| It is the new direction for research and development of the new anticancer drugsto get fewer side effects and effective antitumor drugs from chinese herbal medicineand plant in the new century. Carvacrol has been confirmed to have manypharmacological effects and lower toxicity on normal cells. What there are alreadyseveral experimental studies have shown that carvacrol can inhibit a variety of tumorcells growth. However, it is still not reported that inhibitory effect on humanhepatocellular carcinoma and its mechanism.Objective:To study the effects of carvacrol against proliferation and apoptosis on HepG2cells and investigate its possible molecular mechanism.Methods:The hepatocellular carcinoma HepG2cells and human normal liver LO-2cellswere cultured in vivo containing0,0.05,0.1,0.2,0.4mM of carvacrol. Cell viabilitywas analyzed by MTT assay.The morphological alteration was observed byHoechst33258staining. The apoptosis rates were analyzed by flow cytometry. Bcl-2,caspase-3, PARP and MAPK proteins levels were detected by Western blottinganalysis. To further analyse possible mechanism of carvacrol against apoptosis andproliferation about MAPKs, we examined changes both the cell viability andmorphology due to apoptosis in the presence or absence of specific inhibitors for ERK(U0126) and p38MAPK (SB203580). Results:The treatment of LO2cells with carvacrol (0-0.4mmol·L-1) for24h was withoutsignificant effect on cell viability. However, on treatment of similar doses of carvacrolon HepG2cells, the viability of HepG-2cells was decreased after treatment with0.05,0.1,0.2, or0.4mmol·L-1carvacrol. The IC50for carvacrol was approximatelyestimated to be0.32mmol·L-1for HepG-2cells. Apoptosis was measured by Hoechst33258staining, The results showed that the uniform HepG-2cells with normalmorphology were observed in the control group, whereas HepG-2cells withfragmented chromatin and apoptotic bodies were observed followed by the treatmentwith0.05,0.1,0.2, and0.4mmol·L-1carvacrol. The results of FCM indicated that theapoptosis rate was increased from13.5to25.8%,30.5and50.6%respectively by thetreatment of carvacrol for24h. Western blot analysis showed that carvacrol downregulated the expression of Bcl-2protein levels which were reduced with increasingconcentrations of carvacrol and a significant activation of caspase-3occurred at0.2mmol·L-1carvacrol after24h of incubation, and the Caspase-mediatedPARP-cleavage by carvacrol-induced apoptosis also was a concentration-dependentprocess. Moreover, carvacrol selectively altered the phosphorylation state of membersof the MAPK superfamily, increasing phosphorylation of ERK1/2significantly in adose-dependent manner, and activated phosphorylation of p38but not affecting JNKMAPK phosphorylation. According to the MTT analysis, SB203580prominentlyreversed carvacrol (0.4mmol·L-1)-induced cell death, which was concomitant with adecrease in the appearance of apoptotic bodies induced by carvacrol. Pretreatmentwith U0126significantly promoted carvacrol-induced anti-proliferative activity onHepG2cells, but it was not shown the appearance of the apoptotic body increaseinduced by carvacrol was observed. Conclusion:1. It demonstrated that carvacrol could cause obvious growth inhibition ofHepG2cells via a dose-dependent manner at the tested range of concentrations;2. Carvacrol suppression hepatocellular carcinoma HepG-2cells growth byinhibiting proliferation and inducing apoptosis;3. Mitogen-activated protein kinase pathway was confirmed to be involved in thecarvacrol-induced apoptosis and anti-proliferative on HepG-2cells: ERK pathwaymay be involved in the anti-proliferative mechanism, while the p38pathway mightparticipate in the induction of apoptosis caused by carvacrol on HepG2cells. |
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