Cancer Metabolism: The Role of ROS in DNA Damage and Induction of Apoptosis in Cancer Cells

Cancer is a huge challenge for people worldwide. High reactive oxygen species (ROS) levels are a recognized hallmark of cancer and an important aspect of cancer treatment research. Abnormally elevated ROS levels are often attributable to alterations in cellular metabolic activities and increased oxidative stress, which affects both the development and maintenance of cancer. Moderately high levels of ROS are beneficial to maintain tumor cell genesis and development, while toxic levels of ROS have been shown to be an important force in destroying cancer cells. ROS has become an important anticancer target based on the proapoptotic effect of toxic levels of ROS. Therefore, this review summarizes the role of increased ROS in DNA damage and the apoptosis of cancer cells caused by changes in cancer cell metabolism, as well as various anticancer therapies targeting ROS generation, in order to provide references for cancer therapies based on ROS generation.


Introduction
In the 21st century, cancer has become one of the most important diseases threatening human life and health. Lung cancer is the most common cancer type and the leading cause of death in both male and female patients. In contrast, breast cancer is most common in women, accounting for 11.7% of all female cancer cases in 2020 [1,2]. In addition, the incidence of colorectal cancer in young adults is still rising [3][4][5]. Global colorectal cancer deaths are expected to increase by more than 60% by 2035 [6]. Cancer contributes greatly to the global mortality rate. In today's world, cancer is frequently discussed. Where does the fear of cancer come from? Since cancer became the leading cause of morbidity and mortality worldwide, an increasing number of clinical cancer treatment strategies, such as surgery, chemotherapy drugs, radiotherapy and immunotherapy, and different combinations of these therapies, have been used to address the cancer crisis. However, these therapies have different limitations or produce different degrees of toxicity to the body, such as off-target effects, immunosuppression, and cancer cell resistance [7,8]. Therefore, it has forced us to find more effective anticancer strategies. Targeting ROS production would be a good approach for developing these strategies. At normal physiological concentrations, ROS are signal regulatory molecules for various physiological activities that play an important role in basic metabolism. However, when the concentrations of ROS in the body are not controlled, ROS plays a key role in the process of cancer. The abnormal increase in ROS level causes DNA damage, cancer cell metabolism changes, tumor metastasis, and tumor cancer cells. Under normal circumstances, an appropriate amount of ROS is one of the necessary conditions for maintaining cell signaling and redox homeostasis, but a further increase in ROS levels due to exogenous stimulation or metabolic changes in cancer cells is carcinogenic. High levels of ROS in cancer cells put cancer cells in a state of high oxidative stress and lead to the oxidative stress-induced damage of proteins, lipids, DNA, and mitochondria [30], among which DNA is the most sensitive target of ROS. High ROS levels drive DNA damage and genomic instability, contributing to cancer development. However, when ROS levels in cancer cells accumulate toxic effects on cells, they also show obvious anticancer effects. ROS has become an important anticancer target for clinical cancer therapy based on its dual mechanism of action. Recent studies have shown that when NCX4040 (a unique nitric oxide donor) is used to treat tumors, glutathione (GSH) is consumed and ROS is generated, which results in oxidative DNA damage and the destruction of tumor cells [31]. Oxidative stress and inflammation are closely related to cancer and apoptosis [32]. Subsequent research studies have found that NCX4040 can also induce a TNF-dependent pathway, further increasing oxidative stress through NF-κB-mediated immune responses, leading to increased apoptosis (mediated by NCX4040) [33]. In addition, Popovici et al. revealed that Usnea barbata extract in canola oil showed anticancer potential through ROS generation leading to DNA damage and autophagy [34]. Therefore, there is a relatively low or high threshold for ROS accumulation to promote or inhibit cancer.
Over the past few decades, the relationship between oxidative stress, ROS, and cancer has been elucidated. Current research reviews on cancer mainly focus on the adaptive metabolism of cancer cells and cancer development, the abnormal metabolism of cancer cells and the shaping of the tumor immune microenvironment, and the relationship between metabolic phenotypes and cancer. However, the relationship between ROS accumulation caused by cancer metabolism and DNA damage and the apoptosis of cancer cells has not been systematically elaborated. Therefore, this review article aims to systematically summarize the dual mechanism of promoting and inhibiting cancer via ROS accumulation in cancer cells and clarify how ROS levels induce DNA damage and promote the apoptosis of cancer cells. In addition, the various cancer treatment strategies targeting ROS generation that have been used in recent years are also summarized to provide references for further research on drugs, specifically with respect to targeting ROS generation in cancer cells or anticancer therapies.

