Effect Of IPI-926 On Proliferation Of Human Colon Cancer HT-29 Cells And Its Mechanism

Objective: By observing the effect of IPI-926 on the proliferation of human colon cancer HT-29 cells and its effect on the expression of Hh signaling pathway,we aimed to investigate the inhibitory effect of IPI-926 on colorectal cancer and its mechanism of action,for provide scientific evidence and guidance for the treatment of colorectal cancer by IPI-926.Methods: Routine culture of colon cancer HT-29 cells with PRMI-1640 medium.Then,HT-29 cells were randomly divided into experimental group with different concentrations of IPI-926(10?20?30nmol/L)and control group(blank medium added as a blank control group for IPI-926),the experimental group and control group HT-29 cells were cultured for 72 hours respectively,the growth of HT-29 cells and the inhibition rate of HT-29 cells in each group were observed by inverted microscope and MTT assay.And the expression of Shh,Ptch,Smo and Gli-1 m RNA and their related proteins of Hh signaling pathway target genes were detected by RT-PCR(real-time fluorescence quantitative PCR)and Western blotting respectively after 72 hours culture.Results:1.The culture results showed that the control group HT-29 cells grew well and showed adherent growth.The cell bodies were round,oval,triangular-like,cell structure was intact,the cytoplasm was uniform,andthe nucleolus was clear.However,the density of HT-29 cells treated with different concentrations of IPI-926(10,20,30 nmol/L)decreased,and with the increase of drug concentration,the cell density and morphological changes were more obvious.The study found that most of the cell morphology showed irregular,asymmetric growth,increased apoptosis,adherent cells decreased,floating in the solution,and a large number of scattered cell debris.2.MTT assay showed that the inhibitory rates of 10,20 and 30 nmol /L IPI-926 on human colon cancer cell line HT-29 were 17.23% ± 1.32%,38.34% ± 1.82% and 75.81% ± 3.43%,respectively.With the increase of the drug concentration,the inhibition rate was in a dose-effect manner.The difference between the three groups was statistically significant(F =60.290,P = 0.000).3.The results of RT-PCR and Western blotting showed that the m RNA and protein expression of Shh,Ptch and Smo in IPI-926 at 10 nmol / L showed no significant difference compared with the blank control group(t = 0.612,P = 0.573;t = 0.548,P = 0.613;t = 1.039,p =0.357).The results of Gli-1 difference was statistically significant,but the difference was not significant(t = 3.674,p = 0.021).The m RNA and protein expressions of Shh,Ptch,Smo and Gli-1 were significantly down-regulated when IPI-926 was 20 nmol / L and 30 nmol / L compared with blank control group and 10 nmol / L group,and the difference wasstatistically significant.And the expression of Shh,Ptch,Smo and Gli-1 at the IPI-926 concentration of 30 nmol / L(t = 44.929,P = 0.000;t = 22.500,P = 0.000;t = 33.633,p = 0.000;t = 18.558,p = 0.000)was significantly higher than that at 20 nmol / L(t = 14.078,P = 0.000;t = 17.526,P = 0.000;t = 6.124,p = 0.004;t = 6.725,p = 0.003).Conclusion: When the concentration of IPI-926 exceeds 20 nmol/L,the proliferation of human colon cancer HT-29 cells can be effectively inhibited,and the higher the inhibitory rate is,the higher the IPI-926 concentration is.The mechanism may be related to the inhibition of the expression of Shh,Ptch,Smo,and Gli-1 m RNAs and related proteins in Hh signaling pathway target genes.