The role of reactive oxygen species in gastric cancer

Sources of ROS

ROS are highly active oxidizing substances produced in REDOX reactions. ROS are predominantly found in the form of free radicals, including superoxide radical anion (O₂•⁻), hydroxyl radical (•OH), and alkoxyl/alkyl peroxyl (RO•/ROO•)⁸. The other type of ROS is a non-free radical ROS, the most common of which is hydrogen peroxide (H₂O₂) and commonly referred to as ROS in vivo. Because most free-radical ROS contain an unpaired electron in the outer orbitals, free-radical ROS are highly reactive and prone to initiating free radical chain reactions⁹. Most decomposition and synthesis processes in cells involve REDOX reactions, which facilitates the transfer of electrons between highly active substances.

Mitochondria, which are crucial for REDOX and electron transfer, are the primary sites of endogenous ROS production⁷. The electron transport chain (ETC) is a mitochondria-associated process where electrons are transferred through electron carriers, leading to a series of consecutive REDOX reactions. The direction of the ETC typically aligns with the increasing standard reduction potential. Glucose, fatty acids, and other substances enter the tricarboxylic acid (TCA) cycle during catabolism to generate ETC-reducing agents, like nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are vital for sustaining TCA cycle functionality. Oxygen accepts electrons and is then reduced to produce water and adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS)^10,11. The TCA cycle is coupled with OXPHOS, during which oxygen is consumed and large amounts of ATP are produced. Hence, the ETC is also termed the respiratory chain. During the process of oxidative phosphorylation, a large amount of ATP is produced to provide energy for life activities. When a mitochondrion ETC transfers electrons to oxygen via complex I, II, and III, oxygen radicals are generated¹². Oxygen atoms receive a varying number of electrons, which leads to different degrees of oxidation and the formation of oxygen radicals, including O₂•⁻, H₂O₂, and •OH. Therefore, ROS are produced as byproducts of electrons passing through the ETC¹³. Peroxidase-related enzymes and enzyme systems that are associated with biological membranes, such as mitochondria and the endoplasmic reticulum (ER), are involved in ROS generation and metabolism, and intimately associated with these membranes. The endoplasmic reticulum redox reductase (Erol), nicotinamide adenine dinucleotide phosphate oxidase (NOX) family, and other enzymes and enzyme systems are the primary contributors to the production of ROS¹⁴. Some side-reaction enzymes, such as xanthine oxidase (XO) and lipoxygenase (LOX), promote the production of ROS. In addition, some exogenous stimuli, such as ultraviolet radiation, ionization, drugs, chemotherapy, and radiotherapy, also promote the ROS response. The mitochondrial respiratory process, one of the major sources of ROS, is summarized in Figure 1.

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Figure 1

The electron transport chain in the mitochondrial respiratory process is one of the major sources of ROS. Glucose undergoes glycolysis in the cytoplasm to form pyruvate, which is then converted to acetyl CoA and enters the TCA cycle in the mitochondria. The TCA cycle involves the interconversion of reduced and oxidized NADH forms. The transfer of H⁺ generated in the TCA cycle through the four complexes (I–IV) of the respiratory chain is closely associated with ROS production.

Antioxidant enzymes and compounds in cells

Metabolism involves redox reactions and alterations in the oxidation and reduction states of the substances involved in maintaining dynamic equilibrium within the cell¹⁵. Hence, antioxidants and the maintenance of cellular redox homeostasis are essential.

Glutathione peroxidase (GPX), peroxiredoxin (Prx), Superoxide dismutase (SOD), and other substances catalyze ROS reduction to less reactive forms, alleviating oxidative stress in cells¹⁶. For example, GPX catalyzes the reduction of H₂O₂ to water and the conversion of reduced glutathione (GSH) to oxidized GSH (GSSG). GSSG can be reduced by glutathione reductase (GR) until GSSG is oxidized. Prx is converted to oxidized Prx when Prx is catalyzed by H₂O₂, then reactivated by thioredoxin (Trx). Oxidized Trx is then reduced by thioredoxin reductase (TrxR). The above series of redox reactions not only eliminate intracellular ROS but restore the activity of related enzymes. In the above GPX-related antioxidant processes, NAPDH acting as a reducing equivalent is an important antioxidant. Cells can increase NADPH in response to oxidative stress in various ways, including activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) and pentose phosphate pathway enhancers¹⁷.