Mechanisms of ROS Generation in Cancer
Both exogenous environmental stimuli and endogenous factors can lead to ROS generation. When exposed to exogenous stimuli such as air pollutants, heavy metals, radiation, and anticancer drugs, ROS will abnormally accumulate in cells, leading to oxidative stress and gene mutations, which may induce the transformation of normal cells into cancer cells [35]. Air pollution has long been recognized as one of the common factors endangering human health as it induces high levels of ROS generation in cells and leads to oxidative stress, posing a cancer risk to humans [36]. Research studies have shown that oxidative stress in the ROS-mediated pathway may be a key mechanism for the biological effects of ambient fine particulate matter [37]. Niture et al. found that the use of a lowconcentration of Cd (1-10 nM) increases ROS generation, cell proliferation, and cell steatosis while activating fibrogenic/oncogenic signaling in HepaRG and hepatocellular carcinoma (HCC) cells [38]. In the skin of pigmented mice, ultraviolet radiation amplifies ROS generation, potentially increasing the risk of malignant melanoma [39,40]. Clinically, the use of anticancer drugs can induce ROS accumulation either directly or indirectly. In research studies on the antitumor activity of the polyphenol agrimol B (Agr), it was found that Agr could increase the generation of ROS [41]. In addition, the organic arsenic compound Aa-Z2 induces ROS accumulation by targeting pyruvate dehydrogenase kinase 1 (PDK-1) to induce osteosarcoma apoptosis [42]. On the other hand, endogenous factors in the body can also lead to the excessive generation of ROS. Endoplasmic reticulum stress enhances ROS generation [43]. Furthermore, tumor microbiota can regulate immune response and local inflammation through ROS generation, thus promoting tumor progression [44]. In conclusion, ROS levels in the body and cells are affected by internal and external environmental factors.
The metabolic reprogramming characteristic enables cancer cells to generate ROS at higher levels than normal cells through a variety of biological pathways. In tumor tissues, the rapid energy metabolism and proliferation of tumor cells generate a large number of ROS, including superoxide (O 2 − ), hydroxyl radical (−OH), hydrogen peroxide (H 2 O 2 ), and lipid hydroperoxide (LOOH). ROS mainly comes from mitochondria [45]. Mitochondria provide energy for various biological processes in cancer cells, and ROS is produced from oxidative phosphorylation (OXPHOS). However, research studies have shown that elevated ROS levels will induce mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction, which will further aggravate ROS generation, resulting in a vicious cycle of ROS generation and mtDNA damage [46]. In addition, the generation of ROS also depends on the regulation of NADPH oxidases (NOXs), nitric oxide synthase (NOS), and xanthine oxidase. Among them, NOXs are the main regulatory enzymes of ROS regulation [47]. Research studies have shown that NOX1 expression significantly increases the regulation of O 2 − and maintains the proliferative phenotype of colon cancer cells [48,49]. In addition, there are both tumor cells and a variety of normal cells in the tumor microenvironment, in which stromal cell cancer-associated fibroblast-derived H 2 O 2 leads to extracellular oxidative stress and promotes tumor growth and invasion [50]. Therefore, ROS accumulation in cancer cells is mediated by multiple biological pathways ( Figure 1). tocellular carcinoma (HCC) cells [38]. In the skin of pigmented mice, ultraviolet radi amplifies ROS generation, potentially increasing the risk of malignant melanoma [39 Clinically, the use of anticancer drugs can induce ROS accumulation either direct indirectly. In research studies on the antitumor activity of the polyphenol agrim (Agr), it was found that Agr could increase the generation of ROS [41]. In addition organic arsenic compound Aa-Z2 induces ROS accumulation by targeting pyruvat hydrogenase kinase 1 (PDK-1) to induce osteosarcoma apoptosis [42]. On the other h endogenous factors in the body can also lead to the excessive generation of ROS. E plasmic reticulum stress enhances ROS generation [43]. Furthermore, tumor micro can regulate immune response and local inflammation through ROS generation, promoting tumor progression [44]. In conclusion, ROS levels in the body and cell affected by internal and external environmental factors.
The metabolic reprogramming characteristic enables cancer cells to generate RO higher levels than normal cells through a variety of biological pathways. In tumor tis the rapid energy metabolism and proliferation of tumor cells generate a large numb ROS, including superoxide (O2 − ), hydroxyl radical (−OH), hydrogen peroxide (H2O2) lipid hydroperoxide (LOOH). ROS mainly comes from mitochondria [45]. Mitochon provide energy for various biological processes in cancer cells, and ROS is produced oxidative phosphorylation (OXPHOS). However, research studies have shown tha vated ROS levels will induce mitochondrial DNA (mtDNA) damage and mitochon dysfunction, which will further aggravate ROS generation, resulting in a vicious cyc ROS generation and mtDNA damage [46]. In addition, the generation of ROS also pends on the regulation of NADPH oxidases (NOXs), nitric oxide synthase (NOS) xanthine oxidase. Among them, NOXs are the main regulatory enzymes of ROS re tion [47]. Research studies have shown that NOX1 expression significantly increase regulation of O2 − and maintains the proliferative phenotype of colon cancer cells [48 In addition, there are both tumor cells and a variety of normal cells in the tumor m environment, in which stromal cell cancer-associated fibroblast-derived H2O2 lead extracellular oxidative stress and promotes tumor growth and invasion [50]. There ROS accumulation in cancer cells is mediated by multiple biological pathways (Figu   Sources of ROS in cancer cells mainly include mitochondria, NOX, and endoplasmic reticulum [51]. ROS is a natural by-product of cellular aerobic metabolism and plays a significant role in intercellular signaling pathways, redox homeostasis maintenance, and body metabolism. Cancer cells have significantly higher levels of oxidative stress, and they can maintain high ROS levels through various mechanisms to support the development of cancer. Mitochondrial dysfunction and metabolic alterations are known to be the main sources of ROS [52]. Mitochondria are highly dynamic organelles, and their ability to regulate exogenous or endogenous stimuli depends on the close relationship between mitochondrial dynamics and mitochondrial energy metabolism. In the cell, mitochondrial dynamics play a role in regulating mitochondrial fusion and division and the balance between them. When oxidative stress or cell metabolism and other factors cause disorders affecting mitochondrial dynamics (such as the imbalance of the mitochondrial fusion and division), mitochondrial function will become increasingly abnormal, thus inducing an increase in ROS generated by mitochondria and enhancing tumor invasion [53]. In studying the specific mechanism of ouabain (a steroid hormone) against glioblastoma, Yan et al. found that ouabain induced mitochondrial damage and led to increased ROS generation through the phosphorylation of p66Shc (isoform derived from Shc1 gene), mediated by the Src/Ras/ERK signaling pathway [54]. NOX induces the intracellular accumulation of ROS. Research studies have shown that NOX is one of the sources of ROS in melanoma, playing a crucial role in all aspects of melanoma development [55,56]. Brevilin A (Brv-A), a sesquiterpene lactone compound of Centipeda minima induces ROS generation through NOX2 and NOX3 regulation [57]. Baldi et al. showed that the ferroptosis-related gene-activated TGFB pathway could stimulate ROS generation to promote tumor invasion and progression [58]. In addition, Biswas et al. found that MAO-A-mediated ROS accumulation is a key factor in cancer apoptosis [59]. All these facts suggest that the abnormal accumulation of ROS in cancer cells is mediated by multiple mechanisms for tumor response. Table 1 lists the main factors of ROS generation.

