Effect Of Thalidomide On Proliferation And Expression Of Pparγ,nf-κb In Human Colorectal Cancer Cell Line Hct-8

OBJECTIVE:1:To study the effect of the Thalidomide and Thalidomide combined with oxaliplatin on the proliferation and apoptosis of human colorectal cancer cell line HCT-8,2:To study the effect of Thalidomide on the express of PPARγand NF-κB in colorectal cancer cell and explore the new mechanism of Thalidomide in the treatment of colorectal cancer.METHODS:HCT-8 cells were cultured in a humidified 5%CO2/95%air atmosphere at 37℃.1:the effect of Thalidomide group(0,25,50,100,200,400μmol/L),oxaliplatin group(0,2.5,5.0,10.0,20.0μg/ml),Thalidomide combined with oxaliplatin(oxaliplatin2.5,5.0,10,20μg/mL+Thalidomide100μmol/L) on the proliferation of HCT-8 cells was evaluated by MTT when the cells were treated for 24,48,72 hours and the q value was calculated to judge the combined effect.the formula is "q=E(a+b)/(Ea+Eb-Ea×Eb)",E(a+b),Ea and Eb is defined with the inhibition rate of Thalidomide,oxaliplatin and Thalidomide combined with oxaliplatin respectively,In the analysis,there is synergy when the q more than 1.15;additivity when the q is between 1.15 and 0.85,;and antagonism when the q is less than 0.85.2:cell cycle of HCT-8 cells treated by Thalidomide(0,100,400μmol/L) was detected by flow cytometry,the experiment is done three times,and Iteraction between cell cycle of HCT-8 with different group was analysised.3:the protein expression of NF-κB,PPARγin human colorectal cancer cell line HCT-8 treated by Thalidomide with different group(0,100,400μmol/L) was studied by immunohistochemistry staining. RESULTS:1:The results of MTT test showed that Within the concentration of(25～400)μmol/L,Thalidomide can obviously inhibit proliferation of HCT-8 in vitro.The inhibitive ratio was stepping up as the concentration increasing,so Thalidomide could inhibit the proliferation of HCT-8 cells in time-and-dose-dpendent manner,and the highest inhibitive ratio was the group with the concentration of 400μmol/L and 72h. There was significantly statistical significance of inhibitive ratio in different concentration groups of HCT-8 after being treated Thalidomide,and the same result compared with untreated group(P＜0.01).F_time=4111.113,F concentration =3103.643,F_J=65.808,p＜0.01,the oxaliplatin group was significantly statistical significance of inhibitive ratio in different concentration,oxaliplatin could inhibit the proliferation of HCT-8 cells in time-and-dose-dpendent manner,and the highest inhibitive ratio was the group with the concentration of 20μg/mL and 48h,The inhibitive effeet of Thalidomide combined with oxaliplatin on HCT-8 cell lines was significantly higher than that of Thalidomide or oxaliplatin alone in vitro.2:Flow cytometry(FCM) showed that cell cycles of HCT-8 cells treated with Thalidomide was arrested in G0/G1 phase,the number of cells in G0/G1 phase increased gradually,while the number of cells in S phase and G2/M phase decreased grudually.That was,Thalidomide could induce an arrest of cell cycle in G1 phase by a dose-dependent manner.with the concentration of Thalidomide increasing,There were signigiciant differences when Thalidomide groups compared with the control group (p＜0.01),and there was a negative correlation between the G1 phase and S phase(r =-0.998,p=0.045).3:Immunocytochemistry staining results:There were signigiciant differences when Thalidomide groups compare with control group,with the concentration of Thalidomide increasing,the expression of PPARγ,increased significantly,which of NF-κB significantly reduced,and there was a negative correlation(r=-1.0 p=0.012).CONCLUSIONS:1:Thalidomide could inhibit HCT-8 cell lines and associate with apoptosis,it enhanced the oxaliplatin's inhibitve effect on the proliferation of HCT-8 cell lines, and there was synergistic effection;2:cell cycle of HCT-8 cells treated with Thalidomide was arrested in G0/G1 phase;3:The inhibitive effect of Thalidomide on the proliferation of colorectal cancer cell line,Is likely to be associated with the elevated PPARγprotein expression and decreased NF-κB protein expression,and there was a negative correlation;4:Thalidomide may be a PPARγagonist.