In addition, reductive substances, such as vitamins A, C, and E and some metal ions, are involved in maintaining cellular oxidative balance and facilitating normal cellular growth, division, and the activity of vital enzymes and biochemical processes^18,19.

Regulatory mechanism of ROS

In addition to the above-mentioned antioxidant enzymes and compounds, the expression of antioxidant genes and the function of mitochondria are also essential for maintaining redox balance.

A significant portion of these genes encode antioxidants and other cytoprotective proteins. A previous study demonstrated that 3-hydroxybutyrate dehydrogenase 2 (BDH2) reduces the nuclear factor erythroid 2-related factor 2 (Nrf2) protein level by binding to the E3 ubiquitin ligase, Keap1. Specifically, Keap1 facilitates the proteasomal degradation of Nrf2. Upon activation, Nrf2 binds to antioxidant response elements (AREs) in the promoters of target genes²⁰. This binding reduces ARE promoter activity, leading to ROS accumulation. The Keap1-Nrf2 pathway has a pivotal role in ROS regulation^21,22.

Targeted intervention of mitochondria can directly impact the primary sites of ROS production. Mitophagy reduces ROS production by degrading damaged mitochondria, thereby promoting cell survival and preventing cell death²³. Mitophagy is closely related to tumor development and treatment^24,25. Gamma-glutamyltransferase 7 (GGT7) recruits RAB7 by direct binding and drives RAB7 to translocate from the nucleus to the cytoplasm and inhibit ROS production, subsequently mediating mitophagy by increasing mitophagy mediators/inducers²⁶.

Role of ROS in oncogenic functions

Low doses of ROS contribute to the development of cancer. This heightened oxidative state, surpassing that of healthy cells, significantly sustains the cancer stem cell (CSC) phenotype and promotes proliferation, metastasis, and adhesion. The Warburg effect, a result of intracellular metabolism regulation, confers a survival advantage²⁸. Cancer metabolism involves glycolysis and the TCA cycle, lipid metabolism processes, and products that regulate cell function^29,30. This process of using ATP from glycolysis as the primary source for tumor cells is also related to intracellular lipid metabolism. ROS increases the synthesis of fatty acid transport protein (FATP) and other substances help tumor cells obtain ATP via glycolysis through the Warburg effect. The heightened production of lipid peroxidation (LPO) products signifies cancer progression, providing energy for cell proliferation and migration while regulating cellular lipid levels. Tumor cells reside in a hypoxic environment, known as the tumor microenvironment (TME). Under conditions of nutrient deficiency, tumor cells resort to glycolysis to enhance survival³⁰. Upregulation of genes in tumor cells and the synthesis of enzymes promoting lipogenesis contribute to cell cycle progression³¹. These are all ways in which ROS are involved in metabolic regulation.

ROS directly participate in REDOX signal transduction as a second messenger to promote tumor angiogenesis. H. pylori pathogen-associated molecular patterns (PAMPs) interact with pattern-recognition receptors (PRRs) located on immune and epithelial cells to generate ROS, and activate mechanisms underlying angiogenesis and epithelial–mesenchymal transformation (EMT). Pathways favorable for growth and metastasis are activated in tumor cells, which accelerates the original metabolic mechanism in normal cells and effects the mechanisms underlying GC progression.

ROS affects cell migration and invasion. An increase in myoferlin protein expression on the cell membrane leads to GC migration, which is correlated with a significant increase in intracellular ROS levels. When the ROS inhibitor, N-acetylcysteine (NAC), is used to inhibit the ROS accumulation of myoferlin, the redox balance is regulated and the migration of GCs is inhibited³².

Drug resistance to ROS accumulation has also become a new target for Cancer in general treatment. Blocking cancer-associated fibroblast (CAF)-exosomes mediates lipid-ROS inhibition, which leads to increasing ferroptosis levels in cancer cells and results in increasing drug sensitivity.

