Abstract:

Objective:To investigate the effect of cyclopamine on proliferation,apoptosis of colorectal cancer  Caco-2 cells and expounds its mechanism in the mitochondria using proteomic methods.Methods The  colorectal cancer  Caco-2 cells in  logarithmic growth phase  were divided into cyclopamine treatment groups and negative control groups.   The  Caco-2 cells were treated    with  5,10,20 and 40 μmol·L^-1 cyclopamine,MTS  assay and flow cytometric analysis were applied to  detect the inhibitory rate of growth  and the apoptotic  rate of Caco-2 cells. Nano-LC-ESI-MS method was used to detect the protein expression differences in mitochondria between various  groups.Results The growth inhibition rate of Caco-2 cells in treatment groups by 10,20,40 μmol·L^-1 cyclopamine after 24,48 and 72 h were 23.1%±1.8%,46.2%±0.9%,53.4%±2.3%,45.1%±2.8%,73.0%±2.5%,81.2%±1.6%,59.7%±2.3%,87.5%±1.4% and 91.0%±1.06%,they were obviously higher than those in negative control groups(1.8%±0.2%,2.5%±0.1%,and 3.7%±0.3%)（P<0.01）.The apoptotic rates of Caco-2 cell in treatment groups by 5,10,20, 40 μmol·L^-1 cyclopamine after 48 h were 24.1%±1.3%，31.7%±1.6%，50.5%±2.3%, and 64.0%±1.9%，they wer higher than that in negative control groups (14.5%±0.7%)(P<0.01). Proteomics difference analysis showed that Caco-2 cell mitochondrial proteins obviously changed after treated with cyclopamine,the expressions of  32  proteins were down-regulated  and the expressions of 25  proteins were up-regulated. Function analysis showed that these proteins involved in  apoptosis,cytoskeleton,nucleic acid,ribosomes and protein metabolism,etc,among them Clusterin,OPA1,ATF6,RGD1565411 and Cofilin had close relation with apoptosis.Conclusion Cyclopamine can inhibit the proliferation  of  colorectal cancer Caco-2 cells  by blocking HH pathways,its mechanism involve in  promoting apoptosis.Cyclopamine could change the  protein expression of Caco-2 cell mitochondria  obviously,the differences are  related to  apoptosis closely.

Key words: cyclopamine;colorectal neoplasms;Hedgehog signaling pathway;proteomics