By Michał Skrzycki

Excerpt from the article published in Open Life Sciences, vol. 16, no. 1, 2021, pp. 1225-1239. https://doi.org/10.1515/biol-2021-0124

Editor’s Highlights

• The Sigma-1 receptor (Sig1R) is a membrane and cytosol protein with a sequence similar to fungal C-8.7 sterol isomerase but without any other known homology with other mammalian proteins. 
• The Sig1R is most often placed between the ER membrane and the mitochondrion, where it binds to the IP3 receptors and ankyrin.
• The Sig1R might be an additional regulator of the antioxidant system, reactive oxygen species (ROS), and antioxidant enzymes (SOD), in the formation and development of cancer.on which activity might depend on the survival and proliferation of cancer cells.
• The induction of ROS secretion by the Sig1R might be associated with changes in the activity or expression of SOD isoenzymes in reaction to the elevated level of ROS in the cell.
• The Sig1R protein influences the activity of the respiratory chain.
• The Sig1R (stress–response protein) might affect the expression of SOD isoenzymes at subsequent stages of tumor development (UICC) and in various sections of the intestine (colon, sigmoid colon, rectum).
• Changes in the expression of SOD isoenzymes and Sig1R indicate mitochondria as the main cell compartment allowing neoplastic cells to adapt to oxidative stress.

Abstract

This long-term research was designed to evaluate whether superoxide dismutase (SOD) isoenzymes participate in the development of human gastrointestinal neoplasms and the potential influence of the sigma1 receptor (Sig1R) on the regulation of SOD gene expression during the neoplastic process. The experiments included human tissues from selected gastrointestinal tract tumors (liver cancer, colorectal adenocarcinoma, and colorectal cancer liver metastases). Activity, protein levels, and mRNA levels were determined for SOD isoenzymes and Sig1R. Additionally, markers of oxidative stress (glutathione, lipid peroxidation) were measured. The results showed significant changes in the antioxidant system activity in all examined types of tumors. SOD changed both in healthy cells and in neoplastic cells. The activity and expression of all studied enzymes significantly changed due to the advancement of tumor development. The Sig1R might be an additional regulator of the antioxidant system on which activity might depend on the survival and proliferation of cancer cells. Overall, the study shows that SOD1 and SOD2 are involved not only in the formation of neoplastic changes in the human gastrointestinal tissues (healthy intestine – colon tumor; healthy liver – liver cirrhosis – liver cancer) but also in the development of tumors in the sequence: benign tumor – malignant tumor – metastasis.

1 Introduction

The current state of knowledge clearly indicates that oxidative stress is one of the most important causes of the formation and development of gastrointestinal neoplasms. Inflammation and external environmental factors (food antigens, bacteria, viruses, fungi, parasites, and toxins), metabolism of ethanol and xenobiotics, and excess iron in the diet favor the formation of large amounts of free radicals and reactive forms of oxygen and nitrogen (ROS, RNS) in the membrane cells of the intestine, stomach, pancreas, and liver. As a result, the damage of proteins, lipids, and DNA might lead to neoplastic transformation [1,2,3,4].

The first ROS resulting from the single-electron reduction of oxygen is the superoxide anion radical (O₂ ⁻). It undergoes a dismutation reaction to hydrogen peroxide (H₂O₂), which might be converted into a hydroxyl radical (HO*) in a Fenton or Haber–Weiss reaction. Further reactions lead to the formation of less common ROS and reactive forms of nitrogen (e.g., peroxynitrite NOO–). All the above-mentioned compounds strongly react with the molecular components of the cell, but in lower concentrations, they can act as intracellular messengers [5,6].

1.1 Oxidative stress

The disparity between the formation and inactivation of reactive oxygen species leads to oxidative stress. A long-term state of oxidative stress causes disturbances in cellular metabolism, which are related to the damage caused by the action of ROS. This increases the risk of irreversible changes in cellular macromolecules such as proteins, lipids, DNA, and carbohydrates. The loss of activity of some proteins, changes in the fluidity of lipid membranes, mutations, and damage to nucleic acids are the most common consequences of ROS action in normal and neoplastic cells [7,8].

