Induction of apoptosis and the signalling pathways involved by dillenia suffruticosa dichloromethane root extract in mcf-7 and mda-mb-231 breast cancer cells

Dillenia suffruticosa has been used traditionally to treat cancerous growth. Previous study reported that dichloromethane extract of D. suffruticosa root (DCM-DS) was the most cytotoxic towards breast cancer cells. The present study investigated the mode of cell death and the signalling pathways involved in MCF-7 and MDA-MB-231 breast cancer cells treated with DCM-DS. DCM-DS was obtained by sequential solvent extraction. The cytotoxicity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using an inverted light microscope and AnnexinV/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) level were performed by using flow cytometry. The cells were co-treated with DCM-DS and antioxidants α-tocopherol or ascorbic acid to evaluate the involvement of ROS in the cytotoxicity of DCM-DS. Effect of DCM-DS on the expression of antioxidant, apoptotic, growth, survival genes and proteins were analysed by using GeXP-based multiplex system and Western blot, respectively. The compounds in DCM-DS were isolated by various chromatography techniques. The structure of the compounds was elucidated by using nuclear magnetic resonance analysis. DCM-DS was cytotoxic to the MCF-7 and MDA-MB-231 cells in a time-and dose-dependent manner. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 and MDA-MB-231 cells, respectively. DCMDS induced apoptosis and oxidative stress in these two cell lines. Treatment with α- tocopherol reduced the cytotoxicity of DCM-DS at 50 µg/mL in the cells, suggesting that DCM-DS induced lipid peroxidation to destroy the cancer cells. Therefore, DCMDS can be employed as a pro-oxidant agent to treat breast cancer. The induction of apoptosis in MCF-7 and MDA-MB-231 cells by DCM-DS is possibly due to the activation of pro-apoptotic JNK1 and down-regulation of anti-apoptotic ERK1 and AKT1, which in turn down-regulates anti-apoptotic BCL-2 to increase the BAX/BCL-2 ratio to initiate the mitochondrial apoptotic pathway. The induction of cell cycle arrest in MCF-7 and MDA-MB-231 cells is possibly via p53/p21-dependent and p53- independent but p21-dependent pathway, respectively. A total of seven triterpene compounds were isolated. Betulinic acid (BA) appears to be the major and most cytotoxic compound in DCM-DS. Therefore, BA could be used as a mean for standardisation of herbal product from D. suffruticosa. In conclusion, the data suggest the potential application of DCM-DS in the treatment of breast cancer