Objective
Aim Results Reference

Air pollutants
To demonstrate that oxidative stress is the key mechanism by which secondary organic aerosols affect cell health.
To a large extent, cellular health depends on cellular ROS levels, and secondary organic aerosols exposure produce biological effects through oxidative stress. [37] To investigate mechanism of oxidative stress and cytotoxicity induced by DEPs exposure in HBE.
Cells treated with DEPs showed high levels of ROS generation and oxidative DNA damage. [60] Anticancer drugs To investigate whether ononin synergizes with paclitaxel to inhibit non-small-cell lung cancer progression and promote apoptosis in vitro and in vivo.
The combination of ononin and paclitaxel increased the expression of ROS generation and apoptotic markers and inhibited cell proliferation through the PI3K/Akt/mTOR signaling pathways. [61] To investigate mechanism of action regarding redox-active quinone chelators.
Redox-active quinone chelators can be reduced by ETC and GSH while generating ROS. They can also generate ROS upon photoexcitation. [62] To explore the anticancer mechanism of Brv-A.
Brv-A induces ROS generation; however, Brv-A regulates ROS generation through NOX2 and NOX3 proteins and the induction of ER-stress in MCF-7 BC cells. [57] To investigate the antitumor mechanism of Agr.
Agr increased ROS, blocked Bcl-2 expression, and increased Caspase-3 and Bax expression to promote apoptosis in cancer cells. [41]

Objective Aim Results Reference
To investigate the effect of curcumin on the effect of irinotecan on CRC cells.
Curcumin enhances the killing effect of irinotecan on CRC cells by mediating increased ROS production and the activation of the ER stress pathway. [63] To determine the effect and mechanism of action of curcumin analog WZ35 against human prostate cancer.
WZ35 exhibited strong antitumor potential against PC-3 cells by inducing ROS generation and subsequently inducing ER stress-dependent apoptosis and cell cycle arrest. [64] To reveal the mechanism of WZ35-mediated ROS generation and amino acid metabolism regulation that inhibit gastric cancer cell metastasis.
WZ35 can deplete the GSH reserve by increasing ROS generation. The mechanism maintains the GSH consumption phenotype through the ROS-YAP-AXL-ALKBH5-GLS2 loop. [65] To investigate the molecular mechanism of human BC cells apoptosis caused by SH003.
SH003 induced apoptosis in BC cells by increasing ROS generation and activating the ER stress signaling pathways. [66]

Chemicals
To explore the key mechanism of methamphetamine in HCC.
ROS-mediated Ras up-regulation activates the MEK/ERK signaling pathway, which is key mechanism by which methamphetamine promotes HCC progress. [67] To investigate the antitumor mechanism of organic arsenic compound Aa-Z2 on osteosarcoma by targeting cancer metabolism.
Aa-Z2 induces ROS accumulation by targeting PDK-1 to induce osteosarcoma apoptosis. [42] To explore the mechanism of mitochondrial damage after exposure to toxic polypropylene nanoplastic.
Polypropylene nanoplastic stimulation leads to mitochondrial dysfunction and ROS generation and causes lung inflammation through the p38-mediated NF-κB pathway. [68]

Radiation
To evaluate the combined action of EMFs and black carbon particles in the HL-60 promyelocytic cell line exposed to 2.45 GHz RF radiation.
The interaction between black carbon particles and RF leads to ROS generation and triggers oxidative stress to activate necrosis/apoptosis, leading to long-term cytotoxicity. [69] Heavy metals To study the co-carcinogenic effects of UVB and arsenic on mouse epidermal cell line JB6 and its mechanism.
Arsenic enhances UVB-induced ROS generation and causes DNA damage and apoptosis in mouse skin cells. [70] To investigate the ROS generation and related bio-effects of various metal-porphyrin complexes under ultrasonic exposure.
Zn(II) and Pd(II) porphyrins are the most effective in producing singlet oxygen and hydroxyl radicals, and the different patterns of ROS generation depend on the metal moiety. [71] To investigate the role of ROSassociated autophagy in Cd-induced cell proliferation and the invasion of A549 cells.
Exposure to Cd (2 µM) significantly increased ROS accumulation, induced autophagy, and enhanced cell growth, migration, and invasion in A549 cells. [72] To investigate the effects of low concentrations of Cd on the regulation of liver cancer cell proliferation, steatosis, and fibrogenic/ oncogenic signaling.

Objective Aim Results Reference
Cigarette smoke To assess the toxic effects of PAH and HEV light combination in human RPE cells.
Toxic synergistic interaction between IcdP and HEV light. This synergy translates into the disruption of the mitochondrial network, enhanced ROS accumulation, and apoptosis. [73]

Cytokine
To investigate the role of IL-13 and 13(S)HpODE (endogenous product during IL-13 activation) in mediating apoptotic pathways in three different in vitro cellular models: A549 lung cancer, HCT116 colorectal cancer, and CCF52 GBM cells.
13(S)HpODE significantly reduces solid tumor growth through the activation of apoptosis. IL-13 and 13(S)HpODE participate in activating the p53-p21 signaling cascade via MAO-A-mediated ROS and ultimately induce apoptosis by inhibiting Bcl-2 and promoting Bax. [59]

FRGs
To explore and verify the mechanism by which FRGs promotes the progression and invasion of colon adenocarcinoma.
FRGs improves tumor cell survival by activating the TGFB pathway, which can stimulate ROS generation, accelerate ECM decomposition, and promote tumor progression and invasion. [58]

Mitochondria damage
To investigate the molecular mechanism of ouabain-induced ROS generation and apoptosis in human GBM cells.
Ouabain-induced GBM cells apoptosis increased ROS generation through the phosphorylation of p66Shc (mediated by the Src/Ras/ERK signaling pathways). [54]

NOX
To investigate in GBM cell lines that cause TGF-β1 to drive metabolic reprogramming and aggressive cancer by enhancing NOX4 activity.
TGF-β1 up-regulated NOX4 expression accompanied by ROS through Smad-dependent signaling and then induced HIF-1α overexpression and metabolic reprogramming while promoting EMT (modulated by the PI3K/AKT/HIF-1α signaling pathways). [74] To investigate the role of NOX complex proteins in the Cd-induced malignant transformation of prostate epithelial cells and the molecular mechanisms involved.
ROS is known to play a significant role in the development and maintenance of malignant tumors. ROS levels in cancer cells are dynamic due to complex changes in the exogenous environment and changes in the endogenous metabolism of the body, which are regulated by various metabolic ways. The metabolic reprogramming of cancer cells is a prominent feature of cancer that makes ROS generation more favorable. Therefore, it is necessary to study the correlation between ROS levels and cancer metabolism further.

