Inflammation- and Metastasis-Related Proteins Expression Changes in Early Stages in Tumor and Non-Tumor Adjacent Tissues of Colorectal Cancer Samples.

Chronic inflammation can induce malignant cell transformation, having an important role in all colorectal cancer (CRC) phases. Non-tumor adjacent tissue plays an important role in tumor progression, but its implication in CRC has not yet been fully elucidated. The aim was to analyze the expression of inflammatory, epithelial-mesenchymal transition (EMT), and metastasis-related proteins in both tumor and non-tumor adjacent tissues from CRC patients by western blot. Tumor tissue presented an increase in metastasis and EMT-related proteins compared to non-tumor adjacent tissue, especially in stage II. Tumor tissue stage II also presented an increase in inflammatory-related proteins compared to other stages, which was also seen in non-tumor adjacent tissue stage II. Additionally, the relapse-free survival study of Vimentin and VEGF-B expression levels in stage II patients showed that the higher the expression levels of each protein, the lower 10-year relapse-free survival. These could suggest that some metastasis-related signalling pathways may be activated in stage II in tumor tissue, accompanied by an increase in inflammation. Furthermore, non-tumor adjacent tissue presented an increase of the inflammatory status that could be the basis for future tumor progression. In conclusion, these proteins could be useful as biomarkers of diagnosis for CRC at early stages.

Mitochondrial Function Differences between Tumor Tissue of Human Metastatic and Premetastatic CRC.

Most colorectal cancer (CRC) patients die as a consequence of metastasis. Mitochondrial dysfunction could enhance cancer development and metastatic progression. We aimed to evaluate the adaptations associated with mitochondrial function in tumor tissues from stages III and IV of human CRC and whether they could ultimately be used as a therapeutic target in metastatic colorectal cancer (mCRC). We analyzed the protein levels by Western blotting and the enzymatic activities of proteins involved in mitochondrial function, as well as the amount of mitochondrial DNA (mtDNA), by real-time PCR, analyzing samples of non-tumor adjacent tissue and tumor tissue from stages III and IV CRC patients without radio- or chemotherapy treatment prior to surgery. Our data indicate that the tumor tissue of pre-metastatic stage III CRC exhibited an oxidant metabolic profile very similar to the samples of non-tumor adjacent tissue of both stages. Notable differences in the protein expression levels of ATPase, IDH2, LDHA, and SIRT1, as well as mtDNA amount, were detected between the samples of non-tumor adjacent tissue and tumor tissue from metastatic CRC patients. These findings suggest a shift in the oxidative metabolic profile that takes place in the tumor tissue once the metastatic stage has been reached. Tumor tissue oxidative metabolism contributes to promote and maintain the metastatic phenotype, with evidence of mitochondrial function impairment in stage IV tumor tissue.

Antioxidant enzymes change in different non-metastatic stages in tumoral and peritumoral tissues of colorectal cancer.

Antioxidant defences and oxidative stress are related to development, progression and malignancy of colorectal cancer. However, their role in early stages of cancer remains unknown. More and more recent studies have revealed that non-tumour adjacent tissue is not a normal tissue. Thus, our aim was to analyse protein levels of MnSOD (Manganese Superoxide Dismutase), acMnSOD (Acetylated Manganese superoxide Dismutase), SIRT3 (Sirtuin 3), CuZnSOD (Cupper Zinc Superoxide Dismutase), CAT (Catalase), GPx (Glutathione Peroxidase), and GRd (Glutathione Reductase) both in tumour and non-tumour adjacent tissue from colorectal cancer patients by western blot. Non-tumour adjacent tissue seemed to have higher levels of antioxidant enzymes that detoxify hydrogen peroxide compared to tumour tissue. In contrast, tumour tissue had higher levels of MnSOD and acMnSOD. Furthermore, most of the proteins analysed showed significant differences between stage I and II in both non-tumour adjacent and tumour tissue. This could indicate that antioxidant enzymes, especially MnSOD, play a crucial role in early stages of colorectal cancer in both tissues, so they could be analysed as novel biomarkers to improve colorectal cancer diagnosis.

Sirtuin 3 silencing impairs mitochondrial biogenesis and metabolism in colon cancer cells.

Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase and targets several crucial enzymes against oxidative stress. Recent reports suggest that SIRT3 could also participate in the quality and quantity control of mitochondria. The aim of this study was to analyze whether SIRT3 silencing in colon cancer cells could affect mitochondrial biogenesis and impair mitochondrial function. For this purpose, metastatic colon cancer cell line SW620 was transfected with a specific shRNA against SIRT3 to obtain a stable knockdown. Gene expression and protein levels of several proteins related to mitochondrial biogenesis and function were determined by RT-qPCR and Western blotting. Mitochondrial function was studied by analyzing COX, ATPase, and LDH enzymatic activities, oxygen consumption, superoxide levels, and mitochondrial membrane potential. Confocal images were also taken to study mitochondrial morphology, and cell motility and clonogenicity were also studied. SIRT3 silencing resulted in a reduced mitochondrial biogenesis and function, as evidenced by the decrease in proteins such as PGC-1α and mitochondrial transcription factor A and lower levels of OXPHOS complexes. Furthermore, COX activity and oxygen consumption were also diminished after SIRT3 knockdown. Finally, SIRT3-silenced cells showed mitochondrial aggregation compared with control cells as well as reduced motility and colony formation ability. In conclusion, SIRT3 silencing in SW620 cancer cells leads to decreased mitochondrial biogenesis and mitochondrial dysfunction, ultimately affecting cell viability and could be a therapeutic strategy to render cells more sensitive to treatment.

Non-tumor adjacent tissue of advanced stage from CRC shows activated antioxidant response.

Colorectal cancer (CRC) is a leading cause of malignant cancer-related morbidity and mortality, with a higher incidence in developed countries and a high mortality rate mainly attributable to metastases. The aim of the present study was to determine the metabolic adaptations related to oxidative stress in tumor tissue from advanced stages (III and IV) of CRC and whether they could be used as potential biomarkers for clinical applications. To tackle this aim, we have analyzed the protein expression levels related to oxidative stress and the enzymatic activities of MnSOD and catalase, comparing samples of non-tumor adjacent tissue and tumor tissue of CRC patients in stages III and IV. The results showed no differences between stage III and IV in tumor tissues for any of the proteins studied. However, some differences were found between samples of non-tumor adjacent tissue and tumor tissue for some of the antioxidant enzymes. Overwhelmingly, the greatest differences were detected when comparing samples of non-tumor adjacent tissue from stage III and stage IV. To the best of our knowledge, this is the first study where differences between the non-tumor adjacent tissues of CRC patients from different cancer stages were determined. This study suggests that the parameters analyzed should be evaluated as biomarkers for the evolution of CRC. Furthermore, tumor tissue status should not be of sole importance for the prognosis of CRC, as the non-tumor adjacent tissues could also merit consideration.

Sirtuin 3 silencing improves oxaliplatin efficacy through acetylation of MnSOD in colon cancer.

Sirtuin 3 (SIRT3) is the major mitochondria deacetylase and regulates ROS levels by targeting several key proteins, such as those involved in mitochondrial function and antioxidant defenses. This way, SIRT3 balances ROS production and scavenging and promotes cell survival. The aim of this study was to analyze the effect of SIRT3 silencing on the antioxidant response in SW620 colon cancer cell line, and whether this intervention could improve efficacy of oxaliplatin, a common drug used to treat colon cancer. For this purpose, we obtained stable clones of SW620 with SIRT3 knockdown and determined parameters such as ROS levels and ROS production, levels of several antioxidant enzymes, cell viability, and apoptosis. Results showed that after SIRT3 silencing, both ROS levels and production were increased, and antioxidant enzymes gene expression was significantly reduced. Furthermore, manganese superoxide dismutase levels and enzymatic activity were reduced. Combination of SIRT3 knockdown with oxaliplatin treatment further increased ROS production and apoptosis, reducing cell viability. Finally, survival curves on colon cancer patients suggested that SIRT3 expression is related to a poorer prognosis. In conclusion, SIRT3 could be a target for colon cancer, since it regulates the antioxidant response and its knockdown improves the efficacy of oxaliplatin treatment.

Resveratrol induces mitochondrial respiration and apoptosis in SW620 colon cancer cells.

BACKGROUND: The polyphenol resveratrol (RSV) is found in the skin of red grapes and has been reported to exhibit anticancer properties. The antitumor effects of RSV in the gastrointestinal tract have gained considerable interest due to the high exposure of this tissue to this dietary compound. One of the hallmarks of cancer cells is their particular metabolism mainly relying on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. Although RSV has been described to act as a calorie-restriction mimetic, modulating energy metabolism in normal tissues, little efforts have been done to study the effects of this polyphenol in the metabolism of cancer cells. Taking this into account, the aim of this study was to explore metabolic effects of this polyphenol in colon cancer. METHODS: Oxygen consumption, ATP levels, Western blotting and other molecular biology techniques were carried out to characterize the metabolic signature of RSV in SW620 colon cancer cells. RESULTS: Paradoxically, the cytotoxic effects of RSV were associated with an increase in oxygen consumption supported by mitochondrial biogenesis and increased fatty acid oxidation. This partial reversion of the Warburg effect was followed by hyperpolarization of mitochondrial membrane and ROS production, leading to an increased apoptosis. CONCLUSIONS: Our results propose that the anticancer mechanisms of RSV could reside in targeting cancer cell metabolism, promoting mitochondrial electron transport chain overload and, ultimately, increasing ROS production. GENERAL SIGNIFICANCE: These results shed new light into the anticancer mechanism of RSV supporting the ability of this compound in potentiating the effects of chemotherapy.