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Maintaining a balanced redox state within cells is crucial for the sustenance of life. The process involves continuous cytosolic disulfide reduction reactions to restore oxidized proteins to their reduced thiol forms. There are two main cellular antioxidant pathways-the thioredoxin (Trx) and glutathione (GSH)/glutaredoxin (Grx) systems. In the GSH/Grx system, glutathione reductase (GR; GSR) catalyses the reduction of GSH disulfide (GSSG) to its sulfhydryl form (GSH), which can then further reduce oxidized Grxs. GR is an essential enzyme that helps in maintaining the supply of reduced glutathione-GSH, which is a significant reducing thiol found in most cells and known for its antioxidant properties. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of GR protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of GR and tumour histological grade, depth of invasion, regional lymph node involvement, staging, and PCNA immunohistochemical expression. It was found that 95% of patients with stage I had low levels of GR expression, whereas 89% of patients with stage III had high levels of immunohistochemical expression. A high level of expression was also detected in the patients with stage II of the disease, where almost 63% were characterized by a high expression of GR. The Western blot method revealed that the highest level of expression was found in the LS 174T cell line, which corresponds to stage II. The results of our study indicate that the immunohistochemical expression of GR may act as an independent prognostic factor associated with colon adenocarcinoma patients' prognosis.

Glutaredoxin 2 (Grx2; Glrx2) is a glutathione-dependent oxidoreductase located in mitochondria, which is central to the regulation of glutathione homeostasis and mitochondrial redox, and plays a crucial role in highly metabolic tissues. In response to mitochondrial redox signals and oxidative stress, Grx2 can catalyze the oxidation and S-glutathionylation of membrane-bound thiol proteins in mitochondria. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of Grx2 protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of Grx2 and tumor histological grade, depth of invasion, regional lymph node involvement, angioinvasion, staging, and PCNA immunohistochemical expression. It was found that 87% of patients with stage I had high levels of Grx2 expression. In contrast, only 33% of patients with stage II and 1% of patients with stage III had high levels of Grx2 expression. Moreover, the multivariate analysis revealed that the immunohistochemical expression of Grx2 protein apart from the grade of tumor differentiation was an independent prognostic factors for the survival of patients with colon adenocarcinoma. Studies analyzing Grx2 levels in patients' blood confirmed that the highest levels of serum Grx2 protein was also found in stage I patients, which was reflected in the survival curves. A higher level of Grx2 in the serum has been associated with a more favorable outcome. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western Blot.

Glutaredoxin 1 (Grx1) is an essential enzyme that regulates redox signal transduction and repairs protein oxidation by reversing S-glutathionylation, an oxidative modification of protein cysteine residues. Grx1 removes glutathione from proteins to restore their reduced state (protein-SH) and regulate protein-SSG levels in redox signaling networks. Thus, it can exert an influence on the development of cancer. To further investigate this problem, we performed an analysis of Grx1 expression in colon adenocarcinoma samples from the Polish population of patients with primary colon adenocarcinoma (stages I and II of colon cancer) and those with regional lymph node metastasis (stage III of colon cancer). Our study revealed a significant correlation between the expression of Grx1 protein through immunohistochemical analysis and various clinical characteristics of patients, such as histological grade, depth of invasion, angioinvasion, staging, regional lymph node invasion, and PCNA expression. It was found that almost 88% of patients with stage I had high levels of Grx1 expression, while only 1% of patients with stage III exhibited high levels of Grx1 protein expression. Furthermore, the study discovered that high levels of Grx1 expression were present in samples of colon mucosa without any pathological changes. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western blot.

Glutathione peroxidase 2 (Gpx-2) is a selenoenzyme with antioxidant capabilities that may play a role in cancer development. Hence, we investigated the immunohistochemical expression of Gpx-2 protein in colon adenocarcinoma samples derived from patients with colon adenocarcinoma who did not receive any form of treatment prior to the surgical procedure. The associations between the immunohistochemical expression of Gpx-2 and clinical parameters were analysed using the Chi 2 test and Fisher's exact test. A Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Gpx-2 expression and the 5-year survival rate of patients. In total, 101 (80.80%) samples had strong Gpx-2 protein expression and 24 (19.20%) samples were characterized with low expression. The high expression of Gpx-2 was correlated with the histological grade of the tumour ( p < 0.001), PCNA immunohistochemical expression ( p < 0.001), depth of invasion ( p = 0.001) and angioinvasion ( p < 0.001). We can conclude that high expression of Gpx-2 is correlated with reduced survival of colon adenocarcinoma patients (log-rank, p < 0.001).

Several studies revealed that expression levels of glutathione peroxidase 1 (Gpx-1) can be associated with cancer development, mainly through its role in hydroperoxide scavenging by regulating intracellular reactive oxygen species (ROS) levels. Therefore, our aim was to investigate the expression of Gpx-1 protein in a population of Polish patients with colon adenocarcinoma in the absence of any therapy prior to radical surgery. The study was carried out using colon tissue from patients with adenocarcinoma of the colon confirmed by histopathological examination. Gpx-1 antibody was used to determine the immunohistochemical expression of Gpx-1. The Chi 2 test or Chi 2 Yatesa test were used to analyse the associations between the immunohistochemical expression of Gpx-1 and clinical parameters. The relationship between Gpx-1 expression, and 5-year patient survival was examined using Kaplan-Meier analysis and the log-rank test. Intracellular localisation of Gpx-1 was detected by the use of transmission electron microscopy (TEM). Western blot analysis was used for the evaluation of Gpx-1 protein expression levels in cancer cell lines in vitro. Immunohistochemical study revealed that the high expression of Gpx-1 was associated with the tumour's histological grade, proliferating cell nuclear antigen (PCNA) immunohistochemical expression, depth of invasion, and angioinvasion (all p < 0.001) (4). The high immunohistochemical expression of Gpx-1 is correlated with poor prognosis of colon adenocarcinoma patients.

The Notch signalling pathway is one of the most conserved and well-characterised pathways involved in cell fate decisions and the development of many diseases, including cancer. Among them, it is worth noting the Notch4 receptor and its clinical application, which may have prognostic value in patients with colon adenocarcinoma. The study was performed on 129 colon adenocarcinomas. Immunohistochemical and fluorescence expression of Notch4 was performed using the Notch4 antibody. The associations between the IHC expression of Notch4 and clinical parameters were analysed using the Chi 2 test or Chi 2 Yatesa test. The Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Notch4 expression and the 5-year survival rate of patients. Intracellular localisation of Notch4 was detected by the use of the immunogold labelling method and TEM. 101 (78.29%) samples had strong Notch4 protein expression, and 28 (21.71%) samples were characterised by low expression. The high expression of Notch4 was clearly correlated with the histological grade of the tumour ( p < 0.001), PCNA immunohistochemical expression ( p < 0.001), depth of invasion ( p < 0.001) and angioinvasion ( p < 0.001). We can conclude that high expression of Notch4 is correlated with poor prognosis of colon adenocarcinoma patients (log-rank, p < 0.001).