Evaluation of the Anticancer Activity of Calcium and Gemcitabine Combination in Pancreatic Ductal Adenocarcinoma Cells

Project no. 24166 · Status: Kapanmış · 2022

Summary

Pancreatic cancer, one of the most fatal malignancies globally, poses a complex challenge to clinical approaches and prognosis, with a death rate nearly equivalent to its incidence rate. The 5-year survival rate after diagnosis ranges between 1% and 14%. Surgical resection remains the only curative approach, but it is applicable to only 10-20% of patients.Gemcitabine (2',2'-difluorodeoxycytidine / dFdC), utilized as the primary therapeutic solution in adjuvant chemotherapy after surgical resection, is a nucleoside analog that pharmacologically exerts its effects by inhibiting DNA synthesis in cells.E-cadherins are calcium-dependent cell-cell adhesion proteins, and a decrease in E-cadherin expression is known to contribute to the progression of solid tumors. A study on breast cancer has demonstrated that calcium can positively alter E-cadherin expression, preventing tumor cells from evolving towards a more malignant phenotype.In this proposed research project, aiming to mitigate the aggressive progression of pancreatic cancer considering issues such as the inadequacy of chemotherapeutics and existing treatments, a pancreatic cancer model using MIA PaCa-2 cells (pancreatic ductal adenocarcinoma cell line) was initially employed. To investigate the effects of calcium alone, gemcitabine alone, and their combination, four groups were created, and all planned analyses were conducted based on these four groups. The WST-8 test assessed cytotoxicity, FITC Annexin V evaluated apoptosis, and the Cell Cycle Test Plus DNA Reagent kit analyzed the cell cycle for cells in the experimental groups. For the analysis of gene expressions of apoptosis regulators and E-cadherin, RNA isolation, followed by cDNA synthesis, was performed, and real-time polymerase chain reaction (RT-PCR) was conducted. Gemcitabine's IC50 values for MIA PaCa-2 cells at 24, 48, and 72 hours were determined as 149.17 µM, 47.25 µM, and 21.43 µM, respectively, while calcium's values were 468.30 µM, 383.33 µM, and 54.25 µM. Using the synergyfinder program and the ZIP method, the combination of gemcitabine and calcium showed a decrease from 47.25 µM to 12.5 µM for gemcitabine and from 383.32 µM to 75 µM for calcium at 48 hours. The dose reduction index was calculated as 3.78 for gemcitabine and 5.11 for calcium. Compared to the control group, apoptosis induction was 4.34-fold in the gemcitabine group, 1.62-fold in the calcium group, and 7.46-fold in the combination group. Cell cycle progression revealed S and G2/M arrest in all three groups compared to the control. Increased expression was observed in genes of the intrinsic pathway of apoptosis, and alterations were noted in cell cycle regulators in the gene expression analysis. Additionally, E-cadherin expression increased with the combination application.These results indicate the therapeutic potential of simultaneous application of calcium with gemcitabine in pancreatic cancer.

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