In addition to the activity of antioxidant enzymes, the most significant markers of oxidative stress in the body are the level of lipid peroxidation, the concentration of glutathione (GSH), carbonyl groups, and 8-hydroxy-2′-deoxyguanosine. Lipid peroxidation is a chain of reactions in cell membranes under the influence of O₂ ⁻ and/or HO*. The end products of this process (e.g., malondialdehyde [MDA], 4-hydroxynonenal) are highly reactive compounds that can last in cells longer than reactive oxygen species and the ability to diffuse from where they are formed to distant regions of the cell.

GSH is the most common, low molecular weight antioxidant. The GSH/GSH from oxidized glutathione (GSSG) redox balance is sustained by the activity of antioxidant (GSHPx, glutathione S-transferase [GST]) and complementary (glutathione reductase [GSHR]) enzymes in its concentration. GSH and its dependent enzymes play a key role in the detoxification of peroxides, hydroperoxides, xenobiotics, and drugs [9].

1.2 Superoxide dismutase (SOD) as the component of the antioxidative system

The amount of ROS present in the cell depends on the activity of both the enzymes that generate them and the enzymes removing them, that is, antioxidant enzymes. Small-molecule antioxidants also play an important role in passive ROS inactivation, for example, GSH, tocopherols, vitamins C and A, and others [10,11].

Numerous enzymes are involved in removing ROS from the cell. The SOD is the first and key antioxidant enzyme responsible for the dismutation reaction of the superoxide radical anion to H₂O₂. In humans, there are three SOD isoenzymes: cytoplasmic (SOD1/CuZnSOD – copper–zinc), mitochondrial (SOD2/MnSOD – manganese), and extracellular (ECSOD) [12,13,14].

The product of the SOD reaction – H₂O₂ is decomposed in two ways: at high concentrations by catalase (CAT) and at lower concentrations by selenium-dependent glutathione peroxidase. Both reactions lead to the formation of water but glutathione peroxidase uses GSH as a cofactor for the reaction. This enzyme belongs to the group of GSH-dependent enzymes. GSH-dependent enzymes also include selenium-independent glutathione peroxidase (GSHPx), which removes organic peroxides. In turn, GST couples toxins and xenobiotics with GSH, whereas GSHR is responsible for the renewal of GSSG. The excess of transition metals catalyzing the Fenton and Haber–Weiss reactions is removed by chelating proteins called metallothioneins [15,16,17,18].

1.3 Sigma1 receptor (SigR) as the regulatory element of the antioxidative system

The Sig1R is a membrane and cytosol protein with a sequence similar to fungal C-8.7 sterol isomerase but without any other known homology with other mammalian proteins. The SIGMAR (Sigma1 receptor) gene is located on human chromosome 9p13. This gene is expressed as a single 30 kDa polypeptide containing 223 amino acids. Sig1R is located in the plasma membrane or the endoplasmic reticulum (ER) due to the presence of two transmembrane domains [19,20]. The Sig1R might be involved in the pathogenesis of many diseases (neurodegenerative, depression, addiction, arthritis, Crohn’s disease, and tumors), but there is not enough data to fully elucidate its function.

The Sig1R is most often placed between the ER membrane and the mitochondrion, where it binds to the IP3 receptors and ankyrin. The Sig1R is involved in numerous processes:

1. –regulating the ion channel function for calcium, potassium, sodium, and chlorine [21,22,23],
2. –inhibiting the nitric oxide synthase activation [24],
3. –acting as a chaperone protein – might be a cell stress receptor [25], and
4. –influencing apoptosis and cell proliferation by regulating the functioning of signaling pathways for Akt and extracellular signal-regulated kinases (ERK) [26].

Moreover, it exhibits chaperone activity and translocation ability from the ER membrane to the cell membrane. As Sig1R appears to have a considerable effect on the levels of numerous ions needed by the cell, it influences significant cell metabolism pathways [27].

