Cytotoxicity Of Zuojinwan On Eight Kinds Of Human Cancer Cells

Objective: To investigate the cytotoxicity of Zuojinwan on8kinds of humancancer cells, and then to study the possible mechanism.Methods: In this study, different concentrations of Zuojinwan (19,38,76,152and304μg/ml, final concentration) under different treatment times (24,48and72h) wereinvestigated to inhibit the growthes of8kinds of human cancer cell lines, includinghuman hepatocellular carcinoma cell lines SMMC-7721, BEL-7402, BEL-7404andHepG2, human lung cancer cell lines A549, NCI-H446and NCI-H460, and humancolon cancer cell line HCT-116, by MTT assay. Apoptosis and cell cycle weremeasured by flow cytometry and propidium iodide staining. Meanwhile, single-cell gelelectrophoresis (SCGE) was employed to detect cell death and nuclear DNAfragmentation caused by Zuojinwan. Acridine orange (AO)/ethidium bromide (EB)staining,4′,6′-diamidino-2-phenylindole (DAPI) staining, rhodamine123staining andtransmission electron microscope (TEM) were used to evaluate the morphology changesin cell apoptosis. In mechanism investigation, protein expressions of Bax, Bak, Bcl-2and Bcl-xl were assayed by western blotting. Furthermore, the activities of Caspase-3and Caspase-9after Zuojinwan treatment were also detected.Results: The growthes of8kinds of hunman cancer cells were strikingly inhibitedby Zuojinwan in a dose-dependent manner. Flow cytometry assay found that Zuojinwancould block SMMC-7721and HepG2cells at G0/G1phase, NCI-H460cells at S phase,while no significant cell cycle interference could be found on other cell lines. Cellsexposured to Zuojinwan (38、76and152μg/ml) exhibited a typical sub-G1fraction,representing the apoptotic cell population,. DNA damages caused by Zuojinwan (152μg/ml) were found by SCGE assay, and the tail length and tail DNA%weresignificantly increased. Cell morphology and ultrastructure changes were found toverify the apoptosis effect caused by Zuojinwan. Compared with control group, the expressions of Bak in SMMC-7721, A549and HCT-116cells treated by Zuojinwan(152μg/ml) for24h were up-regulated with3.05,10.45and3.50times, respectively.The expressions of Bax were up-regulated with2.23,5.50and2.86times. Meanwhile,the expressions of Bcl-2were down-regulated to18.75%,47.17%and21.80%, and theexpressions of Bcl-xl were down-regulated to25.74%,30.70%and7.82%, respectively.In addition, when the SMMC-7721, A549and HCT-116cells were treated byZuojinwan at the concentration of152μg/ml for24h, the activities of Caspase-3wereraised to2.14,1.90and2.29times, and the activities of Caspase-9were raised to1.87,1.91and2.04times compared with control group, respectively. These results indicatedthat the mitochondrial pathway was activated by Zuojinwan.Conclusion: This is the first time to verify the anti-tumor activities of Zuojinwanon a variety of human cancer cell lines, and the actions was related to activemitochondrial pathway. This study provides useful information for the deepinvestigation of Zuojinwan and new drug exploitation of this traditional Chinese herbalformula.