The Dual Role of ROS in Promoting and Suppressing Cancer Cells
The abnormal metabolic behavior of cancer cells is to support the need for cancer cells to maintain their development in harsh environments such as lack of oxygen and nutrition [22]. Because cancer cells are constantly meeting their increased energy and biosynthesis needs, they must "broaden" their metabolic pathways autonomously. All cells, including cancer cells, require glucose metabolism for energy, and glucose metabolism is closely related to redox homeostasis. However, the energy metabolic pathway of cancer cells is classic aerobic glycolysis, even when oxygen is plentiful. The rapid uptake of glucose is accompanied by an increased rate of glycolysis. In precursor B-cell acute lymphoblastic leukemia cells, the glucose analog 1,5-anhydroglucitol interrupts mitochondrial respiration by up-regulating pyruvate dehydrogenase kinase 4 (PDK4), thereby increasing the rate of glycolysis, and enhanced glycolysis leads to oxidative stress in tumor cells by increasing ROS levels [76,77]. Additionally, the decreased O 2 − level under hypoxia conditions inhibits the AMP-activated protein kinase (AMPK) activation and shifts metabolism from glycolysis to oxidative phosphorylation [78]. Overall, there is a close relationship between ROS homeostasis and the regulation of metabolism and cancer development.
An imbalance between redox homeostasis and oxidative stress will inevitably result from the accumulation of abundant metabolites from the hyperactive metabolic activities of cancer cells. Continuous oxidative stress will lead to a large accumulation of ROS within cancer cells, and the effect of ROS on cancer progression has been shown to be paradoxical [79][80][81]. High levels of ROS that do not cause cell death can promote cancer cell growth, metastasis, and angiogenesis. However, when ROS generation reaches a level that cancer cells cannot withstand (toxic level), it will kill cancer cells, that is, it has an antitumor effect. Therefore, the amount of ROS accumulation affects both the development and maintenance of cancer. The level of ROS in cancer cells is a double-edged sword because, as of now, no clear value that defines whether levels of oxidative stress are low or high has been established. The ROS generation mechanism in cancer cells is complex and key to maintaining redox homeostasis.

High Levels of Steady-State ROS Play a Role in Promoting Cancer
The moderate accumulation of ROS is conducive to tumor maintenance and development [80]. Increased ROS generation or decreased ROS clearance due to exogenous or endogenous factors will put cells in a state of oxidative stress [82]. Oxidative stress can cause oxidative damage to biological macromolecules, among which DNA oxidative damage can cause mutation accumulation and genome instability, which is a favorable inducing factor for tumorigenesis. Oxidative DNA damage includes base oxidation, deoxyribose oxidation, single-strand breaks (SSBs), and DNA double-strand breaks (DSBs) [83]. Among the four bases, guanine is the most easily oxidized DNA base because it has the lowest redox potential [84], and its extensive accumulation of oxidation product 8-oxoguanine (8-oxoG) is closely related to tumorigenesis [85]. The base excision repair pathway (BER) is a key repair mechanism for oxidative DNA damage. When the important base excision repair mechanism is inhibited or cannot repair DNA damage in time, it will lead to increased gene mutations and greatly increase the probability of tumor induction [86]. Equally important, due to the strong adaptive capacity of cancer cells, cancer cells will respond to moderately high levels of ROS and further enhance the antioxidant system by inducing an increase in antioxidant enzymes and antioxidant proteins, thus continuing to maintain the development of cancer. Therefore, moderately elevated ROS is beneficial for cancer transformation.
Many research studies have shown that excessive ROS generation has a pro-cancer effect [79,87]. High levels of ROS are associated with the development and progression of many malignancies. Adaptation to hypoxia, metabolic rate, oncogenic mutations, and the activation of pro-tumor signaling can all be inductors of tumorigenesis. It has been reported that hypoxia-induced ROS up-regulates the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 through the NF-κB signaling pathway, thus playing a role in promoting aneurysms [88]. Hypoxia-induced ROS can also enhance the proliferation, migration, and invasion of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, thus exacerbating tumor progression [89]. In addition, Gao et al. suggested that the sterol-regulatory element binding protein 1 (SREBP1) promotes colon cancer cell invasion via the NF-κB-MMP7 axis by increasing ROS [90]. O 2 − acts as a positive regulator of the Rac1-PI3K signaling pathway, which is associated with stimulating the migration and invasion of Nex10C mouse melanoma cells [91]. On the other hand, high levels of ROS homeostasis and glycolysis are hallmark features of cancer. In leukemia, ROS over generation affects glycolysis. It promotes the proliferation of acute myeloid leukemia by up-regulating the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a key glycolytic regulator [92]. Furthermore, elevated glycolysis levels can promote the progression of precursor B-cell acute lymphocytic leukemia (pre-B AML) by activating MAPK/ERK pathways by increasing intracellular ROS levels [77]. The above research studies have shown that increased ROS accumulation can promote tumor progression through a variety of signaling pathways (Figure 2).