Numerous scientific studies reported an increased expression of the Sig1R in various types of cancer (breast, prostate, lung, melanoma, and glioma), which might be necessary for regulating SOD isoenzymes. Increased level of Sig1R changes the functioning of the Akt and ERK pathways. The Akt pathway activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factor, and the ERK pathway activates the c-Fos transcription factor (AP-1 subunit). Both of these factors directly participate in the regulation of transcription of all SOD isoenzymes. In addition, the activity of the Sig1R might change depending on the presence of ROS in the cell, and it might directly affect the amount of NF-kB protein [28,29].

The goal of this study was to analyze multiple data from a number of previous researches done in our department. Such complex analysis and collation of different data allowed for new and comprehensive conclusions concerning the activity of the antioxidant system during tumor development.

…

3 Results and discussion

In the studies on the antioxidant barrier status in gastrointestinal neoplasms (benign and malignant liver cancer, colorectal adenocarcinoma, metastasis of colorectal cancer to the liver), a significantly intensified lipid peroxidation and disturbances in GSH levels and GSH-dependent enzymes activity were found in patient’s serum. The highest level of lipid peroxidation was observed in gastric cancer, whereas the lowest level characterized liver cancer. Lipid peroxidation was also increased in colorectal cancer and its metastases to the liver. Results of this research point to the oxidative stress accompanying the formation and development of gastrointestinal neoplasms. An important result of these studies is the demonstration that increased peroxidation of lipids in the blood serum of patients was characteristic for all studied neoplasms of the gastrointestinal tract. No such changes in the level of GSH and the activity of GSH-dependent enzymes were found. It is not clear whether the systemic disorders of GSH red/ox in subjects with gastrointestinal neoplasms might be the causative agent of cancer or if the neoplastic disease itself is a cause of such disorder (Table 2) [39].

Table 2

Oxidative stress markers (GSH and lipid peroxidation) in blood serum of patients with various types of gastrointestinal tract cancers

Clinical diagnosis	Healthy donors	Hepatic cancer	Colorectal cancer	Colorectal cancer to liver metastases
Blood serum	Control	Before surgery	After surgery	Before surgery	After sugery	Before surgery	After sugery
N	30	11	10	11	12	11	14
TBARS nmol/L	1.2 ± 0.2	2.2 ± 0.4*	1.5 ± 0.2**	3.1 ± 0.5*	2.8 ± 0.5*	3.5 ± 0.3*	1.7 ± 0.1**
GSH µmol/L	15.2 ± 4.1	18 ± 4.6	12.1 ± 0.9	18.5 ± 2.4	16 ± 2.1	37.3 ± 10.3*	34 ± 10.6*

TBARS (lipid peroxidation) level in blood serum of patients with gastrointestinal tract tumors. * Statistically significant versus control serum (p < 0.05). ** Statistically significant versus serum before surgery (p < 0.05). GSH level in blood serum of patients with gastrointestinal tract tumors. * Statistically significant versus control serum (p < 0.05). ** Statistically significant versus serum before surgery (p < 0.05).

Because the adaptation of neoplastic cells to oxidative stress relies on modifying their own antioxidant systems [40,41,42], the activity and expression of antioxidant enzymes (in particular SOD – the key antioxidant enzyme) at various stages of cancer development were tested. First, the level of reduced GSH and the activity of GSH-dependent enzymes (GSHPx, GST, and GSHR) in neoplastic tissues originating from various types of gastrointestinal neoplasms were analyzed. Significant changes in the activity of GSH-dependent enzymes and the level of GSH have been observed in gastric cancer, benign and malignant liver cancer hepatocellular carcinoma, primary colorectal cancer, and colorectal cancer liver metastases [43].

Our earlier research results indicate a disturbance of the antioxidant system based on GSH and GSH-dependent enzymes in tumors of the gastrointestinal tract. That is probably due to the generation of large amounts of ROS in tumor cells. Changes in the level of GSH and the activity of GSH-dependent enzymes in gastrointestinal neoplasms are connected with the altered ability to detoxify ROS, which might act as an adaptation mechanism of neoplastic cells to oxidative stress conditions.

