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iNOS / NOS2 Back At It - Colorectal Cancer - FOXO4, ALAS1 (Heme), and iNOS/NOS2..GPX1, GPX2 Too:)

Updated: Dec 20, 2023

This article is not intended to be medical or healthcare advice. Please consult your Primary Care Physician or an M.D. prior to embarking on any health related regimen.

Some published research related to colorectal cancer - given the prevalance of this - wanted to make sure this was shared broadly. Many of you have heard me talk or write about iNOS/NOS2 before. I sometimes call it the flamethrower in jest. Observationally, families that have NOS2 up regulations, seem to have uncommonly high GI issues ranging from diverticulitis to colon cancer. Is it any surprise, knowing that arginase is a substrate during NOS Uncoupling and causes more oxidative stress - if inhibited is implicated in reducing the spread of Colorectal Cancer[16]. Phenols inhibit Arg1, while fish oils, retonic acid, arginine, all upregulate it. [17,18,19]

FOXO4 Gene

".....The progression of several kinds of cancer is closely associated with Forkhead box O4 (FOXO4). However, the function of FOXO4 in CRC is unclear. ....FOXO4 was downregulated in CRC tissues compared with normal tissues and positively correlated with APC2 and p(S37)-β-catenin....

This research demonstrates that overexpressed FOXO4 inhibits the migration and metastasis of CRC cells by enhancing the APC2/β-catenin axis, suggesting that FOXO4 is a potential therapeutic target of CRC......Additionally, FOXO4 reduced the migration and metastasis of CRC via the APC2/β-catenin axis, identified by in vitro and in vivo experiments. Our results suggested that FOXO4 might play an important role in tumor deterioration and is a potential treatment target in CRC."[1]

ALAS1: Proliferation and metastasis of CRC cells could be inhibited by suppressing ALAS1

"The upregulation of ALAS1 was closely related to the depth of tumour invasion, N stage, and tumour size. Further analysis showed that inhibition of ALAS1 activity could inhibit the proliferation and metastasis of CRC cells, suggesting that ALAS1 might be a novel target for the treatment of CRC..... Commonly, 5'-aminolevulinic acid synthase1 (ALAS1) is the rate-limiting enzyme for haem biosynthesis. Recent studies have shown that ALAS1 is involved in a number of cellular functions and has significant effects on non-small cell lung cancer (NSCLC). However, current concepts of disease pathogenesis fail to fully explain the role of ALAS1 expression and biological functions in CRC.....Using HCT116 cell lines, we studied the impact of ALAS1 on biological function by knocking down or inhibiting ALAS1.....We found an increase in the levels of ALAS1 in cancer tissues compared to adjacent colorectal tissues. The increase in ALAS1 expression was closely related to the invasion depth, N staging and tumour size of CRC patients. The proliferation and metastasis of CRC cells could be inhibited by suppressing ALAS1....The abnormal expression of ALAS1 is closely related to the proliferation and metastasis of CRC cells, suggesting that ALAS1 may be a novel therapeutic target for the treatment of CRC.[2]

NOS2 and Colorectal Cancer

"An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ." [4]

More NOS2 and Colorectal Cancer

"....To investigate the nitric oxide synthase 2 (iNOS/NOS2) gene expression in colorectal carcinoma (CRC) and its association with the patients’ prognosis....iNOS/NOS2 mRNA expression level between cancer and normal tissue of colorectal cancer subjects were compared through the Cancer Genome Atlas (TCGA). Correlation between OS, DFS and iNOS/NOS2 expression was analyzed by cox proportional hazard regression analysis in the TCGA database....iNOS/NOS2 expression of cancer tissue was significantly higher compared to corresponding normal tissue of CRC subjects (P < 0.05). 51 nodes and 366 edges with the average node degree of 14.4 was constructed which indicated that the PPI enrichment was statistically significant (P < 1.0e-16). And peroxisome, carbon metabolism, PPAR signaling pathway were enriched in the KEGG pathway analysis. Kaplan-Meier plot showed that high expression of iNOS/NOS2 was associated with the poor overall survival (HR=0.6, P=0.019) and disease-free survival (HR=0.65, P=0.049) of colorectal cancer patients. [5]

Conclusion: iNOS/NOS2 was up-regulated in CRC and associated with poor prognosis of CRC which maybe a potential biomarker for this disease."[5]

NOS Uncoupling Present in Colorectal Cancer - BH4 Levels Important

"Increased levels of reactive oxygen/nitrogen species are one hallmark of chronic inflammation contributing to the activation of pro-inflammatory/proliferative pathways. In the cancers analyzed, the tetrahydrobiopterin:dihydrobiopterin ratio is lower than that of the corresponding normal tissue, leading to an uncoupled nitric oxide synthase activity and increased generation of reactive oxygen/nitrogen species. Previously, we demonstrated that prophylactic treatment with sepiapterin, a salvage pathway precursor of tetrahydrobiopterin, prevents dextran sodium sulfate–induced colitis in mice and associated azoxymethane-induced colorectal cancer. Herein, we report that increasing the tetrahydrobiopterin:dihydrobiopterin ratio and recoupling nitric oxide synthase with sepiapterin in the colon cancer cell lines, HCT116 and HT29, inhibit their proliferation and enhance cell death, in part, by Akt/GSK-3β–mediated downregulation of β-catenin. Therapeutic oral gavage with sepiapterin of mice bearing azoxymethane/dextran sodium sulfate–induced colorectal cancer decreased metabolic uptake of [18F]-fluorodeoxyglucose and enhanced apoptosis nine-fold in these tumors. Immunohistochemical analysis of both mouse and human tissues indicated downregulated expression of key enzymes in tetrahydrobiopterin biosynthesis in the colorectal cancer tumors. Human stage 1 colon tumors exhibited a significant decrease in the expression of quinoid dihydropteridine reductase, a key enzyme involved in recycling tetrahydrobiopterin suggesting a potential mechanism for the reduced tetrahydrobiopterin:dihydrobiopterin ratio in these tumors. In summary, sepiapterin treatment of colorectal cancer cells increases the tetrahydrobiopterin:dihydrobiopterin ratio, recouples nitric oxide synthase, and reduces tumor growth. We conclude that nitric oxide synthase coupling may provide a useful therapeutic target for treating patients with colorectal cancer."[6]

NOS2 Matters In Tumor Initiation

Despite differences in etiology, initiation, and progression, chronic inflammation has been shown to be a common element within these cancers showing interactions of numerous pathways. NO generated at the inflammatory site contributes to the initiation and progression of disease. The amount of NO generated, time, and site vary and are an important determinant of the biological effects initiated. Among the nitric oxide synthase enzymes, the inducible isoform has the most diverse range, participating in numerous carcinogenic processes. There is emerging evidence showing that inducible nitric oxide synthase (NOS2) plays a central role in the process of tumor initiation and/or development.[3]

