In silico identification of natural anticancer product and their efficacy in breast cancer cells and cancer stem like cells

Abstract

Breast cancer is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to breast cancer recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Literature survey and in silico study identified Bulbine frutescens (Asphodelaceae), Kurarinone (KU) and 3-O-(E)-p- coumaroylbetulinic acid (CB) as lead plant product/phytochemicals. Methanolic and hexane extract of B. frutescens (BME and BHE respectively), KU and CB were studied for their anticancer activity and notch signaling pathway inhibitory potential in breast cancer cells. Moreover, KU and CB were also studied for their effect in mammosphere. Literature-based identification of methanol soluble phytochemicals of B. frutescens and in silico docking study revealed Bulbineloneside D as a potent notch signaling inhibitor (ϒ-secretase). In silico docking potential of KU and CB were equal to standard gamma secretase inhibitor DAPT (-8.74 kcal/mol). KU-gamma secretase complex showed lower RMSD value, marginal fluctuation in Radius of gyration (Rg), more number of inter hydrogen bonding, and stable secondary structure of the protein which indicates KU as candidate gamma secretase inhibitor (GSI). B. frutescens extracts (IC50 4.8– 28.4 μg/ml), Kurarinone (IC50 0.43-3.42 µM) and CB (IC50 0.99-5.88 µM) significantly decreased cell viability in MDA-MB-231 and T47D cells in time dependent manner. B. frutescens, KU and CB induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in test extract/phytochemicals treated breast cancer cells. Western Blot analysis showed reduced expression of cyclin D1 and increased procaspase 3 protein expression in extract/phytochemicals treated breast cancer cells in time dependent manner. Fluorescence spectrophotometry and confocal microscopy showed extract/phytochemicals induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells at IC50 and sub IC50 concentration. Flow cytometric apoptosis analysis of extract/phytochemicals treated MDA-MB-231 cells showed significant increase in early apoptotic population in comparison to non-treated cells at IC50 and sub IC50 (half of the IC50) concentration. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens, Kurarinone and CB in HEK293 transfected cells at IC50 concentration. Moreover, RT-PCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract/phytochemical treated breast cancer cells in time dependent manner. Western Blot analysis showed reduced notch responsive protein (Hes1, Hey1 and E-cadherin) expression in extract/phytochemical treated breast cancer cells. KU and CB treatment decreased the mammosphere formation ability in MCF-7 cells at IC50 concentration by lowering the notch signaling target proteins (Hes1, Hey1, and E-cadherin) and proteins involved in cancer cell self-renewal (c-Myc, SOX-2, CD44). In conclusion, extract/phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. KU and CB have the ability to downregulate the notch signaling pathway in breast cancer and cancer stem like cells.