Toxic Levels of ROS Play a Pro-Apoptotic Role
Since ROS levels play a role in promoting cancer, a further increase in ROS levels can promote antitumor signaling and trigger oxidative stress, thus inducing tumor cell apoptosis through the pro-apoptotic pathway. The imbalance of ROS induces adverse cancer outcomes by affecting signal transduction within the DDR [93] because the accumulation of ROS can be toxic to cells, and toxic levels of ROS have been shown to affect replication and transcription by inducing DNA strand breaks and base oxidation, which may lead to the accumulation of mutations in organisms and the transformation of tumors [94]. It was found that due to the increased expression of the tumor-suppressant gene deleted in liver cancer 1 (DLC1), ROS could further inhibit the DYRK1A-EGFR axis, triggering DNA damage, while the DNA double-strand break marker protein γH2AX was up-regulated and the DNA repair-related proteins p-BRCA1 and RAD51 were down-regulated, which contributed to tumor inhibition [95]. Irreversible DNA damage is a key factor in tumor cell apoptosis. For example, through ROS inducers such as chemotherapy, severe DNA damage causes irreparable damage to cells and eventually leads to tumor cell death [80]. Moreover, ROS can not only cause oxidative DNA damage and directly drive the apoptosis of lung cancer cells, but also cause endoplasmic reticulum stress in cancer cells to promote the translocation of calreticulin (multifunctional soluble protein) to the cell membrane, resulting in DNA damage and the expression and release of nuclear high mobility group box (HMGB1) and heat shock proteins (HSP90), thus inducing immunogenic cell death [96]. In addition, research studies have shown that metal-based anticancer chemotherapeutic agents can cause lethal damage to malignant tumors by inducing cytotoxic effects caused by multiple modes, including ROS-induced DNA damage and angiogenesis [97]. Therefore, DNA damage is a key ROS-mediated apoptosis signaling pathway in tumor cells.
Apoptosis is an important homeostatic mechanism of the body that plays an important role in maintaining the homeostasis of normal cells. Cancerous cells can break through the age limit and have the ability to continue to proliferate, which is also the reason for the occurrence of tumors. The apoptosis of cancer cells allows for the development of tumors to be controlled, which also brings greater hope to cancer treatment. At present, there are three major apoptotic pathways: (i) the endogenous mitochondrial apoptosis signaling pathway, (ii) the death receptor pathway, and (iii) the endoplasmic reticulum signaling pathway. It has been demonstrated that ROS generated in cancer cells induces apoptosis through the activation of these apoptotic signaling pathways and plays a role in tumor inhibition [98,99].
Toxic levels of ROS can activate members of the mitogen-activated protein kinase (MAPK) family, among which the activation of the c-Jun N-terminal kinase (JNK) signaling pathway (one of the major signaling cassettes of MAPK signaling pathway) can lead to intrinsic or extrinsic apoptotic signaling to initiate apoptosis [79,100]. Research studies have shown that JNK phosphorylation plays a role in inducing the apoptosis of human hepatocellular carcinoma BEL-7402 and HepG2 cells, specifically through the ROS-JNK-P53 signaling pathway [101]. In addition, the activated JNK signaling pathway is a key link in the apoptosis of intrahepatic cholangiocarcinoma cells by MLN2238 [102]. Furthermore, the ROS-mediated PI3K/Akt/mTOR and MAPK signaling pathways are the mechanisms by which CBL0137 promotes ROS generation and induces the apoptosis and autophagy of aggressive B-cell non-Hodgkin's lymphoma cells [103]. Therefore, the occurrence of cancer cell apoptosis largely depends on the JNK signaling pathway.
Among the many apoptosis signaling pathways, the mitochondrial apoptosis signaling pathway plays an irreplaceable role in cancer cell apoptosis. Mitochondria are not only the "energy factories" of cells, but also the regulatory centers of cell survival and death. Mitochondrial dysfunction leads to excessive ROS generation, which leads to oxidative stress and aggravates mitochondrial dysfunction, leading to apoptosis. In addition, when cytokines, death receptors, cellular metabolites, and other factors stimulate mitochondria and affect the mitochondrial electron transport chain and energy metabolism, it is likely to activate the endogenous mitochondrial apoptosis signaling pathway, leading to cell apoptosis. Caspases play a special role in the mitochondrial intrinsic apoptosis signaling pathway and death receptor apoptosis signaling pathway, and its activation is one of the signs of cell apoptosis. ROS causes mitochondrial membrane damage and releases cytochrome c into the cytoplasm, which forms a complex with apoptotic protein activating factor 1 (Apaf-1) and pro-caspase-9 to induce the cleavage of caspase-3 and caspase-7, eventually leading to DNA fragmentation and cell death [80,104]. In the death receptor pathway, the ligand binds to the death receptor and causes the activation of caspase-3, which leads to the cleavage of downstream caspase 3 and the cleavage of Bid (Bcl-2 family protein) to Bid, then truncated Bid is transported to the mitochondria to mediate the release and transport of cytochrome c [105]. In addition, the excessive accumulation of ROS can also act by affecting the activity of apoptosis-related factors such as Bcl-2 family proteins. Bcl-2 family members also regulate the mitochondrial apoptosis pathway through protein-protein interactions [106]. Any pro-apoptotic factor or signal can disrupt mitochondrial membrane potential by reducing the Bcl-2/Bax ratio in cancer cells, causing the mitochondria to release cytochrome c into the cytoplasm [57] and ultimately mediating apoptosis through the caspase-dependent death pathway.
It has been confirmed that ROS-induced apoptosis is related to endoplasmic reticulum stress [107,108]. The abnormal accumulation of ROS can induce endoplasmic reticulum stress-mediated apoptosis in various malignancies [57]. The endoplasmic reticulum is the site of protein synthesis. High levels of ROS accumulation can lead to endoplasmic reticulum stress and increase protein folding errors in the endoplasmic reticulum lumen, thereby amplifying unfolding protein response (UPR) signaling and inducing apoptosis [109,110]. Research studies have shown that C/EBP-homologous protein (CHOP) is highly expressed under endoplasmic reticulum stress and has a pro-apoptotic effect [111]. ROS-induced endoplasmic reticulum stress plays an important role in the apoptosis of gastric cancer cells [112]. Similarly, ROS levels regulate endoplasmic reticulum stress and mitochondrial dysfunction in oral cancer to induce PERK/eIF2α/CHOP signaling pathway activation and inhibit cell growth and apoptosis [113]. Several members of the TNF receptor family are capable of inducing apoptosis because of intracytoplasmic (death) domains that bind proteins in the cell death pathway. These include receptors for TNF-α, the FAS ligand (FASL), and the TNF-related apoptosis-inducing ligand or TRAIL. At least two receptors have been described for TRAIL: R1 and R2. R1 does not interact with the common adaptor molecule FAS-associated death domain (FADD), which binds to TNFR1 and FAS. The second receptor, R2, was also found to be widely distributed on tissues and, in contrast to R1, induced apoptosis via mechanisms that involved interactions with FADD [114]. It can be concluded that ROS-induced endoplasmic reticulum stress is also an important pathway of cancer cell apoptosis (Figure 2.2