While NOS1 and NOS3 are constitutively expressed (constitutive NOS, cNOS), NOS2 activity is induced by inflammatory cytokines, endotoxin stimulation, and hypoxic conditions. NOS isoforms generate NO at different levels. Under physiological conditions, cNOS generates NO in the picomolar (pM) to nanomolar (nM) concentration range upon transient increases in intracellular Ca2+ levels. The inducible isoform can produce NO at micromolar (μM) concentrations and its activity is sustained.[3]

Numerous studies showed that NOS2 and COX2 expression levels are coordinately induced in tumor cells indicating interplay between these enzymes in cancer. Furthermore, NOS2 can augment COX2 expression and vice versa. Moreover, both NOS2 and COX2 are regulated by the NFκB pathway and induced by interferon gamma (IFNγ). NFκB is considered to be the primary inflammation-associated transcription factor due to its activation by multiple cytokines and pathogens.[3]

NOS2 Expression In GI Cancer

Many studies have shown a relationship between NOS2 expression and cancer risk and development. To better examine and understand the NOS2 gene expression status in cancer versus their normal counterpart, the FireBrowse gene expression viewer was used to visualize expression data collected from various whole genome RNA-Seq studies. The RSEM (RNA-Seq by Expectation Maximization) mRNASeq expression profiles for each The Cancer Genome Atlas (TCGA) disease chart. Using this approach, we found that NOS2 gene expression was detected in all cancer types and most corresponding normal tissues, which represent nearly all organs/systems. Interestingly, majority of cancer data appeared to have larger error bars in their expression levels compared to their normal counterparts, which are likely a reflection of the heterogeneous nature of the cancer itself. The transcriptome data would be more accurate and represent the real expression level of NOS2 gene in cancer if those RNA-Seq studies were performed on laser-captured microdissected cancer samples. In GI tumors, our analysis shows that NOS2 gene expression is downregulated in colon adenocarcinoma (COAD) and colorectal adenocarcinoma, but in rectum adenocarcinoma, NOS2 expression shows an upward trend when compared to the corresponding controls. Expression is also upregulated in esophagus adenocarcinoma, stomach adenocarcinoma, and stomach and esophageal carcinoma. In the case of hepatocellular carcinoma (HCC), there is an elevated expression level of NOS2 relative to normal tissue. The high expression of NOS2 in liver cancer is not surprising since high NOS2 levels have been found under a variety of conditions in hepatocytes.[3]

NOS2 In Inflammatory Bowel Disease and Ulcerative Colitis

Chronic, remittent, or progressive inflammation of colon and rectum is classified as IBD, and this includes CD and UC. CD may affect the entire GI tract, whereas UC is related to inflammation of the colonic mucosa. IBD, being derived from a chronic inflammatory process, is associated with increased risk of colon cancer.[3]

The increase of both forms of IBD is associated with multiple factors, including genetic susceptibility, and environmental factors such as microbial flora and immune dysregulation. Very early onset inflammatory bowel disease (VEO-IBD) is described as IBD in children under the age of 10 years. Dhillon et al. found that NOS2 has increased activity in VEO-IBD associated with a NOS2 variant.[3]

Studies conducted in the early 1990s with samples from patients suffering from IBD showed increased activity of NOS2, levels of its substrate, l-arginine, and product, citrulline in UC,. Other studies showed increased luminal NO gas sampled from patients with UC and increased NOS2 mRNA, NOS2 protein expression, and nitrotyrosine marker in both UC and CD.[3]

Hofseth et al. showed that colon tissues from patients with colon cancer-prone chronic inflammatory disease had increased phosphorylated and total p53 associated with increases in NOS2 expression. Furthermore, they showed that NO derived from decomposition of NO donors promotes DNA damage, phosphorylation, and accumulation of p53. Moreover, tissues from patients with UC and CD had shown increased macrophage infiltration associated with increased inflammation. The increase in general inflammation and prolonged increase in NO concentrations contribute to the rise and development of colon cancer.[3]

NOS2 and Gastic Cancer

The American Cancer Society estimated more than 26,000 new cases of gastric cancer and 10,700 deaths resulting from this disease in 2016. The majority of gastric cancers are adenocarcinomas, that is, it begins in glandular cells responsible for the release of mucus and other fluids. The etiology of gastric cancer is unknown, but several risk factors have been associated with the development of this disease, including advanced age, male gender, diet, gastric adenomatous polyps, chronic inflammation, family history of gastric cancer, and Helicobacter pylori infection.[3]

One of the strong risk factors in the development of gastric cancer is H. pylori infection, which affects more than half of the world's human population. H. pylori is a gram-negative bacterium and it has in its outer membrane LPS, which can interact with Toll-like receptors (TLRs) leading to initiation of innate immune responses. Inflammation caused by infection with H. pylori has an important role in development and progress of gastric and esophageal cancers. Approximately 10% of infections are associated with the development of peptic ulcers, chronic atrophic gastritis, gastric cancers, or mucosa-associated lymphoid tumors.[3]

Gastric epithelium expresses TLR5 and TLR4 that can recognize different components from H. pylori and initiate multiple cell signaling responses against the infection. H. pylori, through activation of TLR4, which has a site that recognizes LPS as a ligand, can induce the expression of host inflammatory genes, such as TNFα, which in turn activate NFκB leading to increased COX2 and NOS2 expression. H. pylori infection also augments proliferation associated with activation of MEK1/2-ERK1/2 pathway and Ras-mediated activation of AP-1, a transcription factor associated with regulation of COX2 and NOS2 expression. Furthermore, levels of NOS2 and nitrotyrosine in the gastric mucosa are significantly higher in H. pylori-positive than in H. pylori-negative patients with gastric cancer, showing that high NOS2 is an important determinant of carcinogenesis of gastric cancer following H. pylori infection.[3]

NO has an important role in normal gastric function by controlling gastric blood flow and maintenance of gastric mucosal barrier integrity. NO controls the gastric blood flow through the activation of its second messenger cGMP. Changes in NO generation and NOS expression driven by various agents have been associated with gastric cancer onset and progression. Mutations within the NOS2 gene or its promoter are associated with NOS induction and correlate with increased rates of cancer, including gastric cancer, colorectal cancer, and esophageal cancer.[3]

