Investigation Of The Role And Mechanism Of Transient Receptor Potential Canonical Channel 5 In Colorectal Cancer Chemoresistance

Part â… ,The role of transient receptor potential canonical channel 5 in colorectal cancer chemoresistanceObjective:To investigate the role of transient receptor potential canonical channel 5（TrpC5）in colorectal cancer chemoresistance.Methods:We retrospectively analyzed 72 advanced colorectal cancer patients with unresectable metastasis who underwent a biopsy and/or surgery for a primary lesion.All the patients received postoperatively 5-Fu based first-line systematic chemotherapy.Tumor assessment was performed according to the Response Evaluation Criteria in Solid Tumors criteria,and the assessment was classified as acomplete response（CR）,a partial response（PR）,stable disease（SD）and progressive disease（PD）.Immunohistochemistry staining was performed to examine the TrpC5 expression in colorectal cancer tissue.Pearson’s chi-squared test was performed to analysize the correlation between TrpC5 level and clinical and pathological parameters and chemotherapy outcome.Real-time PCR and western blot were performed to examine the mRNA and protein level of TrpC5 in human colorectal cancer wild type cells（HCT-8,LoVo）and 5-Fu-resistant cells（HCT-8/5-Fu,LoVo/5-Fu）.La³⁺was applicated to potentiate TrpC5 activity in 5-Fu-resistant cells.MTT assay was performed to evaluate the cytotoxic effect of 5-Fu on human colorectal cancer cells and 5-Fu-resistant cells treated with TrpC5-specific shRNA.Results:After the first 2 cycles of chemotherapy,28 patients achieved CR or PR（these were considered responders）and the remaining 44 patients achieved SD or PD（these were considered non-responders）.Immunohistochemistry staining showed TrpC5expression in colorectal cancer tissues.A Pearson’s chi-squared test showed a higher TrpC5expression was closely related with chemotherapy failure in the advanced colorectal cancer patients,while no significant correlation with patients age,sex,tumor location and tumor grade.High TrpC5 expression was observed in 9 responders and in 31 non-responders,while low TrpC5 expression was observed in 19 responders and in 13 non-responders（p<0.05）.Real-time PCR and western-blot showed much higher expression of TrpC5 at the mRNA and protein levels in HCT-8/5-Fu and Lo Vo/5-Fu cells,whereas only a low level of TrpC5 expression was detected in their parental lines HCT-8 and LoVo（p<0.05）.Application of La³⁺（100μM）elicited a rise in intracellular Ca²⁺in 5-Fu-resistant cells but not in wild type cells,which could be inhibited by TrpC5-specific blocking antibody,indicating the involvement of TrpC5 in the process.The MTT assay showed that HCT-8/5-Fu and LoVo/5-Fu cells were much more resistant to 5-Fu-induced cell death than HCT-8 and LoVo cells.The half-maximal inhibitory concentrations of 5-Fu(5-Fu IC₅₀)in HCT-8/5-Fu and LoVo/5-Fu cells were 186.6 mg/L（95%confidence interval（CI）:39.6-878.3 mg/L）and 38.7 mg/L（95%CI:34.6-43.3 mg/L）,more than in HCT-8（IC50:3.1mg/L,95%CI:2.2-4.3 mg/L）and LoVo（IC50:0.4 mg/L,95%CI:0.4-0.5 mg/L）（p<0.05）.Administration of TrpC5-shRNA caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells,with the 5-Fu IC₅₀ decreased to 17.5 mg/L（95%CI:5.8-53.3 mg/L）and 5.5 mg/L（95%CI:3.9-7.6 mg/L）respectively（p<0.05）.Conclusion:we demonstrated an essential role of elevated TrpC5 expression in human colorectal cancer chemoresistance.Part â…¡,The mechanism of TrpC5 in colorectal cancer chemoresistanceObjective:To investigate the mechanism of TrpC5 in colorectal cancer chemoresistance.Methods:Western blot was performed to examine the protein level of TrpC5,nuclearβ-catenin and nuclear Cyclin D1 in wild type cells（HCT-8,LoVo）and 5-Fu-resistant cells（HCT-8/5-Fu,LoVo/5-Fu）.XAV939（an inhibitor of the Wnt/β-catenin signal pathway）and LiCl（an activator of the Wnt/β-catenin signal pathway）were used in this study.MTT assay was performed to evaluate the cytotoxic effect of 5-Fu on human 5-Fu-resistant cells treated with XAV939.Western blot was performed to examine the protein level of TrpC5in wild type cells treated with LiCl and 5-Fu-resistant cells treated with XAV939.Nuclearβ-catenin and nuclear Cyclin D1 protein level were determined by western blot