Changes in the Adaptation of Cancer Cells to ROS
Cancer cells employ multiple adaptive mechanisms in response to elevated ROS and oxidative stress [115]. In order to maintain genome stability under oxidative damage, cells have evolved a variety of DNA repair methods, such as BER, nucleotide excision repair, and mismatch repair. Experiments have shown that mice lacking the BER enzyme have a higher mutation rate and tumor incidence [116,117], and 8-oxoguanine DNA glycosylase1 (OGG1) initiates BER to remove 8-oxoG to avoid the accumulation of 8-oxoG and induce mutations [85]. In addition, mitochondrial homeostasis, antioxidant response, and hypoxia adaptation are also regulatory pathways in cancer cells when they are under ROS stress.
As we all know, endogenous ROS mainly comes from mitochondria and is a byproduct of the oxidative phosphorylation of mitochondria. Research studies have demonstrated that uncontrolled mitochondrial ROS (mROS) affects normal cell survival and carcinogenic transformation [118]. However, there is also growing evidence to suggest that increased and sustained ROS leads to severely elevated oxidative stress, which leads to the accumulation of cellular damage at DNA, RNA, and protein levels, ultimately leading to cancer cell death. In order to maintain their survival, cancer cells are able to resist oxidative stress through a mitochondrial oxidative stress checkpoint mechanism, which is involved in metabolic regulation. DNA-PKcs (a multi-faceted player in DNA damage response) is not only a key regulator of DNA double-strand break repair but also a direct regulator of mitochondrial homeostasis. DNA-PKcs can directly interact with mitochondrial proteins ANT2 and VDAC2. The resulting DNA-PKcs/ANT2/VDAC2 (DAV complex) regulates mitochondrial ADP-ATP exchange and OXPHOS, thereby accelerating cellular oxidative stress repair [119].
The maintenance of redox homeostasis in cancer cells under high ROS levels supports cancer cell survival and proliferation. Cancer cells can make adaptive changes to oxidative stress, such as metabolic reprogramming, enhanced antioxidant capacity, and resistance to apoptosis, to meet the needs of their own continuous growth. As tumor-initiating cells, cancer stem-like cells (CSCs) have a more powerful antioxidant defense system to counteract, clear, and maintain tumorigenesis [120]. Research studies have shown that in order to adapt to high ROS levels during tumor progression, tumor cells survive high ROS levels through the high expression of antioxidant genes, increased GSH synthesis, and increased NADPH generation (through activation of AMPK, the pentose phosphate pathway, and reduced glutamine and folate metabolism) [121].
The antioxidant response is one of the key cellular defense mechanisms used by cancer cells to prevent oxidative stress, and nuclear factor erythroid 2-related factor 2 (Nrf2) is the core of the antioxidant reaction [122]. Nrf2 activation can not only participate in cancer metabolic reprogramming by promoting the high expression of metabolic enzymes but also maintain cellular redox homeostasis under oxidative stress. As one of the most important proteins involved in the control of the antioxidant response, Nrf2 has the ability to maintain cell homeostasis and support the metabolic process to cope with oxidative stress caused by abnormal accumulation of ROS. Nrf2 overactivation can up-regulate the expression of antioxidant enzymes, thereby increasing GSH synthesis to balance the high level of ROS accumulation. Research studies have shown that melanoma redox homeostasis mainly depends on Nrf2 to regulate the expression of antioxidant enzymes such as HO-1, SOD, CAT, and GPX [123]. In addition, the abnormal activation of the Nrf2/Keap1 (Kelch-like ECH-associated protein 1) signaling pathway is crucial to maintain cell homeostasis. The mechanism of its action is that oxidative stress leads to the release of Nrf2, which was originally coupled with the inhibitor Keap1. Nrf2 is then translocated to the nucleus to regulate the up-regulation of downstream antioxidant enzymes, thus playing a role in oxidative stress defense [124,125]. At the same time, the decrease in Nrf2 activity or its downstream targets can inhibit the apoptosis of cancer cells by mediating various intracellular signaling pathways to avoid their death [126,127]. Overall, these results indicate that cancer cells can both balance ROS by increasing the expression of antioxidant proteins and maintain their survival by resisting the apoptosis of cancer cells.
Cancer cells adapt to hypoxia through changes in energy metabolism. The maintenance of the continuous high proliferation state of cancer cells requires the consumption of a large amount of oxygen, so the hypoxic environment is considered to be a sign of the tumor microenvironment [128]. Hypoxic conditions lead to increased mROS generation, and long-term high ROS levels will cause oxidative damage to cells. Cancer cells adapt to proliferation and migration under hypoxic conditions through energy metabolism [89]. The Warburg effect (oncology) has pointed out that cancer cells have a different way of metabolizing energy compared to normal cells, namely, via aerobic glycolysis [23]. It is currently believed that glycolysis plays a role in the redox homeostasis of cancer. The intermediates produced by glycolysis enter other metabolic pathways to produce reducing agents, such as GSH, to maintain cell homeostasis [80]. In addition, cellular hypoxia adaptation depends on hypoxia-inducible factor-1α (HIF-1α) signaling, and ROS promotes the stability and activation of HIF-1α [129,130]. Thus, hypoxic adaptation also favors the survival of cancer cells under high ROS levels.
The occurrence and progression of cancer are closely related to bidirectional regulatory mechanisms below or above the threshold ROS level. Cancer cells also have the sufficient adaptive capacity to cope with the high level of ROS pressure and can maintain their survival and development through a variety of adaptive mechanisms, which is a favorable obstacle to tumor treatment.

Cancer Therapeutic Strategies Targeting ROS Generation
Recently, cancer treatment strategies that target ROS regulation in cancer cells have attracted increasing attention. The occurrence of cancer is a serious threat to human health. The current cancer treatment methods include radiotherapy, chemotherapy, surgery, immunotherapy, and molecular targeted therapy, and their cancer treatment effects have also been recognized in the clinical treatment of cancer patients. However, due to nonspecific targeting, various adverse reactions, and the continuous emergence of tumor drug resistance, the application of traditional anticancer strategies has been greatly limited [131]. High levels of ROS and oxidative stress are significant markers of cancer. The adaptive metabolic reprogramming of cancer cells leads to increased ROS levels in cancer cells, and elevated ROS also regulates tumor metabolism and progression [80]. In recent years, more and more research studies have been conducted on the relationship between ROS and cancer. ROS is considered to be an important anticancer target due to the different roles of different levels of ROS in various stages of cancer development, and many studies have shown that therapeutic strategies targeting ROS are significantly effective in the treatment of various diseases, including human lung cancer, colorectal cancer, breast cancer, and glioblastoma [61,[132][133][134]. Therefore, based on the effectiveness of excessive ROS for cancer treatment, new anticancer therapies targeting ROS generation can be regarded as a good solution to the current problems of cancer drug resistance and the low efficacy of traditional anticancer therapies.