Studies of resected tumors showed a correlation with increased NOS2 expression and reductions in patient survival and disease stage. Furthermore, NOS2 expression was associated with increased metastasis and angiogenesis in patients with gastric cancer. Zhang et al. showed that in over 50% of gastric cancer patients, increased NOS2 expression was associated with elevated rates of lymph node metastasis, vascular invasion, distant metastasis, and tumor node metastasis stage. Moreover, NOS2 is also associated with increased density of intratumor microvessels and angiogenesis in gastric cancer. In addition, NOS2 has been shown to be positively correlated to lymphangiogenesis and lymphatic metastasis in gastric cancer.[3]

NOS2 and COX2: GI Microbiome and Cancer

Microbiota participate in the production of NO from and and may act in the control of blood pressure and cardiovascular health. Furthermore, the GI microbiota also has an important role in digestion of food, development of resistance against pathogens, development of mucosa-associated lymphoid tissue, and in overall systemic immune homeostasis.[3]

Recent studies have shown a relationship between pretumor microbiota composition and development of gastric cancer. Interestingly, some studies found differences in microbiota composition between healthy subjects and patients affected by digestive diseases. Furthermore, chronic H. pylori infection can shift the gastric microbiota by altering stomach pH. Besides H. pylori, other bacteria, such as Escherichia coli, Fusobacterium nucleatum, and Bacteroides fragilis, are associated with colon cancer pathogenesis. Presence of bacteria can change the GI tumor microenvironment, thus effecting the carcinogenic process. E. coli colonization of tumor can influence the protumor activity of macrophages by sustaining increased levels of COX2 expression. Furthermore, commensal microbiota can modulate the tumor response by altering the levels of cytokine (TNF) production by myeloid cells within the tumor. Commensal microorganisms are important regulators of the immune system and inflammation and may be addressed in therapies for cancer treatment.[3]

Chronic inflammation plays a key role in initiation and progression of GI cancers. NOS2 and COX2 are important mediators of inflammation-driven cancer progression. The role of COX2 in various GI cancers is well established and NSAIDs have been shown to be a viable chemopreventive option. Recent observations show that majority of patients with colon, gastric, esophageal, and liver cancers have elevated expression of NOS2 in their lesions as well. Furthermore, NOS/NO levels are often associated with increased metastasis, leading to poor patient prognosis. The association of elevated NOS2 expression with cancers arising due to bacterial, viral, and fungal infections suggests an important relationship of the same with tumor immune response and chronic inflammation. The cross talk between NOS2 and COX2 may increase risk and determine patient survival.[3]

GPX2 - Glutathione Peroxidase 2 - Primarily Expressed In......The GI Tract

"Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity."[9]

"The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (−Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the −Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to −Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation."[10]

"GPx2, the gastrointestinal glutathione peroxidase, is a selenoprotein predominantly expressed in the intestine. An anti-inflammatory and anticarcinogenic potential has been inferred from the development of colitis and intestinal cancer in GPx1 and GPx2 double knockout mice. Further, induction by Nrf2 activators classifies GPx2 as a protective enzyme. In contrast, enhanced COX-2 expression is consistently associated with inflammation. The antagonistic roles and an intriguing co-localization of GPx2 and COX-2 prompted us to investigate their possible mutual regulation. Both enzymes were upregulated in tissues of patients with colorectal cancer and colitis, and co-localized in the endoplasmic reticulum. A stable knockdown of GPx2 in HT-29 cells by siRNA resulted in a high basal and IL-1-induced expression of COX-2 and mPGES-1, enzymes required for the production of the pro-inflammatory PGE2. Accordingly, si-GPx2 cells released high concentrations of PGE2. Observed effects were specific for GPx2, since COX-2 and mPGES-1 expression was not affected by selenium-deprivation which resulted in the disappearance of GPx1. It is concluded that GPx2 by compartmentalized removal of hydroperoxides silences COX-2 activity and suppresses PGE2-dependent COX-2 expression. Thus, GPx2 may prevent undue responses to inflammatory stimuli and, in consequence, inflammation-driven initiation of carcinogenesis."[12]

GPX1 (The Most Ubiquitous GPX Enzyme) In Play , but not SOD2

"Oxidative stress plays a role on the development of colorectal cancer. Manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPX1) are crucial in regulating oxidative balance and its stabilization. Possible mechanisms of action of these enzymes in various types of cancers require further investigation. We aimed to determine expression levels of these genes and their effects on protein levels in serum of patients with colorectal cancer. Expression levels of genes were determined using Real Time-Polymerase chain reaction in 35 patients with colorectal cancer. We used enzyme-linked immunosorbent assay to determine MnSOD and GPX1 levels. We found significant differences in GPX1 expression between tumor and normal tissues, with a 2-fold decrease in tumor tissues (p<0.05). However, although no significant difference was found between the expression of MnSOD gene in tumor and that in normal tissues, there was a 1.13-fold change in expression. We observed no relationship between expressions of either gene and their levels in serum.

The GPX1 gene may play a critical role in the development of colorectal cancer."[13]

"Selenium has been shown to reduce cancer incidence in animal models and more recent data indicate that it may be protective in humans as well. However, little is known about the mechanism by which selenium prevents cancer. Cytosolic glutathione peroxidase (GPX1), a selenium-containing antioxidant enzyme, has been implicated in the development of cancer of the head and neck, lung, and breast, in part because of allelic loss at the GPX1 locus. The study of allelic loss at the GPX1 locus in colon cancer was investigated by examining loss of heterozygosity (LOH) in DNA extracted from both tumor and adjacent histopathologically normal tissue obtained by laser capture microdissection. Tissue samples were obtained from 53 colon cancer patients. Two highly polymorphic markers, alanine codon repeats and a proline-leucine polymorphism (198P/L) present in the GPX1 gene, were used to examine LOH at this locus. Analysis of both polymorphisms identified LOH at GPX1 in a significant percentage of colorectal cancer (42%). These results indicated that LOH at the GPX1 locus is a common event in cancer development and that GPX1 or other tightly linked genes may be involved in the etiology of this disease."[14]

Selenium plays an important role in human health and disease, including colorectal inflammation and cancer. A decreased Se status is often correlated with increased CRC risk. Although the exact mechanisms in many cases remain to be elucidated, selenoproteins appear to affect multiple signaling pathways that reflect those properties of cancer cells often referred to as ‘hallmarks of cancer’ . Especially those selenoproteins that link directly or indirectly to redox homeostasis via regulation of oxidative stress, apoptosis, or inflammation and immune responses (e.g., GPX1-4, TXNRD1, SELENOF, SELENOP), but also those that have been linked to the canonical WNT/β-catenin signaling pathway (e.g., DIO3, GPX2, TXNRD3, SELENOP), appear to have a direct impact on CRC risk and development. This is likely because signaling pathways in redox homeostasis network with classical signal transduction pathways in every cancer hallmark and enabling characteristic. Imbalances in these pathways may be the driving force of tumor initiation and progression, thus ultimately affecting colorectal tumorigenesis. In recent years, evidence of the contributions of the intestinal microbiome to colorectal pathogenesis has emerged. Because both dietary Se as well as selenoproteins expression appear to regulate intestinal microflora, this further intertwines selenoproteins with redox homeostasis and inflammation. Human genome studies have linked various SNPs in selenoproteins genes with CRC risk, and there is strong evidence that a number of selenoprotein SNPs have functional consequences (e.g., GPX1-2, SELENOP, TXNRD1). Thus, even though the functions for some selenoproteins remain to be elucidated, strong links have been provided for several selenoproteins and the development or progression of CRC by in vitro, in vivo, and evidence from human clinical trials. Thus, selenoproteins could provide targets for cancer treatment or prevention strategies, and additional in vitro, animal, and clinical research is necessary to elucidate such potential.[15]