in5-Fu-resistant cells treated with TrpC5-shRNA and wild type cells transfected with the pCI-TrpC5 plasmid.Results:Western blot showed moreβ-catenin and cyclin D1 protein expression in the nucleus of 5-Fu-resistant cells than wild type cells.Administration of XAV939 decreased the nuclearβ-catenin accumulation and the nuclear expression of cyclin D1 in5-Fu-resistant cells.On the contrary,exposure to LiCl increased the nuclearβ-catenin accumulation and the nuclear expression of cyclin D1 in wild type cells.The MTT assay showed administration of XAV939 caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells,with the 5-Fu IC₅₀ decreased to 27.2 mg/L（95%CI:24.8-29.7 mg/L）and 3.7 mg/L（95%CI:2.7-5.0 mg/L）respectively（p<0.05）.Western blot showed no significant change of the protein level of TrpC5 in wild type cells treated with LiCl and 5-Fu-resistant cells treated with XAV939.Suppressing TrpC5 expression with TrpC5-specific shRNA in 5-Fu-resistant cells resulted in decreased nuclear accumulation ofβ-catenin and nuclear expression of cyclin D1,and on the contrary,enforcing TrpC5expression in wild type cells increased the nuclear accumulation ofβ-catenin and nuclear expression of cyclin D1.Conclusion:We identified the critical role of the elevated TrpC5 expression via activating Wnt/β-catenin signal pathway in colorectal cancer chemoresistance.Part â…¢,The impact of Trp C5 on aerobic glycolysis in colorectal cancerObjective:To investigate the impact of TrpC5 on aerobic glycolysis in colorectal cancer.Methods:We retrospectively analyzed 72 advanced colorectal cancer patients with unresectable metastasis who underwent a biopsy and/or surgery for a primary lesion.All the patients received postoperatively 5-Fu based first-line systematic chemotherapy.Tumor assessment was performed according to the Response Evaluation Criteria in Solid Tumors criteria.Glucose transporter 1（GLUT1）expression and glucose consumption were used to represent the glycolytic level.Immunohistochemistry staining was performed to examine the TrpC5 and GLUT1 expression in colorectal cancer tissue.Pearson’s chi-squared test was performed to analysize the correlation between TrpC5/GLUT1 level and clinical and pathological parameters and chemotherapy outcome.Real-time PCR and western blot were performed to examine the mRNA and protein level of TrpC5 and GLUT1 in human colorectal cancer wild type cells HCT-8 and 5-Fu-resistant cells HCT-8/5-Fu and HCT-8/5-Fu treated with TrpC5-shRNA.Western blot was performed the protein level of GLUT1 and nuclear c-Myc in HCT-8/5-Fu treated with XAV939.Results:After the first 2 cycles of chemotherapy,28 patients achieved CR or PR（these were considered responders）and the remaining 44 patients achieved SD or PD（these were considered non-responders）.Immunohistochemistry staining showed that TrpC5 and GLUT1 were mostly localized near the cell surface.TrpC5 and GLUT1 were found to be highly co-expressed in 21 of the 44 non-responders and in only 4 of the 28responders.A Pearson’s chi-squared test showed a significant correlation between TrpC5expression and GLUT1 expression,and a higher TrpC5/GLUT1 expression was closely related with chemotherapy failure in the advanced CRC patients.Real-time PCR and western-blot showed much higher expression of TrpC5 and GLUT1 at the mRNA and protein levels in HCT-8/5-Fu,whereas only low levels of TrpC5 and GLUT1 expression were detected in their parental lines HCT-8（p<0.05）.Additionally,higher glucose consumption rates were found in HCT-8/5-Fu and LoVo/5-Fu than in HCT-8 and LoVo cells（p<0.05）.Real-time PCR and western-blot demonstrated that,when the HCT-8/5-Fu cells were treated with a TrpC5-shRNA,the TrpC5 and GLUT1 mRNA levels and glucose consumption were both reduced compared with the cells treated with a scrambled shRNA.A Western blot showed decreased nuclear c-Myc expression and a dramatically decreased GLUT1 repression in the HCT-8/5-Fu cells treated with XV939.Conclusion:We found the significant correlation between TrpC5 expression and GLUT1 expression and demonstrate the essential role of TrpC5 in GLUT1 induction through Wnt/β-catenin signal pathway in colorectal cancer.High TrpC5/GLUT1expression is associated with chemoresistance in colorectal cancer.