression of STAT3 may be correlated to increasing mtROS at complex I [8]. TNF-α that causes the shedding of TNF-α receptor-1 reducing the severity of microvascular inflammation, has been found to induce a calcium-dependent increase in mt ROS [10]. Studies have shown that many ROS-producing enzymes, like NADPH oxidase, xanthine oxidase, and uncoupled eNOS, can stimulate mtROS production in a process called “ROS-induced ROS” ^[11][12][13][11,12,13]. Another transcription factor hypoxia-inducible factor 1α (HIF-1α) also plays a prominent role in bringing about a reduction in ROS by a number of mechanisms including induction of pyruvate dehydrogenase kinase 1 (PKD1), which shunts pyruvate away from the mitochondria; triggering mitochondrial selective autophagy; and induction of microRNA-210 blocking OXPHOS [14]. Low levels of mtROS regulate the stability of HIF-1α leading to hypoxia adaptation while moderate levels of mtROS have been found to regulate the production of proinflammatory cytokines by directly activating the inflammasome and mitogen-activated protein kinase (MAPK). However, high levels of mtROS are capable of inducing apoptosis by oxidation of the mitochondrial pores and autophagy by the oxidation of autophagy-specific gene 4 (ATG4) [5]. Depending on the tumor cell microenvironment, the c-Myc gene controls apoptosis by inducing aerobic glycolysis and/or OXPHOS which is required for the activation of certain tumor suppressor proteins, such as Bax and Bak ^[15][16][17][15,16,17]. Mitochondria also play an important role in the loss of caveolin 1 (cav-1) in the tumor-associated fibroblast compartment, which is related to the early tumor recurrence, metastasis, tamoxifen-resistance, and aggravated increase in tumor growth [2]. Cav-1 loss induces autophagy and mitophagy, [18] driving the “Reverse Warburg Effect” by a feed-forward mechanism. This onset of inflammation, autophagy, mitophagy, and aerobic glycolysis in the tumor microenvironment is triggered by activation of the transcription factors NFκB and HIF-1α ^[19][20][19,20]. Mitochondria-generated ROS plays an important role in cell proliferation and quiescence. The pro- or anti-tumorigenic signaling is controlled by a mitochondrial ROS switch of the antioxidant SOD2/MnSOD [21]. Cell proliferation is favored by decreased SOD2/MnSOD activity resulting in increased O₂⁻ production whereas proliferating cells transit into quiescence when SOD/MnSOD activity increases resulting in increased H₂O₂ activity [22]. Inactivation of mitochondrial antioxidant responses like the Thioredoxin reductase (TrxR); which causes reduction of oxidized Trx to produce reduced Trx that reacts with ROS, contributes to increased oxidative stress in cancer cells. Studies have shown that the cellular redox status is impacted by the recruitment of mitochondria by the expression of hTERT. This observation is supported by the presence of hTERT in the mitochondria and since mitochondrial-dependent apoptosis in target cells can be carried about by introducing hTERT inhibitors [23].

