Study On The Mechanism That Germercrone Inhibits Proliferation Of HepG2Cells

Rhizoma curcuma, which is the dry root or the dry stem of the Curcuma phaeocaulis,Curcuma kwangsiensis and Curcuma wenyujin ascribed to the Zingiberaceae, has thefunction of bloating the broken blood, reducing swelling and relieving pain. Germacroneis the main active ingredient of the volatile oil of the R. curcuma. Some research hasreported that the volatile oil of the R. curcuma has the function of anti-tumor，anti-inflammatory and anti-virus, and the research demonstrated that the germacrone has anobvious therapeutic effect with less mechanism reported. Taking human HepG2cell lineas the test material, this thesis explains the anti-tumor mechanism of germacrone, by usingthe molecular biology tools Western blotting, RT-PCR and flow cytometry. The mainresults include：（1）Germacrone inhibited human HepG2cell line’s proliferation in a dose-and time-dependent manner. MTT assay showed that the higher the drug concentration, the strongerthe inhibition on cell growth. In addition there are no significant growth-inhibitory effectdifferences between incubation with germacrone for24h and48h, and no time-depententinhibition was observed.The IC50was160μM. There was no statistical difference whentreated with germacrone more than160μM.（2）Germacrone induced G2/M arrest in cell cycle progress. The flow cytometryassay showed that germacrone induced an obvious G2/M arrest. Western blotting andRT-PCR assays indicated that germacrone inhibited CyclinB1and CDK1which are thekey genes of the G2/M phase. It means that Germacrone inhibited the proliferation of theHepG2cell through regulating the CyclinB1and CDK1.（3）Expression levels of p53, Bax, Bcl-2were detected by western blotting. Theresults showed that p53and Bax were up-regulated by germacrone, while Bcl-2wasdown-regulated.（4）Cells were stained with Hoechst33258and the results showed that the blue lightspot were getting more with germacrone treatment. Also the cells were stained withPI/Annexin V with the results that the percentage of living cells was getting lower, while the percentage of apoptosis cells was getting higher. All the results indicated thatgermacrone induced apoptosis of HepG2Cells.（5）Expression of p-JAK2and p-STAT3was detected by Western blotting. Theresults showed that both of them were down-regulated by germacrone. It indicated thatgermacrone inhibited the excessive activation of the JAKs/STATs signaling pathway.In conclusion, this study successfully determined that germacrone significantlyinhibited the proliferation of human HepG2cells. With the further understanding ofapoptosis on HepG2cell, this research can provide new idea and methods for supplyingbetter anti-tumor treatment.