Effect Of Curcumin On P21^WAF1/CIP1 And P27^KipI Expression In Human Hepatoma Cell Line HepG2

Objectives: Hepatocellular carcinoma ranks fifth in global incidence ofmalignancies with an estimated number of700,000people who die of itannually. With the highest incidence all over the world, China accounts for55%of the new cases of liver cancer worldwide. As the second-leading causeof cancer-related deaths in China after lung cancer, the mortality of livercancer accounts for45%at the global level. Despite the newly achievedbreakthroughs in treatments such as surgical removal, liver transplant,comprehensive and sequential therapy, detection of PLC is still difficult forliver cancer and palliative treatment is the only option because it occursstealthily and proceeds rapidly. Additionally, the sensitivity of PLC is low toradiotherapy and chemotherapy. Therefore, it’s urgent to seek new therapiesand medications to increase the patient’s survival time and improve the livingquality of patients with PLC.Recent studies indicated that curcumin has the functions of anti-oxidation,anti-inflammation, anticoagulation, anti-atherosclerosis and scavenging action,inhibiting the growth of tumors. On animal models it also displayed goodeffects of inhibition and inducing apoptosis of a variety of tumor cells such asthose of carcinoma of skin, hepatocellular carcinoma, colon cancer, lungcancer, ovarian cancer, breast cancer and gastric cancer. However, themechanism of its inducing apoptosis remains unclear and there are fewresearches on it so far.p21^WAF1/CIP1, p27^KipIare members of the CIP/KIP family of cell-cycleinhibitors，which form cell-cycle regulatory network together with cyclin andCDK. They have similar inhibitory domain, can inhibit the G1cyclin-CDKcomplex and cyclinB-CDK1, the configuration and the change of the activityof the catalytic subunit of CDKs and the inhibition.This gene plays an important role in cell-cycle regulation. According to the latest studies,p21WAF1/CIP1and p27^KipIdisplayed the function of inhibiting tumor genes duringthe occurrence and development of a wide range of tumors like liver cancer,gastric cancer, breast cancer, cholangiocarcinoma, lung cancer and so on. Anumber of studies and analyses indicated that high expression of p21^WAF1/CIP1and p27^KipIare closely linked to the prognosis of tumors.By examining the effect of curcumin on both the cell cycle of humanhepatic carcinoma cell line （HepG2） and the expression of p21^WAF1/CIP1andp27^KipI, this experiment explores the possible mechanism of curcumin’sinhibitory effect on cell proliferation of HepG2.Methods: Routine culture for human hepatic carcinoma cell line （HepG2）was performed. Distribution of cell cycle was examined through FCM: HepG2administered （48h） with various concentrations of curcumin （0、10、20、30、40、50μmol/L） and HepG2with concentration of30mol/L for different periods（0h,24h,48h,72h） were measured with flow cytometry and analyzed bysoftware; Western Blot analysis was performed: after HepG2testing wasconducted in the first procedure, the effect of curcumin on the expression ofp21WAF1/CIP1and p27^KipIin HepG2cells was observed.Results:1Analysis by FCM showed that with the increasing of the concentrationand the extension of administration time, the numbers of HepG2cellsincreased obviously in G0/G1phase while the proportion of HepG2cells in Sphase and G2/M phase decreased. Thus the cells were arrested in G0/G1phase.Moreover, they displayed significant time and dose dependence （P<0.05）.2After HepG2cells administered with curcumin of differentconcentrations （10,20,30,40,50μmol/L） for48h were examined byWestern-Blot analysis, the p21^WAF1/CIP1expression of groups with differentconcentrations was significantly higher than the blank control group （P<0.05）and significantly increased with the increase of concentration, while there wasno significant difference between the group with40and50μmol/L （P>0.05）;the difference among other groups was significant （P<0.05）; 3After HepG2cells administered with curcumin of differentconcentrations （10,20,30,40,50μmol/L） for48h were examined byWestern-Blot analysis, the p27^KipIexpression of groups with differentconcentrations was significantly higher than the blank control group （P<0.05）,and there was no significant difference between group with10μmol/L and20μmol/L as well as group with40μmol/L and50μmol/L （P>0.05） whilesignificant difference was displayed among other groups （P<0.05）;4After HepG2cells administered with curcumin of30μmol/L fordifferent periods （24h,48h,72h） were examined by Western-Blot analysis,both p21^WAF1/CIP1and p27^KipIexpressions increased significantly comparedwith control group （P<0.05）, and this effect was more distinct with theextending of time （P<0.05）.Conclusions:1Curcumin can induce arrest of the human hepatic carcinoma cell line（HepG2） in G0/G1phase.2Curcumin can raise the expression of p21^WAF1/CIP1and p27^KipIin hepaticcarcinoma cell line （HepG2）, thus it may be inferred that proliferation oftumors could be inhibited and the cells could be arrested in G0/G1phase bymeans of this. What’s more important, the apoptosis of tumor cells could beachieved as a therapy for prevention and treatment in hepatocellularcarcinoma.