Apoptosis induction and cell cycle arrest of Micromelum Hirsutum extracts in human B-lymphoma cells

Abstract

This study aimed to evaluate cytotoxic activity of Micromelum hirsutum solvent extracts on human B-lymphoma cells, Ramos cells. The dichloromethane, hexane, and methanol extracts from branches (BD, BH, and BM) and leaves (LD, LH, and LM) of M. hirsutum were primarily screened for their cytotoxicities against Ramos cells. The dichloromethane and the hexane extracts from both branches (BD and BH) and leaves (LD and LH) of the plant demonstrated cytotoxic effects on Ramos cells higher than on normal human peripheral blood mononuclear cells (PBMCs). These extract were further evaluated for their apoptotic induction activities on Ramos cells. BD and BH induced Ramos cell death mainly by apoptosis in a concentration dependent manner after 12 h exposure. LD and LH also induced Ramos cell death mainly by apoptosis after 12 h exposure, but their effects were concentration independent. BD and BH were further studied for their mechanisms of cytotoxic actions. They induced Ramos cell apoptosis mainly by caspase activation. A pan caspase inhibitor, Z-VAD-FMK, almost completely blocked the effects of BD and BH. It is possible that these extracts induce apoptosis via the intrinsic pathway. Anti-Fas ligand antibody which blocks Fas-Fas ligand interaction of the extrinsic pathway in lymphocytes did not inhibit their apoptotic effects. BD and BH had effects on the expression of Bcl-2 family proteins which involve in the intrinsic pathway of apoptosis. BD significantly inhibited the mRNA expression of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xl and significantly increased the mRNA expression of pro-apoptotic protein, BAX. BH also had similar effects on the mRNA expression of these Bcl-2 family proteins but its effect was statistically nonsignificant. The effects of BD and BH on the cell cycle of Ramos cells were also investigated. BH induced cell death without changing the cell cycle pattern of Ramos cell. BD changed the cell cycle pattern of the cancer cells. It caused the cells accumulation in the S and G2-M phases. The molecular effects of BD on several proteins involve in the cell cycle progression were also evaluated. BD profoundly inhibited the mRNA expression of the cyclin D1, slightly inhibited the cyclin E. Both cyclins play positive role in the cell cycle progression. However, BD also inhibited the mRNA expression of p53 and p21 which negatively regulated the cell cycle. These results demonstrate that dichloromethane and hexane extracts from branches of M. hirsutum (BD and BH) contain active compounds which potentially induce cancer cell death mainly by apoptosis.