However, the most important enzymes defending cells against oxidative stress are SOD isoenzymes – it depends on them how many and what free radicals and reactive oxygen species will be formed from the superoxide radical anion [44].

Due to the fact that superoxide anion is a precursor of the remaining ROS and RNS, studies were conducted to determine changes in the activity and level of the protein of SOD isoenzymes (SOD1/CuZnSOD and SOD2/MnSOD) in liver cancers. The applied research model in setup, the liver cirrhosis (precancerous stage) – benign tumor – malignant liver tumor, gave an answer to the question of whether the level of antioxidant enzymes depends on the extent of tumor development [45].

The obtained results showed significant changes in SOD1 and SOD2 levels during the formation and development of liver tumors (Figure 1).

The activity of SOD1 and SOD2 varied depending on both the stage of the tumor and its cellular location. In the case of the cytoplasmic isoenzyme (SOD1), its activity was significantly reduced in the cirrhotic liver and benign liver tumors but increased in malignant ones. And in case of mitochondrial enzyme (SOD2), its activity did not change in the cirrhotic liver but was increased in both benign and malignant liver tumors. In all examined tissues except for benign liver tumors, SOD1 protein level was decreased, whereas SOD2 protein level increased. The low SOD activity in the cirrhotic liver leads to ROS accumulation, leading to oxidative damage in hepatocytes. That leads to a cascade of events starting with inflammation, further exacerbation of oxidative stress, protein damage caused by it, peroxidation of cell membrane phospholipids and mutations, followed by impaired functioning of genes and signaling pathways resulting in an increase in the rate of cell proliferation, and finally as a result of progressive damage to cellular structures for neoplastic transformation. The opposite situation in malignant liver cancer (increased activity and level of SOD protein) might indicate two-way effects: on the one hand, high activity of SOD causes the generation of large amounts of H₂O₂, which activates many signaling pathways (in particular those responsible for cell proliferation), whereas on the other hand, cancer cells with elevated SOD activity are more resistant to the oxidative stress associated with neoplastic disease. SOD protects cancer cells against the damage caused by ROS by sustaining the equilibrium among the elimination of O₂ ⁻ and the formation of H₂O₂ while simultaneously providing the conditions for their increased proliferation. Consequently, instability of this equilibrium might determine whether cancer cells grow reluctantly and possibly apoptosis is activated or whether there would be an increase of proliferation, consecutively increasing their malignancy. The obtained outcome might point to the potential mechanism of malignant cancer cells bypassing oxidative stress effects, which permit their growth in a hostile environment (excess of reactive oxygen species).

In contrast to malignant tumors, in benign liver cancer, the compensatory effect of SOD isoenzymes (decreased SOD1 vs increased SOD2) was observed for the first time. This action allows the oxidant–antioxidant balance to be maintained in liver benign cancer cells. Cells with a balanced redox state are characterized by a low proliferation rate. This might be one of the causes why benign cancers develop slowly and are mild in their disease.

Assessment of the activity and protein expression of SOD isoenzymes between benign and malignant liver cancers illustrates an increase of both factors in malignant cancers. Increase of the activity and level of SOD1 and SOD2 proteins in malignant liver cancers above the level appropriate for normal cells implies improved resistance against oxidative stress in contrast to benign liver cancers. The obtained results for liver cancer indicate that various antioxidant mechanisms act more efficiently, particularly in malignant cancers. The tumor malignancy appears to depend on the cell redox status, which is largely regulated by SOD.

The conducted research has shown that the activity and level of SOD are associated with the degree of tumor development.

This was also confirmed by studies on the activity and level of SOD in colorectal cancer. It is a relatively well-described neoplasm with clearly classified stages of its clinical development and degrees of cell differentiation. Therefore, studies conducted on colorectal cancer allowed a detailed analysis of the antioxidant system permutations during the development of neoplastic disease. The activity and protein level of SOD isoenzymes and the level of lipid peroxidation (oxidative stress marker) were tested in primary colorectal cancer, which is distinguished by four clinical stages of cancer advancement (UICC – according to TNM classification) and three stages of maturity/differentiation of neoplastic cells (G) [46,47,48].