Oxidative Stress in Colorectal Cancer Pathogenesis

CRC is a multifactorial disease in which several factors play a significant role. Although the cause of CRC is not yet defined, research results confirm the influence of lifestyle factors, including diet, smoking, stress, alcohol and toxins. Oxidative stress leads to inflammatory reactions of the intestinal mucosa, genetic predisposition, altered intestine immune reaction, and, last but not least, dysbiosis—changes in the composition of the intestinal microbiota , which are considered an integral part of the CRC development.

Many studies confirm the influence of free radicals on the initiation, promotion and formation of IBD, and also in the process of multistage carcinogenesis . Oxidative stress in intestinal mucosal cells almost certainly plays a key role in the pathogenesis of CRC. Free radical-induced oxidative damage can result in the activation of metabolic pathways, during which other proteins affecting the processes of cell proliferation and inflammation are created.

The effects of oxidative stress on the cells of the colon mucosa could be divided into three levels: (a) the level of biological membranes (oxidation of lipids), (b) the level of the nucleus (oxidative DNA damage), and (c) the level of proteins and carbohydrates. At the same time, products of oxidative damage by free radicals represent potential indicators or markers of CRC outcome [15].

Lipid peroxidation, the main feature of oxidative stress, promotes cell destruction at the level of phospholipid cell membranes. The endoplasmic reticulum is a reservoir of calcium ions, which escape into the cytoplasm due to the peroxidation of membrane lipids. As a result, there is a loss of control over the activity of Ca2+-dependent enzymes, whose activity is controlled by the levels of calcium ions in the cytoplasm . Moreover, increased levels of Ca2+ ions in the cytoplasm stimulate the NO synthase (NOS) to produce the NO, which induces oxidative damage. Mitochondrial lipids are extremely important for maintaining structural integrity and mitochondrial functions, where oxidative damage to mitochondrial membranes disrupts cell energy metabolism. Damage to the phospholipid bilayer of the cytoplasmic membrane of colon cells leads to malfunctions of membrane receptors, the release of small molecules into the extracellular environment, and subsequent membrane rupture. As a result of lipid peroxidation by free radicals, the structure of fatty acids is damaged and their function is lost. In addition to the formation of by-products such as gaseous alkanes—ethane, propane, pentane, and hexane, lipid peroxidation produces highly toxic aldehydes, ketones, hydroxy aldehydes and epoxides. Elevated levels of ethane, methane and pentane have been detected in patients with Crohn’s disease and ulcerative colitis .

The final product of lipoperoxidation is malondialdehyde (MDA), which reacts with DNA to form MDA–DNA complexes. MDA–DNA complexes have been shown to have pro-mutagenic properties and induce mutations in oncogene/tumor suppressor genes in human tumors .

Enzymes such as lipooxygenase and cyclooxygenase are involved in oxidative stress as well. Lipooxygenase ensures the synthesis of hydroperoxides, while cyclooxygenase ensures the synthesis of endoperoxides, from which prostaglandins are formed. Cholesterol derivatives have significant pro-inflammatory and pro-apoptotic effects. Free radicals oxidize cholesterol to form oxysterols (7α-OH or 7β-OH), which are further oxidized to 7-keto-cholesterol and toxic C-5 and C-6 oxygenated derivatives of cholesterol.

As oxidation products of lipids and carbohydrates, ROS, RNS, and metal ions participate in protein oxidation. Proteins with a side chain composed of amino acids containing sulfur atoms (methionine, cysteine) are easily oxidizable. While the oxidation of cysteine produces disulfides, the oxidation of methionine produces methionine sulfoxide. Hydroxyl radicals activate the peptide bond, forming carbon radicals that react with oxygen, which creates an alkyl peroxyl radical, an alkyl peroxide or an alkyl radical. These radicals also oxidize other places on the polypeptide chain . In the absence of oxygen, carbon radicals of two different proteins due to the formation of cross-links are responsible for breaking the secondary structure of proteins, which gives rise to protein aggregates resistant to degradation by proteolytic enzymes. Significant markers of oxidative damage are carbonylated proteins with incorporated carbonyl groups. Increased levels of carbonylated proteins have been detected in patients with ulcerative colitis, diabetes mellitus , and Alzheimer’s disease . Protein oxidation leads to a gradual loss of the structure and biological function of enzymes, receptors and structural proteins [68].

Free radicals (ROS/RNS), ionizing radiation, and transition metals may directly damage the DNA/RNA. Oxidative DNA damage results in DNA strand breaks, DNA fragmentation, and base mismatches, leading to unwanted mutations . These are subsequently repaired by a system of repair enzymes that cut out and simultaneously replace the damaged bases with new bases. Non-specific endonucleases remove the entire chain. Specific DNA glycosylases remove one specific damaged base. The oxidation of DNA also changes the primary structure of DNA, the exchange or loss of bases and the formation of cross-links.

4.3. Mechanisms of CRC Development Induced by Oxidative Stress

Preclinical and clinical research has identified the primary mechanisms by which free radicals contribute to the development of CRC. The development of CRC is a multistep process of transforming a healthy intestinal cell into an abnormal one, where one mutation is not enough to cause the CRC. The direct oxidizing of bases, sugar components, and proteins associated with DNA causes mutations where free radicals, via transcription factors Nrf2 and NF-κB, intervene with inflammation and carcinogenesis. The activation/inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2), activated by free radicals, is considered effective in CRC prevention and treatment . Its activation inhibits oxidative stress and inflammation, resulting in the prevention CRC development . The primary function of Nfr2 is the regulation of cytoprotective and antioxidant gene expression. Under normal conditions, Nrf2 is in a complex with the inhibitory proteins Keap1 via the ETBE and DLG domains. Keap1 proteins enable the ubiquitination of the Nrf2 protein and, subsequently, its degradation in the proteasome. Keap1 proteins represent a regulatory mechanism by which the amount of Nrf2 in the cell’s cytoplasm is regulated. Protein modifications play a key role in adaptation to oxidative stress by activating antioxidant or metabolic programs to counteract ROS metabolism .