Drug Therapies That Target ROS Generation
Currently, ROS-generation-based cancer treatment strategies have been widely used. Anticancer drugs are the traditional methods of cancer treatment. Some anticancer drugs are based on the inhibition of cancer cell proliferation and cell death caused by ROS generation, which has advantages over the resistance-based shortcomings of traditional drugs (such as 5-fluorouracil and cisplatin) [135]. On the other hand, many chemotherapeutic agents are able to alter redox homeostasis in cancer cells by increasing ROS generation to exert cancer therapeutic effects [136]. For example, the highly selective inhibitor NCT503 enhances the efficacy of temozolomide (a first-line chemotherapeutic agent for glioblastoma treatment) through ROS-mediated DNA damage and the Wnt/β-catenin pathway's inhibition of key proteins in the de novo serine synthesis pathway, up-regulated in many cancers [134]. Moreira et al. demonstrated that celastrol, a natural pentyclic triterpenoid, exerted pro-oxidative activity based on the excessive accumulation of ROS to induce DSBs, apoptotic/necrotic cell death, and the inhibition of cell proliferation, therefore showing promise as a drug for the treatment of drug-resistant colon cancer [137]. In addition, urolithin B, a gastrointestinal final metabolite of polyphenols, inhibits the growth of colon cancer cells by remodeling the intestinal microbiome and tumor immune microenvironment [138]. Meanwhile, plumbagin, a natural compound whose anticancer principle is ROS generation, has been used in the treatment of various cancers, including oral cancer and breast cancer [114,139]. Selenium is considered to be a multi-target drug, and highdose sodium selenite has increased ovarian cancer cell death by inducing ROS generation and GSH depletion to mediate iron death [140]. Therefore, drug therapies targeting ROS generation also play an important role in modern cancer treatment.
In addition to pharmacotherapy, ROS generation is also widely used in several novel anticancer therapies, such as photodynamic therapy (PDT), sonodynamic therapy (SDT), chemodynamic therapy (CDT), and the combination of multiple therapeutic therapies.

Novel Therapies Targeting ROS Generation
PDT is a new type of anticancer therapy in which ROS generated during treatment induces cell death through oxidative damage to cells (such as mitochondria, DNA, proteins, and cell membranes). Existing research studies show that PDT has a good effect on the treatment of cervical cancer, colon cancer, skin cancer, and lung cancer and that it has fewer adverse effects than surgery, chemotherapy, and radiation treatment [141,142]. PDT is a photochemical reaction. Its anticancer principle is that, in an environment containing molecular oxygen, photosensitizers entering tumor cells are activated by the light irradiation of a specific wavelength to stimulate the generation of toxic levels of reactive oxygen species and induce cell death [143]. In PDT therapy, photosensitizers smoothly reach specific tumor cells and are slowly eliminated as a key step of effective treatment. Effective delivery methods, such as ones that include nanoparticles (the key carriers of photosensitizers), have become indispensable means to improve the efficiency and specificity of PDT, such as bimetallic nanoparticles [144,145]. Therefore, the unique working principle of PDT makes it highly specific to cancer treatment.
Although PDT has the ability to accurately target ROS generation, it has certain limitations, such as limited light penetration depth, which is not ideal for the treatment of deep tumors [146]. SDT uses ultrasounds to penetrate deep into biological tissues and overcome penetration barriers for the treatment of deep tumors, showing good selectivity [147]. In addition, the combination of multiple therapeutic therapies is also an effective way to control cancer. The combination of PDT and chemotherapy can increase the toxicity of drugs to cells through photo-dependent ROS generation and thus exert increased synergistic antitumor effects [146,148]. Zhan et al. used nanopotentiators to reshape adenosine metabolism for SDT and CDT in combination with immunotherapy for deep tumors [149]. Moreover, Zhu et al. established a starvation-assisted, photothermal-thriving chemo/chemodynamic combined therapy to enhance CDT therapy by providing H 2 O 2 [150]. Overall, it can be seen that the combination of multiple therapeutic therapies are also a development direction for modern cancer treatment.

Pro-Oxidative Anticancer Agents Targeting ROS Generation
Pro-oxidative anticancer agents (PAAs) targeting redox imbalance are effective for the treatment of cancer patients [151]. PAA is an effective means of directly targeting cellular ROS generation to inhibit tumor development and induce cell death. The metabolic pathways that distinguish cancer cells from normal cells place cancer cells in a state of oxidative stress, which makes them more susceptible to the influence of PAA, thus generating high levels of ROS. It was found that the key to the anticancer activity of Brevilin A was to induce ROS-dependent apoptosis [57,152]. There are already PAAs that selectively generate ROS in cancer cells. Various ROS-responsive drug delivery systems developed using ROS-responsive materials and connectors hold promising prospects for specific therapies targeting cancer [153]. In addition, a dietary curcumin mono-carbonyl analog designed by Liu et al. can selectively generate ROS in NCI-H460 cells of nude mice [154]. Therefore, the development of PAA with cancer-specific ROS generation can reduce the adverse effects caused by non-selective PAA and improve its safety, which is a very promising cancer treatment strategy. At present, targeting the generation of high levels of ROS is an effective strategy for cancer treatment. Specifically, the development and application of new specific anticancer therapy methods such as PDT, SDT, and multiple combination drug therapies make the treatment of various types of cancer more promising ( Figure 4).
However, compared with contemporary conventional anticancer therapies and some other novel anticancer therapies, anticancer therapies targeting ROS generation within cancer cells have unique advantages but also certain limitations. Traditional surgery and chemotherapy drugs may have good efficacy in the early treatment of cancer, but the effectiveness of the treatment of malignant tumors is insufficient, and there are side effects and a high risk of recurrence. Cancer cells are resistant to treatments such as chemotherapy and radiotherapy and cannot be eradicated because of their properties such as rapid metabolic adaptation, high proliferation, and other resistance mechanisms. Cancer stem cells (CSC) exert resistance in radiotherapy and chemotherapy, which is the resistance mechanism of cancer treatment. For example, breast CSCs have traditional hyperthermia and radiation resistance [155]. Photothermal therapy (PTT) has been reported to sensitize CSCs to radiotherapy because of the ability of photothermal agents to affect ROS generation through local temperature control, and combination therapy (PDT/PTT) has shown high efficiency in the eradication of cancer stem cells [156]. However, this therapy has high requirements for the stability and accurate delivery of the photosensitizer. In addition, immune checkpoint blockade (ICB) therapy has been shown to be promising for cancer treatment. The principle is that immune checkpoint inhibitors block negative immune regulators and reactivate the antitumor immune system to increase the effect of cancer treatment. At present, it has been approved for the treatment of some advanced cancers. However, following its increasing application, clinical treatment has shown that this therapy has great drawbacks, such as non-specific targeting and serious tissue, organ, and systemic damage [157]. Fang et al. used topical hydrogel combined with ICB therapy to induce cancer cell death by providing continuous ROS and RNS and to minimize the systemic toxicity of ICB therapy [158]. However, different types and different stages of tumor development require effective delivery systems to deliver ROS to specific tumor sites. Therefore, there is an urgent need to develop effective and safe delivery systems to provide a basis for the universal application of this combination therapy. The challenge of targeting ROS generation-related therapies remains.
generating high levels of ROS. It was found that the key to the anticancer activity of Brevilin A was to induce ROS-dependent apoptosis [57,152]. There are already PAAs that selectively generate ROS in cancer cells. Various ROS-responsive drug delivery systems developed using ROS-responsive materials and connectors hold promising prospects for specific therapies targeting cancer [153]. In addition, a dietary curcumin mono-carbonyl analog designed by Liu et al. can selectively generate ROS in NCI-H460 cells of nude mice [154]. Therefore, the development of PAA with cancer-specific ROS generation can reduce the adverse effects caused by non-selective PAA and improve its safety, which is a very promising cancer treatment strategy. At present, targeting the generation of high levels of ROS is an effective strategy for cancer treatment. Specifically, the development and application of new specific anticancer therapy methods such as PDT, SDT, and multiple combination drug therapies make the treatment of various types of cancer more promising (Figure 4).

