Induction of Apoptosis by Tithonia diversifolia in Human Hepatoma Cells

BACKGROUND: Traditional Chinese herb Tithonia diversifolia, belonging to the Compositae family, has long been applied for the treatment of liver diseases. In recent years, many reports also indicated that it possesses hepato-protective, anti-inflammatory, and anti-cancer activities. OBJECTIVE: In this study, we evaluated whether T. diversifolia is an effective therapy for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Dry leaves of T. Diversifolia were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane-ethyl acetate (8:2→0:1) or methanol as fractions 1-6 (Td-F1 to Td-F6), respectively. We first showed that the ethyl acetate extracts of T. diversifolia leaves (Td-L-EA) exhibits growth inhibition on human hepatoma HepG2 cells. To further check the extracts-induced apoptosis, microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, flow cytometry, and Western blot analysis were performed. RESULTS: After isolating the effective fractions from Td-L-EA, we found strong cytotoxic effects of fraction-2 (Td-F2). By further analyzing the mechanisms of cytotoxic activities using microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, and flow cytometry, we found that induction of apoptosis such as DNA fragmentation increased the apoptosis rate and the apoptosis sub-G1 populations in Td-F2-treated HepG2 cells. In addition, we also confirmed Td-F2-induced degradation of caspase-8, caspase-9, caspase-3, and caspase-3 substrate PARP. Besides, Td-F2 also increased the Bcl-2 proapoptotic family protein Bax expression. CONCLUSION: In short, our results clearly showed the induction of apoptosis by ethyl acetate extracts of T. diversifolia leaves in human hepatoma HepG2 cells, suggesting its potential application as an antitumor agent. SUMMARY: T. Diversifolia leaves were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane/ethyl acetate (8:2→0:1) or methanol. These extracts exhibit growth inhibition on human hepatoma (HCC) HepG2 cells. n-Hexane/ethyl acetate (6:4) extract (Td-F2) induces apoptosis of HCC. [Image: see text] Abbreviations used: T. diversifolia, Tithonia diversifolia; HCC, Hepatocellular carcinoma DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OD, optical density; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; PARP, Poly (ADP-ribose) polymerase; PBS, phosphate buffered saline; PI, propidium iodide.