The promoter hypermethylation of Keap1 leads to a reduction of Keap1 expression and Nrf2 accumulation in the nucleus of patients with CRC. As a result of oxidative stress, Keap1 proteins dissociate from Nrf2 and enter the nucleus. Nrf2, together with the small sMAF (small musculoaponeurotic fibrosarcoma oncogene homolog) protein, induces the transcription of antioxidant response elements (ARE) and leads to the expression of more than 500 target genes, including antioxidant enzymes such as NAD(P)H: quinone oxidoreductase-1 (NQO1), heme oxygenase (HO-1), superoxide dismutase 1 (SOD1), and CAT; and enzymes involved in glutathione metabolisms such as glutathione S-transferase (GST), GPX and others. Nfr2 eliminates ROS through the upregulation of enzymes involved in the induction and synthesis of antioxidant molecules . Heme oxygenase 1 (HO-1) catalyzes the degradation of heme to iron, biliverdin and carbon monoxide (CO) . CO suppresses the nuclear translocation of NF-κB p65, which plays a central role in the inflammation process . Its activation leads to the production of pro-inflammatory cytokines (TNFα, IL-1β, IL-6), chemokines (MCP-1, MIP-1, RANTES, eoxantin, IL-8), transcription factors (Jnk, Erk, p38), inflammation mediators (COX-2), antimicrobial peptides and adhesive molecules (ICAM-1, VCAM-1, ELAM). Therefore, by inhibiting the nuclear translocation of NF-κB, there is a decrease in the intracellular level of pro-inflammatory cytokines.

On the other hand, overexpression of Nrf2 can promote colorectal tumor growth. The aberrant activation or accumulation of Nrf2 is connected with malignant progression, chemotherapy resistance, and poor prognosis. Therefore, if the tumor has already occurred, Nrf2 inhibitors are administered as anticancer agents. Effective Nrf2 inhibitors are brusatol, chrysin , trigonelline , ascorbic acid and retinoic acid . Luteolin, as an inhibitor of Nrf2, reverses the sensitivity of colorectal cancer cells to chemotherapeutic agents .

Nrf2 is probably also an important inhibitor of metalloproteinases (MMPs). While in humans, Nrf2 activation inhibits MMP-7, and in Nrf2-deficient mice, the level of MMP-3 is higher than in controls . At the same time, the Nrf2-deficient mice are more susceptible to benzo[α]pyrene-induced tumor formation . The pathogenesis of CRC is closely related to oxidative DNA damage and the production of pro-inflammatory cytokines, overexpression of Nrf2, expression of metastasis-associated colon cancer 1 (MACC1), and stimulation of MMP production via TNFα . Long-term stimulation of the intestinal epithelium by inflammatory cytokines and persistent activation of NF-κB are involved in the development of chronic inflammation and the initiation of carcinogenesis. The immune system responds to signaled inflammation by activating T-cells and infiltration of inflammatory neutrophils into the mucosal layer of the intestine. Neutrophils produce large amounts of ROS/RNS, whose high local concentration damages other cells of the intestinal mucosa. TNFα together with IL-1β stimulates matrix metalloproteinase (MMP) production and simultaneously regulates the COX-2 overexpression in the early stages of carcinogenesis . IL-6 activates the JAK/STAT pathways, leads to the inhibition of apoptosis and, together with TNFα, promotes angiogenesis and tumor growth. Oxidative DNA damage represents the beginning of the transformation of intestinal epithelial cells. The subsequent activation of oncogenic genes provides cells with advantages in the form of unregulated proliferation, growth, resistance to apoptosis and survival .

The inflammatory environment contributes to tumor initiation by producing reactive oxygen/nitrogen species or epigenetic changes (e.g., DNA methylation, histone modifications or changes in chromatin organization) that can play a role in carcinogenesis by silencing the expression of tumor suppressor genes and activating oncogenic signaling . It also promotes tumorigenesis by providing growth factors and pro-inflammatory cytokines . The environment of chronic inflammation as a result of the ROS signaling function provides transformed cells with suitable conditions (energy source and metabolites) to initiate carcinogenesis. The resulting effect of ROS on tumor initiation and promotion is related to quantity, location and duration.

Under normal conditions, ROS regulate many signal transduction pathways. In general, tumor cells have a higher level of ROS than healthy cells. On the other hand, cancer cells tend to produce higher levels of antioxidants to counteract the damaging effects of ROS. This suggests that maintaining a certain level of ROS is essential for cancer cells to function properly . Thus, while low levels of ROS can promote cell proliferation and invasion, excessive levels of ROS cause oxidative damage to proteins, lipids, RNA and DNA, which in turn induces cell death. As a result of metabolic abnormalities and oncogenic signaling, the protective mechanism against the persistent oxidative stress of the tumor cell is activated. This redox adaptation reaction of cancer cells results in drug resistance [15].

Screening of oxidative stress markers and antioxidants in colorectal cancer patients suggests the existence of a protective mechanism for the tumor cell. The study of Burwaiss et al. analyzed ROS in tumor cells in adjacent surrounding tumor tissues from patients with colorectal cancer and adjacent normal tissues. They found that the tumor’s oxidant and antioxidant levels were significantly lower than those in the surrounding tumor tissue and control healthy tissue. In addition, Indran and co-workers reported reduced both basal and H2O2-induced ROS production in HeLa cells with overexpressed human telomerase reverse transcriptase (hTERT), indicating a possible link between hTERT and OS in cancer cells. hTERT is a significant characteristic of CRC and has a crucial role in the maintenance and the synthesis of chromosomal ends—telomeres . Telomerase has non-telomeric function and supports growth factor-independent growth . Elevated telomerase activity is reported in almost all human cancers. The transcription factor YBX1 (cancer-related gene) upregulates the activation of the Nrf2 gene promoter in the presence of hTERT, which reduces ROS in CRC cells, thus promoting cancer progression (Figure 1). Increased telomerase activity in cancer has been shown to promote resistance to apoptosis. In addition to the response to oxidative stress, cell growth and proliferation, hTERT also regulates a wide range of important cellular functions, such as gene expression, signal transduction, and mitochondrial function . These effects are called non-canonical functions of hTERT . In addition to its nuclear localization, hTERT is also found in cytoplasm and mitochondria . hTERT transport into and out of organelles is regulated by a nuclear targeting signal sequence and a mitochondrial targeting sequence . The secretion of hTERT from the nucleus into the mitochondrion is induced by oxidative stress . Ahmed et al. reported in their study that hTERT overexpression improved mitochondrial functions by inhibiting ROS production and increasing mitochondrial membrane potential in MRC-5 lung fibroblast mitochondria. In cancer cells, hTERT translocation and overexpression improves mitochondrial potential, enhances respiratory chain activity, protects mitochondria from environmental damage, and decreases reactive oxygen species production, ultimately leading to survival.