It has been shown that the activity and protein level of SOD isoenzymes changed depending on the clinical advancement stage and tumor maturity. The outcome of these researches showed that the initial stages of clinical advancement and differentiation of colorectal cancer are characterized by increased oxidative stress. However, the oxidative stress level was significantly lower in the subsequent stages of tumor development (UICC I–IV). It seems that oxidative stress in the early stages of cancer development might result from the inflammatory state related to neoplastic disease. Oxidative stress can be considered a kind of selective pressure – therefore, only cancer cells capable of adapting to it will pass into the next stages of development. This is indicated by the observed decreased level of lipid peroxidation in the following stages of colorectal cancer development.

Our research has shown that throughout the development of colorectal cancer, neoplastic cells have a constant level of antioxidant defense due to the cytosolic SOD activity.

In contrast, the mitochondrial enzyme SOD2 activity differed significantly in the following stages of the UICC but did not change due to the degree of differentiation. These results indicate that changes in SOD2 activity in the development of colorectal cancer are cyclical, that is, the activity decreased in stages I and III and increased in stages II and IV (comparing to control) (Figure 2).

An agent responsible for such fluctuations in SOD2 activity might be its reaction product – H₂O₂, which regulates SOD2 activity through feedback inhibition.

On the one hand, the level of the SOD2 protein in the subsequent stages of colorectal cancer differentiation also changed in a cyclical model: G1 – elevated, G2 – low, G3 – elevated again. On the other hand, SOD2 activity remained at a similar level. H₂O₂ appears to inhibit SOD1 as well. However, while the progression of colorectal cancer in subsequent stages of clinical advancement causes inflammation and oxidative stress due to intestinal wall overgrowth, differentiation is a change in cell histology and morphology and does not affect cell redox state. Therefore, changes in SOD protein level through differentiation might depend on the action of transcription factors (i.e., AP-1, NF-kB) and be only a secondary phenomenon that simultaneously controls the expression of SOD and contribute to the regulation of cell differentiation (Figure 3) [49].

The level of protein and the activity of SOD isoenzymes clearly changes in the successive stages of clinical advancement and differentiation degrees of colorectal cancer. These changes seem to depend primarily on the clinical stage of colorectal cancer. It is unclear whether the changes in SOD levels are correlated directly with cell differentiation. These results also indicate that changes in the protein level and activity of SOD isoenzymes might be an adaptive reaction to oxidative stress occurring in the course of the development of colorectal cancer.

The obtained results fully confirmed the assumptions of the working hypothesis, which allowed the scientific community to draw attention to the problem of changes in the expression and activity of antioxidant enzymes during the development of a neoplastic tumor.

Since the adaptive reaction to oxidative stress requires the activation of appropriate signaling pathways in cells, which will, in turn, activate the transcription of genes encoding antioxidant enzymes [36], another research model was developed to assess the expression of SOD isoenzymes both at the protein and mRNA levels in colorectal cancer [50].

Obtained results clearly show that the increased (comparing to control) level of SOD1 and SOD2 in the early stages of colorectal cancer development provides an effective defense against oxidative stress caused by inflammation. However a decreased protein level of the SOD isoenzymes in UICC stage III might indicate a destabilized antioxidant response of cancer cells, leading to further oxidation of cellular environment and, consequently, greater frequency of mutations. It would allow for changes in the phenotype of neoplastic cells required for the further development of cancer. In stage IV of UICC, differences in the levels of SOD1 and SOD2 proteins might indicate an attempt to reduce the level of ROS in the mitochondria to prevent apoptosis from being triggered, but at the same time to uphold the oxidative milieu in the cytoplasm required for proliferation and causing genetic instability.