Role of ROS in DNA damage and repair

Prolonged exposure to high oxidative stress can increase the probability of cells becoming cancerous. This finding is due to ROS damage to DNA inhibiting the activity of enzymes involved in DNA repair. This oxidative damage to DNA is one of the significant sources of DNA genome mutations³³. MHY440, a novel topoisomerase lota inhibitor, can be induced by enzymes and promote ROS production to cause ROS-dependent apoptosis³⁴. ROS not only cause direct DNA damage but also damage the DNA damage repair system, including base excision repair (BER) for base modification, mismatch repair, nucleotide excision repair (NER) for non-homologous end linking, and homologous recombination (HR) for double-stranded DNA processes³⁵.

Role of ROS in apoptosis

ROS accumulation regulates cell proliferation and apoptosis via key signaling pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways^36,37.

Activated MAPK family members, such as c-Jun N-terminal protein kinase (JNK) and p38 MAPK, induce cell apoptosis. Furthermore, MAPKs contribute to apoptosis by downregulating NF-κB, a transcription factor implicated in cell progression^37,38. NF-κB is initially sequestered by IκB in the cytosol. The presence of ROS induces the phosphorylation and degradation of IκB. Consequently, NF-κB translocates to the nucleus, where NF-κB induces cell proliferation or apoptosis in the presence of excessive ROS³⁹. NADPH oxidase is a group of membrane-associated enzymes that generate O₂•⁻ from oxygen, rapidly converting O₂•⁻ into H₂O₂. Lutein, a member of the xanthophyll family of carotenoids, elevates NADPH oxidase activity, consequently increasing intracellular ROS levels. This increase in ROS mediates NF-κB activation and apoptosis in GC cells³⁹.

Role of ROS in autophagy

The phosphoinositide-3-kinase- (PI3K-) and NF-κB signaling pathways are classical autophagy pathways induced by oxidative stress⁴⁰. In addition, new signaling pathways regulating GC autophagy have been discovered in recent years. The organic arsenical-derived ROS-modulating drug, ACZ2, induces ROS accumulation by depleting GSH and TrxR1, triggering a subsequent ER stress response by activating the PERK/EIF2/ATF4/CHOP signaling pathways, which is a crucial step in ACZ2-mediated autophagy⁴¹. CYT997, a novel microtubule-targeting agent, induces cell apoptosis and triggers autophagy. CYT997 induces apoptosis by stimulating intracellular mitochondrial ROS generation through regulation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in GC cells⁴².

Role of ROS in ferroptosis

Iron accumulation in cells increases lipid peroxide levels, which damages vital structural and functional lipid components, alters cellular pathways regulated by redox balance, and triggering cell death pathways. Cystine/Glutamate reverse transporter (System Xc−), glutathione peroxidase (GP), NOX, and voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane influence lipid transport. GPX and xCT are crucial for maintaining intracellular redox balance, inhibiting lipid peroxidation (LPO), and protecting cells from ferroptosis. GPX4 and System Xc− are two of the key systems^43,44.

The Wnt/β-catenin signaling pathway, a crucial regulator of GPX4, is significantly dysregulated in GC patients. Wang et al.⁴⁵ reported that the β-catenin/TCF4 transcription complex, a key transcription factor, directly interacts with the GPX4 promoter region, promoting GPX4 expression and inhibiting ferroptosis in GC cells. Inhibition of System Xc− leads to insufficient substrate synthesis of GSH, reduces the ROS scavenging ability of GPX4, and induces ferroptosis. Ophiopogonin B (OP-B) blocks the GPX4/System Xc− system, causing ROS accumulation and inducing ferroptosis in GC⁴⁶.

Unlike the above-mentioned ROS-related cell death mechanisms, iron death involves a distinct regulatory pathway targeting LPO. Arachidonate lipoxygenase (ALOX), especially ALOX15, is implicated in LPO and catalyzes the synthesis of oxidized lipid products, like 4-hydroxy-2-nonenal (4-HNE). Cell membrane Exo-miR-522 directly targets ALOX15 and is linked to lipid oxidation in GC cells, which inhibits oxidized lipid production and prevents ferroptosis⁴⁷. The key aspects related to ROS in ferroptosis are shown in Figure 3.