In addition to controlling the cellular processes of free radical regulation by inhibiting oxidative stress and inflammation, new Nrf2 target genes have been identified as being involved in the inhibition of cell proliferation and the induction of apoptosis .

The activation of antioxidant enzymes is the primary cell defense mechanism. The overall increase in SOD activity is a response to tissue protection against oxidative damage under conditions of inflammation and oxidative stress in the pathogenesis of IBD. Accordingly, SOD levels in the peripheral blood of IBD patients are already being used as bio-markers of oxidative stress. Both SOD1 and SOD2 protect against spontaneous tumorigenesis, and although they have been described as tumor suppressors, they can also be upregulated during tumorigenesis. In several models, SOD and GPX, by reducing the hydrogen peroxide to water, can protect against tumor initiation induced by carcinogens and ROS. Malinowska et al. have studied the activities of GPX and SOD in the erythrocytes of CRC patients. The results showed a statistically increased activity of SOD and GPX. In mouse models of colon cancer, GPX3 has been found to suppress tumor initiation . Meanwhile, mice with a reduced expression of SOD2, either alone or in combination with a loss of GPX1, showed increased DNA damage and tumor incidence . However, the tumor initiation effect of GPX was also confirmed. GPX2-deficient mice were protected from azoxymethane-induced colorectal tumorigenesis, which is demonstrated by the tumor-initiating activity of the antioxidant GPX2 . Furthermore, other authors reported that GPX2 overexpression also plays a role in the development of prostate cancer.

The study of Kundatepe et al. monitored the oxidative stress parameters protein carbonyl (PCO), advanced protein oxidation products (AOPPs), malondialdehyde (MDA), total nitric oxide (NOx), pro-oxidant-antioxidant balance (PAB), and the ferric reducing of antioxidant power (FRAP), and found that in an impaired oxidative/antioxidant condition in breast cancer (BC) and colon cancer (CC) the oxidative stress is favored. A German study lasting from 2003 to 2012 evaluated two biomarkers of oxidative stress in CRC patient (n = 3361) d-ROMs (Diacron’s reactive oxygen metabolites) and TTLs (total thiol levels). A strong association between higher d-ROMs and lower TTL levels was observed with poorer survival. The ratio of TTL to d-ROM was an even stronger predictor of CRC prognosis than TTL alone. The results in this study showed a significant improvement in the prediction of CRC prognosis for all cancer stages. The study suggests that oxidative stress contributes significantly to premature mortality in CRC patients. Work by Sawai et al. focused on the correlation of d-ROM and the neutrophil-to-lymphocyte ratio (NLR), an inflammatory marker, as possible prognostic markers. The results obtained during the period 2013–2018 indicate that CRC patients (n = 163) with high d-ROM and high NLR had the worst disease-specific survival. Simultaneously, they discovered that tumor size was significantly associated with d-ROM and NLR. The combination of d-ROMs and NLR as prognostic markers in colorectal cancer may effectively predict prognosis in CRC patients.

Many scientific studies state that the failure of antioxidant mechanisms leads to cancer initiation and subsequent promotion, including CRC . Total antioxidant activities in CRC patients suggest this. Zinczuk et al. showed a low activity of CAT, the enzymes responsible for the elimination of hydrogen peroxide, in the blood of patients with CRC. At the same time, they detected a high activity of superoxide dismutase SOD and a higher concentration of uric acid as the most important plasma non-enzymatic antioxidant in patients with colorectal cancer compared to healthy patients. Simultaneously, the markers of oxidative stress such as MDA, advanced glycation end products and advanced oxidation protein products are significantly increased in CRC patients. On the basis of these results, it is suggested that oxidation processes exceed the antioxidant defense.

Cell transformation is associated with the transcription inhibition of apoptosis-related genes, such as cellular inhibitors of apoptosis (cIAPs), caspase-8/FADD-like IL-1beta-converting enzyme inhibitory protein (c-FLIP) and members of the bcl2 family (e.g., A1/BFL1 and bcl-xl).

4.4. Microbial Dysbiosis and Colorectal Carcinoma

Many microorganisms live in the intestinal lumen and on the intestinal mucosa . In addition to a health-promoting effect, certain members of gut microbiota can be a source of oxidants and contribute to the development of CRC . These contribute to mucosal inflammation, which leads to significant changes in the bacterial population of the large intestine . Changes associated with a reduction in the number and a change in the overall diversity of bacterial species could contribute to inappropriate reactions of the intestinal immune system and thus be key in the development of IBD and carcinogenesis .

Some potential bacteria associated with sporadic CRC are Streptococcus bovis, Streptococcus gallolyticus, enterotoxigenic Bacteroides fragilis, Fusobacterium nucleatum, Enterococcus feacalis, Escherichia coli, Peptostreptococcus anaerobius and Salmonella sp. They have been associated with CRC and cause damage to the host DNA by genotoxic agents, including a colibactin secreted by Escherichia coli, a toxin produced by Bacteroides fragilis and a stomach toxin from Salmonella. E. coli are believed to play a primary role in the induction of chronic inflammation . Their lipopolysaccharides are known to increase the expression of Toll-like receptor 4 (TLR4), which leads to the initiation of CRC . Subsequently, there is an overexpression of NF-κB, which contributes to inflammation and the development of CRC. Streptococcus bovis as an early sign of CRC , and Fusobacterium nucleatum as an indicator of a worse prognosis in CRC patients, increase the inflammation level, leading to the development of CRC. Both in vitro and in vivo studies confirm a high risk of CRC in connection with Enterococcus faecalis, which stimulates macrophages to produce superoxides, and together with hydroxyl radicals, significantly damages DNA. Peptostreptococcus anaerobius on intestinal epithelial cells activates TLR2/TLR4 and increases intracellular ROS levels, which promotes cholesterol synthesis and cell proliferation. The relation of tumorigenic bacteria to the development of CRC is thoroughly explained by a review from Li et al. .

Reactive oxygen species represent a risk factor for the development of CRC, as they cause oxidative damage to the intestinal mucosa cells, leading to the breakdown of the intestinal barrier and its dysfunction. Oxidative damage leads to abnormal Paneth cell morphology, the dilatation of the crypt lumen and erosion of its villi, and an increase in the apoptosis of crypt cells and homogeneously electron-lucent granules , which creates an entry gate for pathogenic microorganisms. The disruption of the barrier triggers the invasion of commensal and pathogenic microorganisms and leads to contact between intestinal epithelial cells and components of the microbiota, which may have protumorigenic properties.