The final stage of malignant cancer development is metastasis. The study also included subjects with metachronous metastases of colorectal cancer to the liver. The observed protein level of SOD isoenzymes clearly indicates the adaptation of cancer cells originating from the primary colon tumor to the liver environment. Liver metabolism generates large amounts of ROS as a result of cytochrome P450 activity. Therefore, cancer cells inhabiting the liver must protect their cytoplasm, which is validated by the high level of SOD1 observed in metastases (Figure 4).

The results acquired for the mRNA level of SOD isoenzymes in all clinical stages of colorectal cancer development indicate its inverse relationship to the protein level of SOD isoenzymes. The explanation for this discrepancy might be the regulation of SOD isoenzymes at the posttranscriptional level [51]. According to the literature data, an elevated mRNA level might point to a posttranscriptional reduction of the SOD protein level and activation of transcription by oxidative stress [52,53]. The differences between the mRNA and protein levels of SOD isoenzymes might depend on alterations of the red/ox cell status under the circumstances such as hypoxia, anoxia, and oxidative stress (each of these conditions occurs during tumor development).

The conducted studies indicate that the gene expression of SOD might be controlled on at least two levels: transcriptional and posttranscriptional. In contrast, the intracellular activation seems to be different for each of these levels, depending on endogenous cell conditions. SOD isoenzymes take part in maintaining the equilibrium between the generation and removal of ROS and might be one of the key enzymes determining apoptosis or survival of the cancer cell population.

Seemingly, determination of only SOD isoenzyme activity/protein level might not be sufficient to fully assess the adaptive potential of neoplastic cells to oxidative stress. Increased mRNA level might act as an unknown potential, capable of generating an additional amount of the enzyme in response to oxidative stress, whereas a decreased mRNA level of SOD might be a response to an elevated concentration of H₂O₂ to limit its pro-apoptotic effects.

The research performed in our department has shown that neoplastic cells adapt to oxidative stress by regulating the level of protein and mRNA of SODs. However, neoplastic cells maintain an increased ROS level required for their continued proliferation and play a significant role in providing an environment favoring genetic instability. For this purpose, the synthesis and activity of SOD isoenzymes must be regulated extremely precisely and on a multilevel basis.

The factor that might significantly affect the regulation of SOD isoenzymes expression seems to be the intracellular Sig1R. Recent scientific reports indicate its crucial role in cell metabolism [54,55,56].

So far, the relationship between the Sig1R and the level of SOD isoenzymes has not been studied. Therefore, we have carried out the study to clarify whether changes in Sig1R levels will be somehow related to oxidative stress and the transcription level of SOD isoenzyme genes in gastrointestinal cancers.

The first phase of the research investigated how Sig1R expression changes in colorectal cancer. The preliminary results of studies on the determination of the expression level of Sig1R in colorectal cancer and liver metastases at various stages of cancer progression are consistent with the literature data: increased levels of Sig1R were observed in colorectal cancer and also in metastases. Nevertheless, this was the only information available on this subject at that time (2013) in the literature data.

Sig1R expression level studies were performed for the first time, including clinical tumor advancement (UICC), tumor location in particular sections of the intestine, and patient age [57]. The highest expression level of the Sig1R was observed in the third stage of UICC. The ANOVA test also showed significant interactions between the clinical stage of the tumor and tumor location and the level of Sig1R expression. There was no interaction between the level of Sig1R mRNA, UICC grade, and the age of patients. However, a significant decrease in Sig1R expression was observed in older patients. It seems that the clinical stage, tumor location in the intestine, and age significantly influence the expression of the Sig1R. This is probably due to the two-fold nature of this receptor: under certain conditions, it can act pro- or antiapoptotic agent. Such effect might be due to the expression-dependent Sig1R activity.

Comparative statistical analysis of the results concerning the expression of the SOD isoenzymes and the Sig1R showed that the expression profile of all tested proteins (SigR1, SOD1, and SOD2) significantly depends on the tumor development stage (UICC) and is significantly different in each section of the intestine. This might indicate a relationship between the expression of these three proteins in cancer (unpublished data) (Figures 5 and 6).