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Figure 3

Key aspects related to ROS in ferroptosis. Ferroptosis is an iron-dependent type of non-classical programmed cell death caused by the accumulation of LPO products. GPX4 and System Xc− have important roles in ferroptosis. Iron is transported by TFRC into the cell, where iron participates in the Fenton reaction and produces ROS. ROS accumulate and attack the cell membrane to produce LPO. The cell takes up glutamate via the Xc− system (SLC7A11 and SLC3A2) and subsequently converts glutamate into cystine, serving as a substrate for GSH synthesis. GPX4 regulates the interconversion between GSH and GSSH to regulate intracellular oxidation levels and the accumulation of lipid peroxidation products.

Role of ROS in the immune response

Appropriate levels of oxidative stress enhance the inflammatory response. In immune cells, ROS act as second messengers to increase the degree of the inflammatory response. ROS are intermediates involved in key immune steps, such as phagocytosis, antigen processing, and antigen presentation⁴⁸. A specific degree of oxidative stress can sustain the activation and proliferation of immune cells while triggering inflammatory factors. The regulatory outcome depends on how much ROS exceeds normal levels; modest ROS elevations bolster the immune system, whereas high levels of ROS instigate pathologic inflammatory responses.

Other cell death pathways related to ROS

Parthanatos is a pathway dependent on PARP1 and independent of caspase, which differs from other known cell death forms⁴⁹. ROS, generated by cell stimulation, cause DNA damage, promote gene expression in the PARP family, and trigger parthanatos⁵⁰. Pyroptosis, marked by inflammasome activation and pro-inflammatory cytokine release, is a programmed cell death type. Using sonodynamic therapy (SDT) to transport hydrophilic tirapazamine (TPZ) targets ROS release, exacerbating hypoxia and inducing pyroptosis⁵¹.

Chemotherapy and ROS regulation

Many classical clinical drugs are also related to the regulation of ROS in digestive tract tumors. A therapeutic dose of oxaliplatin causes linear dysfunction, while high concentrations of oxaliplatin increase ROS levels in mitochondria. 5-fluorouracil (5-FU) is a classic antitumor drug. 5-FU induces nuclear damage in the nucleus and mitochondrial nucleic acid. Binding of 5-FU to mitochondrial DNA leads to the instability of mitochondrial DNA and protein synthesis. Therefore, 5-FU also induces mitochondria-mediated damage⁵². Recent studies suggest that baicalin increases intracellular ROS levels, potentially counteracting 5-FU resistance⁵³.

Radiotherapy and ROS regulation

Radiation therapy directly induces DNA damage through ionization or indirectly induces ROS production, which causes DNA damage. The main cause of damage is DNA damage in the nucleus⁵².

Immunotherapy and ROS regulation

The occurrence of immune dysfunction can be improved through immunotherapy. Dysregulated lipid metabolism and heightened ROS production can result in LPO. Elevated cluster of differentiation 36 (CD36) receptor expression can lead to cellular accumulation of oxidized lipids, causing downstream T-cell dysfunction, particularly CD8⁺ T-cell dysfunction in tumors, thereby restraining cell proliferation and limiting the energy supply⁵⁴. All of these changes depend on the interaction of the cancer cell and the TME⁵⁵. ROS act as a defense barrier to eliminate harmful substances, such as foreign bacteria, and eliminate abnormal cells that have a tendency to become cancerous. The release of highly oxidizing molecules by neutrophils destroys the structure of exogenous pathogens by means of immune clearance⁵⁶. In the GC TME neutrophils undergo spontaneous ferroptosis, releasing lipid peroxides that dampen T-cell activity and increase ROS production. Immunotherapy targeting neutrophils, by enhancing ROS production, induce neutrophil ferroptosis in the TME, culminating in immunogenic cell death (ICD)⁵⁷.

Targeted therapy and ROS regulation

During tumorigenesis, various non-coding RNAs and proteins exert distinct effects by modulating the levels of ROS, impacting tumor growth and proliferation. The key regulatory targets of ROS have a pivotal role in the diagnosis and treatment of GC.

