The Effect of Epigallocatechin Gallate on MicroRNA Levels in Pancreatic Cancer Cells

Objective: Pancreatic cancer, a leading cause of cancer-related mortality in developed countries, exhibits a dismal 5-year median survival rate of less than 5%. Gemcitabine (GEM), an FDA-approved pyrimidine antimetabolite, is commonly employed in pancreatic cancer therapy. However, due to its indiscriminate targeting of all dividing cells, severe side effects are frequently observed in pancreatic cancer patients undergoing GEM treatment. Consequently, meta-analyses have demonstrated that combining GEM with other active compounds significantly improves the 1-year survival rate of pancreatic cancer patients. Epigallocatechin-3-gallate (EGCG), an active compound found in green tea (Camellia sinensis), has shown anti-cancer effects in previous studies on pancreatic cancer. Several studies have provided evidence for the efficacy of the GEM and EGCG combination in treating pancreatic cancer. However, the underlying molecular mechanisms remain a subject of investigation. Notably, among the studies conducted thus far, none have explored the impact of altered miRNA expression, a critical epigenetic modulator in pancreatic cancer pathology. In this study, we aim to determine the cytotoxic and apoptotic effects of the Gemcitabine and EGCG combination in PANC1 cells, a pancreatic cancer model. Subsequently, we investigate the effectiveness of this combination on the expression levels of miRNAs involved in cancer progression. Material and Methods: Cytotoxicity of GEM and EGCG in PANC1 cells was assessed using the WST-1 assay, and combination effects were analyzed using isobologram analysis. Apoptosis analysis was performed using the Annexin V method. miRNA isolation was conducted with the miRNeasy Kit, followed by cDNA synthesis using the miScript II Reverse Transcription Kit. Changes in the expression of miRNAs involved in cancer cell proliferation, apoptosis, and metastasis were examined using real-time qRT-PCR analysis. Results: The IC50 values for GEM at 24, 48, and 72 hours were determined as 72.85 μM, 26.55 μM, and 9.38 μM, respectively. EGCG's IC50 values at 24, 48, and 72 hours were determined as 64.36 μM, 48.34 μM, and 19.73 μM, respectively. When combined at a 2:3 ratio (GEM: EGCG) at 24 and 72 hours, a synergistic effect was observed, while at 48 hours, a strong synergistic drug interaction was observed. Treatment with the 48-hour GEM dose resulted in a 4.2-fold increase in apoptosis compared to untreated controls, whereas the combination treatment led to a 12.04-fold increase. After combination treatment, the expression of tumor suppressor miRNAs, miR-137, and miR-130a-3p, increased, while the expression of oncogenic miRNAs, including miR-27a-3p, miR-425-5p, miR-183-5p, miR-187-3p, miR-21-5p, miR-324-5p, and miR-486-5p, decreased. Conclusion: EGCG can sensitize pancreatic cancer to GEM through epigenetic mechanisms, shedding light on novel therapeutic approaches.