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 Ca
2+-dependent enzymes, whose activity is controlled by the levels of calcium ions in the cytoplasm [
53]. Moreover, increased levels of Ca
2+ ions in the cytoplasm stimulate the NO synthase (NOS) to produce the NO, which induces oxidative damage [
54]. Mitochondrial lipids are extremely important for maintaining structural integrity and mitochondrial functions [
55], where oxidative damage to mitochondrial membranes disrupts cell energy metabolism [
56]. 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 [
57].
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 [
69]. 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 [
70].
2.2. 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 [
71,
72,
73]. The activation/inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2), activated by free radicals, is considered effective in CRC prevention and treatment [
71]. Its activation inhibits oxidative stress and inflammation, resulting in the prevention CRC development [
71]. 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 [
74,
75].
The promoter hypermethylation of Keap1 leads to a reduction of Keap1 expression and Nrf2 accumulation in the nucleus of patients with CRC [
76]. 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) [
77] and leads to the expression of more than 500 target genes, including antioxidant enzymes [
78,
79] such as NAD(P)H: quinone oxidoreductase-1 (NQO1), heme oxygenase (HO-1), superoxide dismutase 1 (SOD), and catalase (CAT); and enzymes involved in glutathione metabolisms such as glutathione S-transferase (GST), glutathione peroxidase (GPX) and others [
80,
81]. Nfr2 eliminates ROS through the upregulation of enzymes involved in the induction and synthesis of antioxidant molecules [
80]. Heme oxygenase 1 (HO-1) catalyzes the degradation of heme to iron, biliverdin and carbon monoxide (CO) [
62]. CO suppresses the nuclear translocation of NF-κB p65, which plays a central role in the inflammation process [
82,
83]. 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) [
84]. 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 [
85,
86]. Therefore, if the tumor has already occurred, Nrf2 inhibitors are administered as anticancer agents. Effective Nrf2 inhibitors are brusatol [
87], chrysin [
88], trigonelline [
89], ascorbic acid [
90] and retinoic acid [
91]. Luteolin, as an inhibitor of Nrf2, reverses the sensitivity of colorectal cancer cells to chemotherapeutic agents [
92].
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 [
93]. At the same time, the Nrf2-deficient mice are more susceptible to benzo[α]pyrene-induced tumor formation [
94]. 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 [
95]. 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 [
96]. TNFα together with IL-1β stimulates matrix metalloproteinase (MMP) production and simultaneously regulates the COX-2 overexpression in the early stages of carcinogenesis [
97]. IL-6 activates the JAK/STAT pathways, leads to the inhibition of apoptosis and, together with TNFα, promotes angiogenesis and tumor growth [
98]. 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 [
99].
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 [
100,
101]. It also promotes tumorigenesis by providing growth factors and pro-inflammatory cytokines [
10]. 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 [
74].
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 [
102]. 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 [
103,
104,
105]. 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. [
106] 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 [
107] reported reduced both basal and H
2O
2-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 [
108,
109]. Telomerase has non-telomeric function and supports growth factor-independent growth [
110,
111]. Elevated telomerase activity is reported in almost all human cancers [
111]. 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 [
112]. Increased telomerase activity in cancer has been shown to promote resistance to apoptosis.
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 [
73
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 [
122]. Both SOD1 and SOD2 protect against spontaneous tumorigenesis, and although they have been described as tumor suppressors, they can also be upregulated during tumorigenesis [
123].