Study On Cell Cycle Arrest Induced By Evodiamine And Mechanism In Human HepG₂Cell Line

Evodiamine is one of main active compotent of Chinese traditional medicine Evodia that is tryptamine indole alkaloid.Experimental studies indicated that Evodiamine with significant pharmacological actions, antitumor activity is one of important pharmacological actions. Evodiamine inhibit many kinds of cancer cell lines by some approaches, such as, promote cell apoptosis, specific block cell cycle, inhibit angiogenesis of tumor and so on. But the effect of cell cycle arrest mechanism on human liver carcinoma HepG2is still unknown. So this experimental study in order to elucidate the mechanism, which has a important significance about developing Evodiamine with low toxicity and high efficiency features.Experiments used some methods in vitro to study the effect of mechanism of cell cycle to arrest on human liver cancer cell line HepG2. The colorimetric MTT assay was used to detect the cytotoxic effect. Using colony information experiment to detect the proliferation of Evodiamine to human liver cancer cell line HepG2. Flow cytometry(FCM) analysis on the effect of Evodiamine on cell cycle in HepG2. Methods examining the influence of Evodiamine on tubulin in HepG2,using laser confocal microscope (LSCM). Detecting the protein level of cyclinBl and CDK1in human liver cancer cell line HepG2by method Western Blot.From Experimental results show that (1)Evodiamine shows significantly cytotoxic effect on human liver cancer cell in dose-dependent manner, and calculate from the IC50is19.62μmol/L;Evodiamine also can inhibit the proliferation of cancer cell, the inhibitory effects was associated with the concentration of Evodiamine. The higher dose followed the amount of cell colonies more formating.(2)Through flow cytometry analyzing, treated human liver cell line HepG2with2.5μmol/L,5μmol/L,10μmol/L Evodiamine by24h. The results compare to negative control group, HepG2cellular proportion in G2/M phase increased obviously, in a dose-dependent manner.(3)Using indirect immunofluorescence (IIF) and observing under laser confocal microscope (LCM) to test the tubulin polymerization of HepG2that treated with Evodiamine after24h. The result compared to negative control group, the distribution of tubulin has changed, changing from clear reticular distribution to confusion and diffusion. Therefore infer that Evodiamine induce cell cycle arrest at G2/M phase, which because of intervention of cell mitosis.(4) After different dose of Evodiamine treated for24h, using Western blot to test the expression level of cyclinB1and cyclin dependent kinase in HepG2. The experimental results show that Evodiamine obvious increase the expression of cyclinB1and CDK1in HepG2in dose-dependent manner.Above experiments in vitro prove that Evodiamine inhibite the growth of human liver cell line HepG2and cell mitosis.One approach of Evodiamine exert the anti-cancer effect is cell cycle arrest. Evodiamine block cell cycle at G2/M phase in HepG2, induce cell proliferation inhibition. The possible mechanism is up-regulation of expression of cyclinB1and CDK1in HepG2.These effects further affect the polymerization of tubulin in cell mitosis, that also mean interfere dynamic equilibrium of tubulin. Unlimited proliferation of tumor is stoped, therefore Evodiamine exert an antineoplastic pharmacological action.