Cancer-associated non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are RNAs that are not translated into proteins⁵⁸. MiRNAs are small, single-stranded, short nucleotide sequences (between 19 and 25 nucleotides in length). MiRNAs bind to the 3′ untranslated region (3′ UTR) of messenger RNAs (mRNAs), leading to mRNA degradation or translation inhibition⁵⁹. However, miRNA function can be blocked by lncRNAs. Numerous lncRNAs > 200 nucleotides in length function as miRNA sponges, reducing the suppression of target mRNAs by binding to miRNA molecules⁶⁰. Furthermore, lncRNAs also interact with mRNAs, forming lncRNA‒mRNA complexes that directly regulate the functionality of mRNAs and influence cellular physiologic processes⁶¹.

GC death is promoted by targeting the inhibition of ROS-associated genes and related transcribed and translated products. Some exosomal lncRNAs and miRNAs have a role in regulating cell proliferation via ROS modulation⁶². Zhang et al.⁶³ reported that exosomal ferroptosis-associated lncRNA (exo-lncFERO), which originates from GC cells, induces SCD1 expression in gastrointestinal neoplasms, thereby regulating ROS levels and inhibiting ferroptosis. Exosomal miR-522, derived from CAFs, targets ALOX15 and blocks lipid-ROS accumulation, thus inhibiting ferroptosis in GC cells⁴⁷. The targeted inhibition of ROS targets with the above influences has great anti-GC potential.

Targeted regulation of ROS-related energy production processes. Considering differences in TCA cycle metabolism, targeted inhibition of metabolic energy supplies is needed for cancer cells. Cancer cells are highly dependent on the TCA cycle gene, Myc, and the frequently mutated RAS subfamily genes, including KRAS, NRAS, and HRAS. Myc is a type of proto-oncogene that regulates the process of oxidative phosphorylation and glutamine metabolites. Mutations in similar proto-oncogenes and suppressor genes may induce changes in the activity of important enzymes in the TCA cycle. These mutations include succinate dehydrogenase (SDH), fumarate hydratase (FH), and the IDH family, including IDH1, IDH2, and IDH3. These mutations lead to mitochondrial dysfunction and serve as markers of cellular targeting²⁶. In addition, some LPOs can form covalent interactions with mitochondrial DNA (mtDNA) and mitochondrial proteins, which also leads to mitochondrial dysfunction⁶⁴.

Natural products modulating ROS levels for anticancer efficacy

Further elevation of ROS levels increases the susceptibility of cancer cells to oxidative stress-induced cell death⁶⁵. Natural products and their derivatives represent a major source for anticancer drug discovery and studies have shown that natural products exert antitumor effects by modulating ROS levels. The sources and ROS-related regulatory mechanisms of natural small molecular compounds in GC are shown in Table 1.

Other therapies and ROS regulation

Photodynamic therapy (PDT) has become an ideal and promising therapeutic method for fighting cancer but its common application in clinical practice is limited by the expensive devices and light sources and the phototoxicity of photosensitizers. A previous study explored the antitumor efficacy of a novel 450-nm blue laser (BL) combined with sinoporphyrin sodium (DVDMS)-mediated PDT against human GC in vitro and in vivo, focusing on the autophagy pathway. Nanoparticle-based drug-delivery systems include liposomes, liposome-like vesicles, mito-porters, and designing mitochondria-targeted nanocarriers, which have the ability to selectively deliver drugs to the mitochondria. These carriers selectively deliver drugs to mitochondria with high ROS levels, thereby achieving targeted therapy of tumor cells⁹⁴. The regulation of ROS in antitumor therapies of GC are summarized in Figure 4.

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Figure 4

Regulation of ROS in GC antitumor therapy. ROS regulation in GC antitumor therapy primarily involves chemotherapy, radiotherapy, immunotherapy, and targeted therapy. Some chemical compounds may increase the level of ROS. Some chemical compounds can elevate ROS levels. Some of the drugs carried by the nanodrug delivery system enter the nucleus and target genes associated with ROS regulation. Radiotherapy typically induces DNA damage in the nucleus. Some drugs delivered via nanodrug delivery systems penetrate the nucleus and target genes involved in ROS regulation.