| Ceramide is important second messenger in the cells, take part in modulating multiple biological effects. The most well-known roles of ceramide are proliferation inhibition and apoptosis (including tumour cells). In view of its role in inducing apoptsis, enhancing the concentration of endogenous ceramide or introducing exogenous analogues are the new targets of oncotherapy. Studies have shown that an exogenous ceramide analogue (C2-ceramide) can induce apoptosis in colon cancer cell lines HT-29, Lovo, and HCT-116through multiple pathways; however, there is no reports about the affect of ceramide on apoptosis of Caco-2cells.Chemotherapy has been widely used in colon cancer treatment, but the oncotherapy are often failed due to chemoresistance. GSTs are a group of phase Ⅱ drug metabolism enzymes. Overexpression of GSTs is one of the important factor for producing chemoresistance, tumour cells can protect themselves against anti-tumour drugs and reduce curative effect of anti-tumour drugs by express GST. Evidence shows that GSTA expression is strongly related to the resistance of anti-cancer drugs, such as nitrogen mustard and anthraquinone. GSTA1is an important member of the GSTA family. Study in this enzyme will advance our understanding in the proliferation and apoptosis of the cancer cells.This study detected the effects of exogenous C2-ceramide on proliferation and apoptosis using Caco-2and HT-29cell lines, this research will provide fundamental knowledge for our future study on GSTA1-associated proliferation inhibition and apoptosis of the cell. This study investigated the expression and activity of GSTA1at different stages of colon cancer cells and determined the change of GSTA1expression and activity during exogenous ceramide-modulated proliferation inhibition and apoptosis of colon cancer cells. Gene transfection techniques were used to silence or overexpress GSTA1, and to explore how ceramides affects cell proliferation and apoptosis. This study will reveal if GSTA1expression in colon cancer cells can protect themselves against the effects of ceramide-modulated cell proliferation inhibition and apoptosis and useful to clarify the relationship between GSTA1, proliferation inhibiton/apoptosis of colon cancer cells and chemoresistance. Results can be translated to guide clinical application, and enhance effects of anti-cancer drugs and reduce the drug resistance in new drug development.MTS assay was employed to determine the effects of C2-ceramide on the cell proliferation in in vitro cell lines Caco-2and HT-29. Western blotting and real-time PCR were used to determine the effects of ceramides on apoptosis by measuring the expression levels of actived caspase-3and Bcl-2gene family. Furthermore, Western blotting, real-time PCR, and enzyme activity assay technologies were employed to determine the expression of GSTA1protein, mRNA and activity at different stages of colon cancer cells, effects of ceramide on GSTA1protein expression, mRNA expression, and activity were also detected. In addition, gene transfection techniques were used to confirm the functions of GSTA1in ceramide-modulated cell proliferation and apoptosis. The first approach is to use siRNA technique to knockdown GSTA1gene in Caco-2cells in order to detect the effect of GSTA1silencing on ceramide-mediated proliferation inhibition and apoptosis; and the second was to transfect HT-29cells with plasmid in order to detect the effect of GSTA1overexpression on ceramide-mediated proliferation inhibition and apoptosis.The results of effect of exogenous C2-ceramide on proliferation showed that the survival rate of colon cancer cell progressively decreased with the concentration of ceramide. The results of effect of ceramide on apoptosis showed that ceramide did not induce the expression of actived caspase-3protein. The expression of anti-apoptotic Bcl-2mRNA and pro-apoptotic Bax mRNA did not show any significant difference. Treatment with ceramide resulted in7.142-fold increase of the actived caspase-3protein expression in HT-29cells comparing with control group (P<0.01), accompanied with0.466-fold decrease of Bcl-2mRNA (P<0.001)and6.938-fold increase Bax mRNA levels (P<0.001).The changes of GSTA1protein levels, mRNA expression and activity in different stages of Caco-2and HT-29were detected. Results showd that GSTA1protein levels, mRNA expression, and enzyme activity in Caco-2cells were progressively increased with the confluency. Compared with preconfluent, GSTA1protein, mRNA levels and activity rose to1.888,3.018,1.994-fold in confluent respectively (P<0.05or P<0.01), and rose to2.988,7.135,6.598-fold in6d postconfluent respectively (P<0.001). There were no expression of GSTA1protein and mRNA of all the stages in HT-29cells.The effect on GSTA1protein levels, mRNA expression and activitiy in Caco-2and HT-29cells induced by exogenous C2-ceramide were detected. Results showed that GSTA1protein, mRNA expression and activity induced by ceramide of Caco-2cells increased1.850,3.093,1.764-fold respectively, significant difference comparing with control group (p<0.01or p<0.001), but no expression of GSTA1protein and mRNA expression were seen in HT-29cells.The best transfection conditions,3μl/mL Lipofectamine2000,40nM siRNA, transfected48h,were confirmed by western blotting analysis. These GSTA1-silenced Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in Caco-2cells. Results showed that there was no significant difference in survival rate between control, siRNA transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, siRNA transfection, negative control group, activated caspase-3protein expression was not detected in3groups with ceramide treatment.The construction of recombinant plasmid GSTA1-pcDNA3.1/V5-His TOPO was verified by double-enzyme cleavage and gene sequence analysis. Recombinant plasmids were then transiently transfected into HT-29cells. The best transfection conditions,3μl/mL Lipofectamine2000,3μg DNA, transfected48h,were confirmed by western blotting analysis. These GSTAl-overexpressed Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in HT-29cells. Results showed that there was no significant difference in survival rate between control, plasmid transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, plasmid transfection, negative control group, activated caspase-3protein expression were induced with ceramide treatment, but no statistics difference between groups(P>0.05).In conclusion, ceramides inhibits the proliferations of two colon cancer cells. Ceramides can induce apoptosis of HT-29but not Caco-2cells. The protein levels, mRNA expression, and enzymes activities of Caco-2cells are increased in correlation with cell confluency, the GSTA1expression is very low or not existed in HT-29cells. Treatment with ceramides can increase GSTA1protein levels, mRNA expression, and activity in Caco-2cells, but the protein and mRNA expression is not seen in HT-29cells. Gene transfection experiments manifest that GSTA1does not affect the ceramide-modulated proliferation inhibition and anti-apoptosis, these results suggested no protective role for GSTA1in ceramide-modulated proliferation inhibition and apoptosis. |
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