Study On Inhibiting Human Cervix Cancer Hela Cells Proliferation Of Cedrelone In Vitro

Objective:To investgate the effect of cedrelone on proliferation of human cervix cancer Hela cells in vitro and to explore the molecular mechanism from the two aspects of dose-effect relationship and time-effect relationship.Methods:(1) The test of antitumor activity of cedrelone in vitro:The CCK-8 assay was employed to dertermine the IC50 values of cedrelone on three human esophageal cancer cell lines EC-9706, Eca109 and KYSE180, human ovarian cancer cell SKOV-3,lung adenocarcinoma cell A549 and human cervical cancer cell Hela.(2) Study on inhibiting Hela cells proliferation and mechanisms of different doses of cedrelone:Cells were treated with 0?g/ml,0.21?g/ml,0.42?g/ml,1.06?g/ml, 1.48?g/ml and 1.90?g/ml cedrelone.After 24 h, cells apoptosis were detected by Hoechst 33258 fluorescent staining and Annexin V-FITC/PI double staining assay via flow cytometric analysis,cell cycle was detected by PI single staining through flow cytometric analysis. The expression of p21and p27 was detected by Real-time PCR.The expression of CDK2 and cyclinA, caspase-3 and Bax was detected by Western Blot.(3)Study the time-effect relationship on inhibiting Hela cells proliferation and mechanisms of cedrelone:Hela cells were exposed to cedrelone at the concentration of 1.48?g/ml for 0h,24h,48h,72h,respectively.Then,cells apoptosis were detected by Hoechst 33258 fluorescent staining and Annexin V-FITC/PI double staining assay via flow cytometric analysis,cell cycle was detected by PI single staining through flow cytometric analysis. The expression of p21 and p27 was detected by Real-time PCR.The expression of CDK2 and cyclinA was detected by Western Blot.Results:(1)After the treatment with different concentration of cedrelone for 48 hours,the growth of the six human tumor cell lines was inhibited and the ICso of cedrelone on EC-9706, Eca109, KYSE180, SKOV-3, A549 and Hela was 1.31?g/ml, 2.21?g/ml,1.08?g/ml,1.61?g/ml,4.88?g/ml,1.41?g/ml, respectively. (2)Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found after human cervical cancer cell Hela cells were treated with cedrelone for 24 h. After treated with 0.21,0.42,1.06,1.48,1.90, 4.22?g/ml cedrelone for 24 h, the inhibition rate of Hela cells was 3%,4.1%,6.2%, 7%,8.4%,58.5%, respectively and the control group was 3.4%. The cell cycle was arrested in S phase via the treatment with cedrelone for 24 h, which leads to inhibit DNA synthesis. Compared with control group,1.48?g/ml cedrelone could significantly increase the expression of p27 (P<0.01) and p21 (P>0.05).1.90?g/ml cedrelone up-regulated the expression of p21(P<0.05) and p27(P>0.05).The concentration of 1.06?g/ml could increase p27 remarkablely(P<0.05),but down-regulated p21(P<0.05).Cedrelone 1.48?g/ml,1.90?g/ml could down-regulate the expression of CDK2 and cyclinA(P>0.05),1.06?g/ml cedrelone reduced CDK2 level(P>0.05),however,it couldn't decrese the expression of cyclinA,however,it couldn't decrese the expression of cyclinA.1.06,1.48,1.90?g/ml cedrelone could not decrease the expression of caspase-3 and Bax. (3)Compared with control group,after incubated with cedrelone 1.48?g/ml for 24 h,48 h,72 h,cells exhibited condensation of chromatin and nuclear fragmentations in a time-dependent manner. The inhibition rate of Hela cells was 3.4%,7%,45.2%,78.3% after treated with cedrelone for 0 h,24 h, 48 h,72 h and the ratio of cells in S phase was21.53%,33.3%,33.92%,62.74%, respectively.After incubated with cedrelone for 72 h,the expression of p21 and p27 was significantly up-regulated (P<0.05,P<0.01).Treatment with cedrelone for 48 h could increase p27 level (P<0.05),which leads to influence p21 moderately (P> 0.05).The expression of p21 and p27 was increased via exposed to cedrelone for 24 h (P>0.05).In addition,the exreession of CDK2 was down-regulated in a time-dependent manner(P>0.05),but cylinA had no change after adminstration with cedrelone for 24 h,48 h,72 h.Conclusion:It was indicated that cedrelone could inhibit the proliferaiton of the six kinds of tumor cells invovled human cervical cancer cell Hela effectively and cedrelone-induced apoptosis on Hela cells was ralated to suppress DNA synthesis in S phase via down-regulating the expression of CDK2 and up-regulating the expression of p21 and p27.