In-silico and In-vitro Molecular Analysis of Oleanolic Acid and Cisplatin on Pancreatic Cancer (Panc-1 Cell Line)

Background: Cisplatin (CIS) is a standard chemotherapeutic drug currently used for various cancer treatments. Due to its chemo-resistance and toxic effects, a new combinatorial approach was preferred. Oleanolic acid is one such pentacyclic terpenoid compound that tends to have various anti-cancer properties against a wide range of human carcinoma models. Yet, the final mechanisms of individual and Combinational Treatment of OA and CIS on pancreatic carcinoma persist indescribable. Objective: The Current study analyses the in-silico and in-vitro Molecular efficacy of the combinational dose of OA and CIS in Pancreatic cancer using the Panc-1 cell line. Methods and Material: The preliminary screening of the anti-cancer effect of OA and CIS was evaluated meticulously using docking score with Auto-Dock. For further in-vitro analysis of the ligand, OA was isolated from blueberry through ultrasonication extraction, followed by a comprehensive range of qualitative and quantitative analysis by chromatography techniques and GC-MS studies. Anti-proliferative and cytotoxicity activity of our combinational compounds were determined using the MTT assay and the LDH leakage assay. Cell membrane integrity was analyzed by measuring ROS generation and mitochondrial membrane potential in treated cells using fluorometric detection methods. Detection of the Anti-Apoptotic potential of our target compound was evaluated by DNA fragmentation assay and Caspase activity assay. Quantitative real-time PCR and Western Blotting were used to determine the genes and Protein expression intricated for apoptosis, angiogenesis, cell cycle regulation, and metastasis. Results: Molecular docking analysis suggests that OA and CIS possess a strong binding affinity for hydrogen bond interaction with the highest fitness score for various anti-cancer genes, leading to the drug's significant apoptotic and anti-angiogenic effects. Further preliminary analysis reports of UV spectra and GC-MS data suggested that the OA compound tends to exhibit a peak at 235-288 nm with a GC retention time of 15.45 min with m/z 240 and m/z 280 ratios. The output of In-vitro analysis of the anti-proliferative and cytotoxicity effect of OA and CIS tends to show the significant inhibition of cells in a dose-dependent manner with IC50 value of 5.75 μM OA and 2.95 μM of CIS with significant leakage in LDH was observed in combinational treated cells compare to individual treated cancer cells. The computational CI plot report of OA and CIS report revealed a synergistic dose effect with a CI value<1. Apoptotic effect of combinational dose revealed synergistic effects by down-regulation of angiogenic and metastatic genes and proteins (CDKN2A, SMAD4, VEG-F, MMP-9) stimulates the caspase cascade activation by intrinsic mediated apoptosis, which was further confirmed through DNA fragmentation assay by cleavage of fragments in treated cells compared to control. Conclusion: In conclusion, the present study indicates that the co-treatment of OA with CIS in pancreatic cancer cells exerts strong interaction with synergistic effects on cell growth inhibition, apoptosis induction, and angiogenesis genes through regulating signal-target proteins applicable for pancreatic cancer.