The above observations open up a completely new perspective of research on the relationship of the Sig1R, reactive oxygen species, and antioxidant enzymes (SOD) in the formation and development of cancer.

In previous studies, we described subsequent studies carried out to evaluate the expression of Sig1R and SOD isoenzymes in the in vitro experimental model. We used different oxygen concentrations occurring during tumor development – hypoxia, tissue normoxia, and atmospheric normoxia (most of the studies on cell lines are conducted in these conditions) [58].

Diversified oxygen availability significantly influenced the expression level of all examined parameters, especially SOD2 and Sig1R. The type of cell line studied (primary cancer, metastatic cells) also influenced mainly the expression of Sig1R [59].

Statistical analysis revealed a strong relationship between the level of Sig1R, SOD2 expression, and cell survival under hypoxic conditions (1% oxygen). This points to the protective role of Sig1R and SOD2 in mitochondria. It is related to the regulation of the calcium ions’ cellular concentration and their influx to the mitochondria through ion channels regulated by Sig1R and oxidative stress (Figure 7).

The obtained results indicate the role of mitochondria as the main organelle enabling cancer cells to exist in conditions of oxidative stress and hypoxia. For the first time, due to the applied research model, our results show how oxygen conditions, such as those in the environment of a neoplastic tumor, affect the mechanisms that protect cells against oxidative stress.

The latest literature data confirms the Sig1R effect on the reactive oxygen species generation [60]. It was observed that compounds that are agonists of this receptor, by affecting the activity of the first respiratory chain complex, increased the level of ROS. The effect of Sig1R on this complex was calcium dependent. The induction of ROS secretion by the Sig1R might be associated with changes in the activity or expression of SOD isoenzymes in reaction to the elevated level of ROS in the cell [61].

Not only the activity but also the level of the Sig1R protein influences the activity of the respiratory chain. Some studies indicate that the knockout of the SIGMAR1 gene caused increased production of ROS in cancer cells, and in CHO cells, additionally induction of the NF-kB transcription factor (one of the inducers of SOD2 expression) [62].

Other authors have shown that Sig1R can stimulate the expression of the so-called antioxidant response element and the expression of cytoplasmic SOD (SOD1) [25,63].

The results and literature data obtained in the course of the research confirm direct and indirect relationships between the Sig1R and SOD isoenzymes.

4 Conclusions

The antioxidant barrier is an important element of the body’s overall protection against the development of cancer. The effects of increased oxidative stress might have a long-term impact on the functioning of many organs, especially those that are particularly vulnerable to it, such as the gastrointestinal tract. Our research on gastrointestinal tract tumors has contributed to the understanding and clarification of many issues related to the functioning and regulation of key antioxidant enzymes. The obtained results clearly show that:

1. Oxidative stress accompanies the neoplastic disease
   1. +occurred in all examined types of gastrointestinal tract tumors
   2. +it is systemic (serum, tissues)
   3. +caused changes in the antioxidant enzymes activity, especially SOD, and in the level of small molecule antioxidants (GSH).
2. Activity and protein level of SOD, the key antioxidant enzyme, varied depending on location in the cell (SOD1 and SOD2) and the type of tumor (benign – malignant), as well as in the precancerous stage (cirrhosis).
3. The expression of the SOD isoenzymes (SOD1 and SOD2) changes with the successive stages of tumor development. This correlation occurs both at the stage of transcription and translation of SOD genes.
4. The expression of SOD isoenzymes is influenced by both oxidative stress and the advancement of the neoplastic changes.
5. The gene expression level of SOD isoenzymes is important for the adaptation of neoplastic cells to oxidative stress and, consequently, for the progression of neoplastic disease.
6. The Sig1R (stress–response protein) might affect the expression of SOD isoenzymes at subsequent stages of tumor development (UICC) and in various sections of the intestine (colon, sigmoid colon, rectum).
7. Changes in the expression of SOD isoenzymes and Sig1R indicate mitochondria as the main cell compartment allowing neoplastic cells to adapt to oxidative stress.