Discussion
Adaptive metabolic alterations lead to the abnormal accumulation of ROS in cancer cells, and cancer cells rely heavily on ROS levels to enhance tumorigenesis or induce apoptosis [80,159]. This review article systematically summarizes the dual roles of ROS accumulation in cancer cells in promoting and suppressing cancer, as well as the DNA damage and cancer cell apoptosis induced by toxic levels of ROS in cancer cells, providing basic information on how to find and identify effective anticancer treatments that excel in "targeting ROS generation" in cancer metabolic pathways.
In clinical practice, in order to cope with the highly invasive and metastatic nature of malignant tumors, it is often necessary to take chemotherapy drugs for a long time. However, the long-term and excessive use of chemotherapy drugs will not only increase the risk of harmful side effects and drug resistance of cancer cells, but also cause disrupt the tumor immune microenvironment and immune escape. Therefore, traditional cancer drugs do not have an advantage in the ongoing battle against cancer. At present, PDT, SDT, and CDT, which are commonly used in clinical practice, have certain advantages in traditional cancer treatment. However, the treatment of highly drug-resistant tumors is still a major problem, often requiring the combination of two or more treatments. Cancer drug resistance is a headache, which means that many chemotherapy drugs are not effective enough to kill cancer cells, and anticancer drugs targeting ROS generation can cause the apoptosis of drug-resistant cancer cells. Patchouli alcohol has been shown to have potential in the treatment of non-small cell lung cancer with or without vincristine resistance by inducing ROS-mediated DNA damage and promoting cell apoptosis [160]. However, anticancer therapies targeting ROS generation also have certain limitations, and only ROS levels that reach the toxicity threshold can play a role in killing tumor cells. However, due to tumor heterogeneity, high or low intracellular ROS levels are still not clearly defined, so the threshold for specific toxic levels of ROS is uncertain. To continuously generate high levels of ROS in as many cancer cells as possible, it is necessary to ensure the stable and continuous generation of ROS in the targeted sites, synthesize efficient and stable photosensitizers, and establish a precise delivery system for photosensitizers [161,162].
A variety of anticancer therapies can induce the generation of ROS to trigger the apoptosis of cancer cells; however, at the same time, the intrinsic detoxification mechanism of the body against ROS-mediated oxidative stress will also be activated. Autophagy can remove oxidative damage of organelles and biomolecules and induce the drug resistance of cancer cells, thus creating resistance to the cytotoxic effect of ROS [163]. The aggressive bone-malignant tumor osteosarcoma (OS) still has no significant effect on multimodal treatment. Since autophagy may play a role in maintaining the survival of osteosarcoma cells, Saini H et al. demonstrated that the use of autophagy inhibitors can disrupt multiple links of autophagy and make OS cells sensitive to drugs [164]. Therefore, autophagy regulation is an important factor that prevents toxic levels of ROS from promoting the apoptosis of cancer cells. In the future, part of our efforts will be devoted to the development of autophagy inhibitors to improve the anticancer effect of existing therapies by regulating autophagy.
The rapid metabolic adaptation of cancer cells occurs to meet the needs of endogenous growth, proliferation, and metastasis. The crosstalk between the complex pathways activated in cancer metabolic reprogramming has still not been fully elucidated, and the specific mechanism of metabolic changes regarding the generation and influence of high levels of ROS needs to be further elucidated. In addition, the specific mechanism of action of different ROS types in cancer cells in each stage of cancer development needs to be studied further, and the question of how to accurately characterize and locate each ROS type is also a problem to be solved. Traditional quantitative ROS detection techniques, such as chemical and immunological techniques, cannot meet the requirements of clinical detection standards. Therefore, the development of temporal-and spatial-specific ROS detection techniques to elucidate the complex biological mechanism of ROS-mediated redox in cancer is of great significance [165]. The solution to these scientific issues will promote the formulation of different cancer types and individualized anticancer therapy methods.
Due to the inherent heterogeneity of tumors, current drug treatments and therapies are unable to meet the increasingly severe changes with respect to cancer deterioration situations, and with the continuous adaptation of malignant tumors to the hypoxic microenvironment and the development of drug resistance through changes in material and energy metabolism, other obstacles have become increasingly prominent, posing great challenges to clinical cancer therapy. ROS are effective anticancer targets, and anticancer therapies targeting ROS are proven to be effective means of cancer treatment. However, among the various therapeutic methods used to induce cancer cell apoptosis through ROS generation, the effectiveness and specificity of such treatments still need to be improved. Therefore, it is necessary to ensure that the generation of ROS with therapeutic effects is selective and specific for cancer-type cells. This should be the goal when developing anticancer drugs that target ROS generation. Therefore, based on the important role of ROS levels in the regulation of cancer, the wide application of anticancer therapy methods targeting ROS generation is promising for cancer treatment.