Cytotoxic activity and mechanism of glycosmis parva leaf extracts on human b-lymphoma cells

Abstract

This study evaluated cytotoxic activities of solvent extracts from the leaves of Glycosmis parva on human B-lymphoma Ramos cells. The cytotoxic activities of the hexane, ethyl acetate, butanol and water extracts on Ramos cells were determined by resazurin assay. Only the ethyl acetate extract was chosen to further study because it exhibited potent cytotoxic activity against Ramos cells with IC50 at 15.68 μg/ml after 24 h exposure. This extract has much lower cytotoxic effect on normal human peripheral blood mononuclear cells (PBMCs) than on Ramos cells. The patterns of cell death induced by this extract were determined by staining with annexin V-FITC/ propidium iodide staining and detecting with fluorescence flow cytometer. The extract induced Ramos cell death mainly by apoptosis after 8 h of treatment. This apoptotic effect was mainly dependent on caspase activation. A pan caspase inhibitor Z-VAD-FMK almost completely blocked the apoptotic effect of the extract. This study also demonstrated that the intrinsic pathway of apoptosis was involved in the apoptotic effect of the extract. The extract had effects on the expression of proteins in the BCL-2 family which regulate the intrinsic pathway of apoptosis. It significantly decreased the mRNA expression of anti-apoptotic BCL-XL and increased the expression of pro-apoptotic BAK. It acted as a cell cycle specific cytotoxic agent against Ramos cells. It caused Ramos cell accumulation at G1 and S phases after 1 h of treatment and further cultured the treated cells in a fresh media for 48 h without the extract. This effect was correlated to its effect on the expression of cyclins and a cyclin dependent kinase inhibitor which all regulate cyclin dependent kinases (Cdks), the key regulatory kinases in the cell cycle. The extract decreased the mRNA expression of cyclin D1 and cyclin E which activate Cdk functions at early G1 phase and late G1 to S phase progression, respectively. It significantly increased the mRNA expression of a CKI p21 which binds to and inhibits Cdk activities at G1 and G2 phases. These results demonstrate that ethyl acetate extract from leaves of G. parva can induce cancer cell death mainly by apoptosis and cause cell cycle arrest at G1 and S phases.