In silico and in vitro analysis of coumarin derivative induced anticancer effects by undergoing intrinsic pathway mediated apoptosis in human stomach cancer.

Background: Coumarin plays a vital role in drug discovery process due to its diverse biologically active components. Recently, coumarin derivatives are paying attention to treat various diseases including cancer. The effect of coumarin derivatives on gastric cancer is not well established although gastric cancer being the fourth leading cancer. Therefore, we attempt to study the effect of styrene substituted biscoumarin (SSBC) to induce apoptosis and inhibit cancer proliferation using in silico and in vitro approaches.

Methods: We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to identify the anti-proliferative activity of SSBC in stomach cancer cell lines (AGS) and toxicity of the compared was also assessed using lung normal cell lines (L-132 and MRC-5). A docking study was carried out between anti-apoptotic protein (BCL2) and SSBC compound. Furthermore, we analyzed the drug likeliness by screening pharmacological properties (ADME) and biological activity of SSBC by performing spectrum prediction analysis (PASS). The apoptotic effect of SSBC in AGS cell lines were detected using flow cytometry (FACS), Hoechst staining and DAPI/PI staining. Later, the regulation of apoptotic pathway by SSBC was also confirmed by qRT-PCR and western blotting analysis.

Results: The inhibition concentration (IC) of SSBC was assayed against AGS and lung normal cell lines (L-132 and MRC-5). The IC value of SSBC toward AGS, L-132 and MRC-5 was 4.56, 268 and 285 μg/ml, respectively. In silico analysis predicted SSBC could bind to the active site of BH3 domain of anti-apoptotic protein and thus resulted in apoptotic mediated cell death. ADME prediction of SSBC exhibit strong binding capacity of 99.08% and showed absorption rate about 95.57% in the intestine. In addition, biological activity of SSBC was also predicted using PASS program and we found SSBC exhibit high activity for various cancer related protein expression including apoptosis pathway proteins such as caspase 3 stimulant, apoptosis agonist. Furthermore, apoptosis of AGS was also assessed using Hoechst staining, DAPI/PI analysis, flow-cytometric analysis, qRT-PCR and western blot analysis.

Conclusion: Our study denotes that SSBC could be very effective against AGS by inducing apoptosis through intrinsic pathway and recommended for in vivo and human trials.