The Study Of Growth Inhibitory Effect And Its Mechanisms Of CD40L On Human Colon Cancer Cell Line HCT-116

Objective:To study the anti-tumor effect and its mechanisms of CD40L on human colon carcinoma cell line HCT-116 in vitro.Methods:In vitro,exponential phase human colon carcinoma cell line HCT-116 was treated by CD40L in different concentrations(0.1ug/ml,0.5ug/ml,1.0ug/ml,2.0ug/ml,3.0μg/ml).Cell morphology changes was observed by optical microscopy.By means of MTT assay,the absorbance value was detected and inhibition rate was calculate; cell cycles and apoptosis were analyzed by flow cytometry (FCM);the mRNA expressions of VEGF,bcl-2,fas were analyzed by reverse transcriplase polymerase chain reaction (RT-PCR);Immunohistochemistry was used to detect VEGF,Bcl-2,Fas protein expression.Results:(1)MTT assay indicated that the inhibitory effect of CD40L on the proliferation of HCT-116 cell was found in dose-and-time dependent manner. There was great statistical signification between varies medicated group and control group(P<0.05),and there also were great statistical signification between medicated group each other(P<0.05).(2)Comparing with control group,the cell apoptosis was induced by CD40L at 48 hours, meanwhile,the ratio of G2/M phase was increased and the ratio of G1/G0 phase and S phase was decreased, in dose-dependent manner(P<0.05). (3)RT-PCR assay showed ttat the expression of fas mRNA were upregulated and VEGF,bcl-2 mRNA was downregulated by CD40L at 48 hours with concentration incread(P<0.05).(4)Western Blot showed that the expression of VEGF,Bcl-2 was downrd after 48h. And the expression of Fas protein were upregulated with CD40L concentration incread(P< 0.05).Conclusion:The growth of HCT-116 cell in vitro is inhibited by CD40L and in dose-and-time dependent manner.CD40L also can arrest cell cycle at G2/M phase, induce cell apoptosis.Maybe its mechanism is through upregulating the expressions of Fas mRNA and protein,downreglating the espression of VEGF,Bcl-2 mRNA and protein.