Markers of Oxidative Stress

Teams of scientists are trying to find suitable markers for indicating the degree of damage to the colon cell by oxidative stress and the stage of the disease that the patient is in. In humans, different methods of oxidative stress measurement are used in materials such as tissue, urine, blood or serum. Regarding precancerous conditions, it is essential to detect the level of initial damage to the cells or macromolecules by oxidative stress.

Reactive species may react with DNA, lipids and proteins. Molecules such as 8-oxoguanine, 8-hydroguanine, 8-hydroxy-deoxy-guanosine and others are leading indicators of oxidative DNA damage. For oxidative damage in protein samples, it is necessary to obtain tissue to detect markers such as 2-hexahistidine, carbonyl groups, hydroperoxides, protein-bound DOPA, 3-nitrotyrosine, etc. .

Nevertheless, intensive research on biomarkers as primary indicators of oxidative damage to the body with CRC is underway. CRC-specific markers could also determine the grade/stage of cancer. In 1968, a tumor marker—a carcinoembryonic antigen (CEA)—was discovered while isolating extracts from the liver metastasized by colorectal cancer and normal fetal digestive tract . CEA is associated with many types of cancer, predominantly with gastrointestinal tumors. The study of Chandramathi et al. detected a significantly increased level of the advanced oxidative protein product (AOPP), hydrogen peroxide (H2O2) and MDA in the urine of CRC patients. The determination of CRC markers was also addressed in a study from 2019, in which the level of MDA in the blood was significantly higher in CRC patients compared to healthy controls. The authors stated that blood catalase (CAT) and MDA could be used in CRC diagnostics or as indicators of tumor invasion depth and the presence of lymph node metastasis. Depending on the stage of the disease characterized by the relevant biomarker, the survival of patients diagnosed with CRC could be predicted.

In addition to oxidative damage products, the factors of the antioxidant system are also determined. As part of monitoring the state of antioxidant capabilities, the activities of antioxidant enzymes are also detected. The most important cellular protective mechanisms against ROS are antioxidant enzymes such as catalase, glutathione peroxidase, and Prxs . In another study, the activities of Cu/Zn-SOD, GPx, GR and GSH activities, and the concentration of non-enzymatic antioxidant uric acid (UA), were detected in the serum or plasma in patients with CRC. Results of this study showed a significant increase in both enzymes activity and UA concentration. In contrast, CAT activity was considerably lower in the serum of patients with colorectal cancer compared to the control group . Markers such as CAT, AOPP, H2O2 and MDA could represent non-invasive oxidative stress markers in colorectal cancer.

However, the determination of antioxidant enzyme activities is limited because not all antioxidant enzymes and their mutual antioxidant action are known yet. Due to this fact, the preferred methods for determining the total antioxidant capacity of the investigated material are total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI) and non-enzymatic ferric reducing ability of plasma (FRAP). The results of clinical studies clearly demonstrated significantly lower levels of TAC and FRAP in CRC patients’ plasma and a significant increase in TOS and OSI levels compared to the control group .

Colorectal cancer (CRC) is very common throughout the world. Despite an improved outcome with the current treatment regimen, a huge number of patients relapse and develop drug resistant disease. Excessive production of reactive oxygen/nitrogen species (ROS/RNS) results in the development of oxidative stress that is closely implicated in the development and progression of CRC. Additionally, the tumor cells can also show an adaptive response against persistent oxidative stress which may lead to chemoresistance. Interestingly, this increased oxidative stress could be manipulated to selectively eradicate the tumor cells. This review depicts how an elevated oxidative stress disrupts different cell signaling pathways and presents the redox modulating agents that showed promising efficacy against CRC derived cell lines.[8]

Redox Status and Reactive Oxygen/Nitrogen Species

An altered redox status accompanied by an elevated generation of reactive oxygen/nitrogen species (ROS/RNS) has been implicated in a number of diseases including colorectal cancer (CRC). CRC, being one of the most common cancers worldwide, has been reported to be associated with multiple environmental and lifestyle factors (e.g., dietary habits, obesity, and physical inactivity) and harboring heightened oxidative stress that results in genomic instability. Although under normal condition ROS regulate many signal transduction pathways including cell proliferation and survival, overwhelming of the antioxidant capacity due to metabolic abnormalities and oncogenic signaling leads to a redox adaptation response that imparts drug resistance. Nevertheless, excessive reliance on elevated production of ROS makes the tumor cells increasingly vulnerable to further ROS insults, and the abolition of such drug resistance through redox perturbation could be instrumental to preferentially eliminate them. The goal of this review is to demonstrate the evidence that links redox stress to the development of CRC and assimilate the most up-to-date information that would facilitate future investigation on CRC-associated redox biology. Concomitantly, we argue that the exploitation of this distinct biochemical property of CRC cells might offer a fresh avenue to effectively eradicate these cells.[8]

 8-oxodG (8-oxo-7,8-dihydro-2′-deoxyguanosine)

It is now a well-established theory that DNA damages and genomic instability elicited by ROS play central roles in the initiation and progression of different cancers including CRC . ROS-induced primary DNA lesions are single and double-strand DNA breaks. The oxidative nucleobase modifications in DNA that are known to result in carcinogenesis via mispair/mutagenic potential of the modified base include oxidized adenines, thymines, guanines, and cytosines. Other genes that are susceptible to mutations by ROS include p53, V-Raf murine sarcoma viral oncogene homolog B (BRAF), adenomatous polyposis coli (APC), and Kirsten rat sarcoma viral oncogene homolog (KRAS). One report revealed a direct relation between oxidative stress and p53 mutation in CRC . Compared to normal mucosa, the level of 8-oxodG (8-oxo-7,8-dihydro-2′-deoxyguanosine) is higher in colorectal tumors . This adduct creates G → T transversions during replication and can be metabolized to form 8-oxodGTP, which eventually results in A → C transversions upon incorporation to DNA. The dGTP, being located chiefly in the cytoplasm, is amenable to attack by ROS while dG is shielded by histones due to its presence in the nucleus . Several types of DNA repair enzymes are available that have the potential of repairing the damages induced by 8-oxodG . 8-oxoguanine DNA glycosylase 1 (OGG1) and MutY homolog (MYH) enzymes can be cited as examples that can repair DNA by eliminating the 8-OHdG or mismatched A . However, OGG1 can be negatively regulated by ROS through the oxidation of Cys326 residue.