2.2. Role of Warburg Effect in ROS

The increased metabolic requirements of the cancer cells are met by upregulation of glucose transport and metabolism irrespective of oxygen supply [24]. There is also some evidence that cancer cells decrease mitochondrial respiration in the presence of oxygen, which suppresses apoptosis [25]. Under hypoxic conditions, the accelerated metabolism produces ROS in cancer cells that is countered by the increased NADPH which is met by the upregulated glycolysis ^[26][27][26,27]. NADPH is an essential cofactor for replenishing reduced glutathione (GSH) which is a critical antioxidant. Therefore, not only are cancer cells’ multiple urgent requirements catered to but cancer cell oxidative stress is also controlled by the Warburg effect [8]. Tumor cells have been reported to switch between the isoforms of pyruvate kinase, used in the last steps of glycolysis [28]. PKM2 the isoform found in high levels in tumor cells is slower and leads to the accumulation of PEP which in turn activates PPP by feedback inhibition of the glycolytic enzyme triosephosphate isomerase (TPI). This produces more NADPH which reduces ROS and further amplifies the inhibitory effect of PKM2 ^[26][27][26,27], Therefore ROS and PKM2 form a negative feedback loop to maintain ROS in a tolerable and functional range. The ROS-regulated gene, hypoxia-inducible factors (HIF-1α) regulates hypoxia-associated genes, some of which are associated with the Warburg effect and its accompanying pathways and hence, are a target of cancer therapies. PKM2 has been found to be the prolyl hydroxylases (PHDs)-induced coactivator for HIF-1α ^[8][29][8,29]. HIF-1α also regulates the MYC proto-oncogene which produces MYC protein [30] that regulates genes participating in energy generation and cell growth and proliferation. HIF-1α and MYC activate hexokinase 2 (HK-II) and pyruvate dehydrogenase kinase 1 (PDK1), which inhibits TCA and increases conversion of glucose to lactate [31]. Glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) are also activated by HIF1 and MYC independently, resulting in increased glucose influx and higher glycolytic rates [13]. Warburg effect increases steady-state ROS condition in cancer cells by producing lactate that is extruded through monocarboxylate transporters to the microenvironment of cancer cells which has no antioxidant properties in contrast to pyruvate, citrate, malate, and oxaloacetate together with the reducing equivalents (NADH.H⁺) which are antioxidant intermediates. This increased oxidative stress in cancer cells is stopped from reaching cytotoxic levels by some antioxidant effects exerted by hexokinase II (HK II) and NADPH.H⁺ produced through HMP shunt. Latest studies show tumor cells have the capability to carry about both glycolytic and oxidative phosphorylation (OXPHOS) metabolism which makes them resistant to oxidative stress through enhanced antioxidant response and increased detoxification capacity [32]. The changes related to energy metabolism may be correlated to the expression of certain p53 downstream genes regulated by it, including SCO2, TIGAR, and the p53 inducible gene 3 (PIG3) ^[33][34][35][33,34,35].

3. Mechanism of Oxidative Stress-Related Carcinogenesis

3.1. Role of ROS in Tumor Cell Proliferation, Survival and Tumor Progression

Increased ROS is responsible for the oxidation of negative feedback loop controllers and hence control the actions of other signaling pathways in tumor growth and programmed cell death by the phosphoinositide 3-kinase/protein kinase B (PI3K/PKB) and mitogen-activated protein kinase (MAPK) signaling pathways ^[36][37][45,46] (Figure 1Figure 2). Reactive oxygen species generation in cancer cells leads to the inactivation of PTEN that leads to an increase in PI3K/Akt signaling that promotes proliferation. Moreover, the cancer cell cycle progression is promoted when ROS inhibits phosphatase Cdc14B resulting in the activation of cyclin-dependent kinase 1 (Cdk1). As mentioned earlier, the major ROS-regulated gene HIF activates PDK1 that further activates Akt which inhibits the tuberous sclerosis complex (TSC). This downregulates mTOR which is a major regulator of cell growth by controlling mRNA translation, ribosome biogenesis, autophagy, and metabolism ^[38][39][44,47]. MAPK/ERK1/2 are activated by growth factors and K-Ras stimulated pathways, lead to increased cellular proliferation in cancer cells ^[40][48]. H₂O₂ has also been found to be responsible for the activation of ERK1/2 and pro-survival PI3K/Akt signaling pathway, resulting in increased proliferation ^[41][42][49,50]. Studies on breast, leukemia, melanoma, and ovarian cancer have shown ERK1/2 plays additional roles like cell survival, anchorage-independent growth, and motility ^[43][51]. The Akt pathway inactivates pro-apoptotic Bad, Bax, Bim, and Foxo transcription factors by phosphorylation thereby promoting cell survival ^[44][45][52,53]. Akt is activated by the Epithelial growth factor (EGF)-derived H₂O₂ production, observed in ovarian cancers ^[46][54]. Cell survival is promoted by the oxidation and inactivation of the negative regulators of PI3K/Akt signaling, i.e., the phosphatases PTEN and PTP1B. The tumor suppressor PTEN has been found to be reversibly inactivated by H₂O₂ in a variety of cancers ^[47][48][55,56].