TRPV1 Induces Apoptosis Of Colorectal Cancer Cells By Activating Calcineurin-NFAT2-p53 Signaling Pathway

Objective:To explore the differential expression of TRPV1?transient receptor potential vanilloid 1?in colorectal cancer tissues and adjacent tissues and normal tissues.Does the expression of TRPV1 affects the proliferation and apoptosis of colorectal cancer cells,and whether TRPV1 promotes apoptosis of colorectal cancer cells by activating p53.Whether it regulates apoptosis-related proteins by affecting the expression of intracellular Ca²⁺and NFAT?activated T cell nuclear factor?.So to investigate the role of TRPV1 in the proliferation and apoptosis of colorectal cancer cells induced by Ca²⁺-regulated signal-dependent pathway calcineurin-NFAT2-p53.Methods:275 cases of colon cancer cells and 349 cases of normal tissue samples were found in the TCGA?tumor genome map?database.To investigate whether TRPV1 protein was differentially expressed in colorectal cancer tissues and normal tissues,10 cases of CRC tissues,10 cases of CRC adjacent tissues0.5 cm away from the edge of the tumor,and 6 cases of normal tissues more than 5 cm from the edge of the tumor were collected from May 2018 to August2018 in Sichuan Province.Immunohistochemical technique for detection of TRPV1 protein expression in each specimen.To further explore the biological behavior of TRPV1 in colorectal cancer,explore whether TRPV1 inhibits the proliferation of colorectal cancer cells through p53 and promotes apoptosis of colorectal cancer cells.Functional experiments were performed on human colorectal cancer HCT116 cell line.Colorectal cancer HCT116 cells were cultured with different reagents and divided into blank control group,capsaicin?TRPV1 activator?group,capsaicin+Pifithrin-??p53 inhibitor?group.Cell viability was determined by CCK-8assay,apoptosis was detected by flow cytometry,and expression of apoptosis-related protein was detected by Western blotting.To investigate the effect of TRPV1 activation on intracellular Ca²⁺and NFAT2?activated T cell nuclear factor 2?protein concentrations.The cells were divided into blank control group and capsaicin group.The intracellular Ca²⁺concentration was detected by Fluo-4 AM staining.The expression levels of p-NFAT2?phosphorylated NFAT2?and NFAT2 protein were detected by Western blot.The expression level of NFAT2 protein was detected by immunofluorescence.Finally,we investigated that TRPV1 increases intracellular NFAT2 protein expression via calcineurin.In turn,it affected apoptosis-related proteins,thereby affecting the proliferation and apoptosis of colorectal cancer HCT116 cells.Divided into blank control group,capsaicin+FK506?calcineurin inhibitor?group,capsaicin group cultured colorectal cancer HCT116 cells.The expression level of NFAT2 protein was detected by immunofluorescence assay,apoptosis was detected by flow cytometry,and the expression level of apoptosis-related protein was examined by Western blotting.Results:275 colon cancer cell samples and 349 normal tissue samples in the TCGA database,the expression of TRPV1 protein in colon cancer tissues was significantly lower than that in normal tissues.Clinical specimens of 10 cases of colorectal cancer tumors,10 cases of adjacent tissues,and 6 cases of normal colorectal tissues collected from our hospital.the expression of TRPV1 protein in colorectal cancer tissues was significantly lower than that in CRC adjacent tissues and normal tissues,The expression of TRPV1 protein in CRC adjacent tissues was between CRC and normal tissues,and the difference in expression was statistically significant.TRPV1 inhibited proliferation of colorectal cancer HCT116 cells by activating p53 and promotes apoptosis.colorectal cancer HCT116 was placed in the blank control group,capsaicin group,capsaicin+Pifithrin-?group.Cell viability was found by CCK-8 assay:blank control group>capsaicin group+Pifithrin-?group>capsaicin group;Apoptosis was detected by flow cytometry:capsaicin group>capsaicin group+Pifithrin-?group>blank control group;Western blotting revealed that the proapoptotic factors p53,BAX,cleaved-caspase:capsaicin group>capsaicin group+Pifithrin-?group>blank control group,while the anti-apoptotic factor bcl-2 expression was reversed.TRPV1 increased Ca²⁺influx and NFAT protein expression levels in colorectal cancer HCT116 cells.Colon cancer HCT116 cells were cultured in the blank control group and the capsaicin group,respectively.Fluo-4 AM staining showed that the intracellular Ca²⁺concentration in the capsaicin group was greater than that in the blank control group.The expression of NFAT2protein in Western blot was higher than that in the blank control group.The p-NFAT2 protein expression control group was higher than the capsaicin group.Immunofluorescence assay showed that the expression level of NFAT2 in the capsaicin group was significantly higher than that in the blank control group.TRPV1 promoted apoptosis of colorectal cancer HCT116 cells by activating calcineurin.The blank control group,capsaicin group,capsaicin+FK506?calcineurin inhibitor?group were cultured after colorectal cancer HCT116 cells.The expression level of NFAT2 protein was determined by immunofluorescence:capsaicin group>capsaicin+FK506 group>blank control group.Flow cytometry to detect apoptosis:capsaicin group>capsaicin+FK506 group>blank control group.The expression levels of apoptosis-related proteins p53,BAX and cleaved-caspase3 were examined by Western blotting:capsaicin group>capsaicin+FK506 group>blank control group,while anti-apoptotic protein bcl-2 expression was reversed.Conclusion:TRPV1 is expressed in colorectal cancer tissues lower than CRC adjacent tissues and normal tissues.The expression of TRPV1 in CRC adjacent tissues is between cancer tissues and normal tissues,and the differential expression is statistically significant.TRPV1 can inhibit the proliferation of colorectal cancer cells and promote the apoptosis of CRC cells.It also induces apoptosis of colorectal cancer cells through Ca²⁺regulation of calcineurin-NFAT2-p53 signaling pathway;TRPV1 is an effective tumor suppressor and may provide a new target for the treatment of colorectal cancer.