Moreover, mitochondrial DNA is highly vulnerable to ROS damage and is more relevant in CRC . Another striking feature is that DNA damage could generate ROS as well . This was evidenced by the fact that H2A histone family member X (H2AX) displayed NOX1-mediated ROS generation after DNA damage . DNA methylation that plays a central role in gene regulation (overexpression or silencing) has been reported to be compromised when oxidation occurs at either the methylated cytosines or guanines in CpG sequences. The formation of a new adduct obstructs the binding of the DNA methyltransferase to the cytosine residue, resulting in hypomethylation of DNA, a characteristic observed in various cancers including CRC . Concomitantly, the level of antioxidant expression is very robust in CRC that aids their adaptation in a highly oxidized milieu. When this type of redox adaptation gets prolonged, oncogenic signaling becomes activated, leading to carcinogenesis. A study conducted by Van der Logt et al. involved the measurement of ROS production in the whole blood under both unstimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions. Blood sampling was performed at least three months after the surgery of CRC. They concluded that ROS levels under both conditions were significantly higher in patients with a history of sporadic CRC, implicating that ROS may have a pivotal role in the etiology of sporadic CRC . Certain oxidative stress markers have been reported to be found in a greater amount in CRC. These include enhanced levels of overall ROS, 8-oxodG in DNA, lipid peroxides, GPx, and nitric oxide (NO) .

4-hydroxy-2-nonenal (HNE)

ROS-mediated oxidation of polyunsaturated fatty acids (PUFAs) elicits lipid peroxidation, the major products of which are malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) as well as some other minor derivatives such as hydroperoxides, lipoperoxides, conjugated dienes, etc. . Studies showed that oxidative stress is a risk factor of CRC and the levels of MDA and HNE considerably increase in CRC with its staging . HNE participates in CRC through dual mechanisms. First, HNE-induced cyclooxygenase-2 (COX-2) activation leads to PG synthesis that stimulates angiogenesis, cell migration and inhibits apoptosis in CRC . Second, the upregulation of COX-2 by HNE induces APC loss that activates the wingless-related integration site (Wnt)/β-catenin signaling pathway . Another important mutagen, MDA, stimulates DNA damage by interacting with DNA.

Several intracellular proteins contain thiol (cysteine) residues, and redox modification of these thiols has been reported to regulate numerous protein activities associated with transcription, translation, and biological functions . A good example is the activation of nuclear factor erythroid 2–related factor 2 (Nrf2) through the thiol oxidation of Kelch-like ECH-associated protein 1 (Keap1). Nrf2 that functions as a major regulator of the cellular antioxidant response is sequestered by Keap1 in the cytosol under normal conditions . Under oxidative stress, thiol oxidation of Keap1 results in the dissociation of Nrf2–Keap1 complex. This ultimately leads to the nuclear translocation of Nrf2, where it regulates the expression of antioxidant genes . The oxidative cysteine modification is mostly carried out by hydrogen peroxide (H2O2), which is the most prominent intracellular ROS. The stepwise oxidation results in thThe development, progression, and prognosis of CRC involve various redox-sensitive proteins such as phosphatase and tensin homolog (PTEN), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) that necessitate the evaluation of redox status of cysteine residues . e production of sulfenic acid (R-SOH), sulfinic acid (R-SO2H), or sulfonic acid (R-SO3H) . All these products except sulfonic acid can be reduced back to thiol state by reducing agents such as thioredoxin, glutaredoxin, peroxiredoxin, and dithiothreitol . Hence, the glutathionylation of reactive cysteines is very crucial by which intracellular redox alteration could be transduced into a functional response. However, it is still not clear how thiolation of proteins related to CRC development might be exploited for better therapeutic response and more studies are warranted to discover the exact mechanisms behind this.

Hypoxia, a salient feature of TME, is characterized by an imbalance between increased oxygen consumption and inadequate oxygen supply . The overexpression of HIF-1α protein is reported in multifarious solid malignancies including colon cancer . The tumor cells exposed to hypoxia harbor increased levels of ROS . The hypoxic tumor cells exhibit adaptation for their survival by upregulating antioxidant capacity and result in more invasive and chemoresistant phenotypes . Gao et al. reported that vitamin C and N-acetyl cysteine-mediated antitumor effects are HIF-1-dependent in the murine models of Myc-mediated tumorigenesis . Essentially, ROS can stabilize HIF-1α under basal oxygen condition as illustrated by Haddad et al. where they reported that cytokine-mediated HIF-1α stabilization and activation entails a ROS sensitive mechanism . Schmitz et al. also reported a similar finding where they showed that redox modification of HIF-1α promoted its target gene expression that ultimately resulted in the development of CRC .

Apart from hypoxia, other cells of TME such as cancer-associated fibroblasts (CAFs) also furnish ROS that can impact the pathology of cancer . CAFs harbor heightened levels of H2O2 that emerge due to a disruption in transforming growth factor beta (TGF-β) signaling . This aberrant TGF-β signaling leads to an elevated production of intracellular ROS by impairing mitochondrial function and inhibiting GPx1 and overproduction of extracellular ROS by inducing NOX . CAF-induced extracellular ROS generation is also augmented by Caveolin-1 that serves as a negative regulator of NOX-derived ROS . Upon exposure to H2O2 or CAF-conditioned medium, normal fibroblasts get transformed into an oxidative, CAF-like state that possesses increased CAF biomarkers, namely, fibroblast activation protein (FAP) and α-smooth muscle actin (αSMA) .

There are numerous cellular antioxidants such as GPx, GR, SOD, catalase, etc. that are the major sources of defense against oxidative stress. DNA repair proteins are also involved in this defense mechanism. They encompass endo- and exonucleases, glycosylases, DNA ligases, DNA polymerases, and so forth. For example, DNA glycosylases participate in the repair and removal of the oxidized base containing DNA mainly through the base excision repair (BER). Other types of oxidative lesions are repaired by nucleotide excision repair (NER) and mismatch repair (MMR) . The initiation of CRC is closely associated with a disruption in these repair proteins. Genetic alterations like single-nucleotide polymorphisms (SNPs) were observed in SOD2, myeloperoxidase, and eosinophil peroxidase genes . Another report showed a correlation between selenoprotein gene modification with CRC. According to this report, SNPs in selenoprotein P plasma 1 (SEPP1), glutathione peroxidase 4 (GPx4), and selenoprotein S (SELS) genes had demonstrated a greater risk of CRC development . A similar pattern of risk was confirmed in another study that had displayed SNPs in SEP15 and SELS genes with a concomitant risk of CRC . Moreover, a disparity in the MAPK signaling pathway was also found to be associated with elevated CRC risk . Other studies also lent support on mutation in selenoprotein genes with CRC development . From the perspective of micronutrients, vitamins C and E were found in a diminished amount in CRC patients . In GPx-3-deficient mice, inflammatory colon carcinoma was evident with increased inflammation, proliferation, and DNA damage


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