Mechanism Of Endoplasmic Reticulum Stress In Apoptosis Of Colorectal Cancer Cells Induced By 3-Bromopyruvate

Background3-bromopyruvate is a strong alkylating agent,which inhibits tumor cell ATP production by inhibiting glycolysis-related kinase HK2,thereby inhibiting tumor cell proliferation and promoting tumor cell apoptosis.In recent years,research on3-bromopyruvic has made great progress,which provides a new direction for clinical anti-tumor research.The endoplasmic reticulum is an important organelle in the cell and the main place for storing Ca²⁺in the cell.Impairment of endoplasmic reticulum function will cause the generation of ER stress.ER stress occurs when cells face external or internal stimuli,and plays a protective role in the body.These effects can provide the body with opportunities for repair and damage.Endoplasmic reticulum stress can promote cell apoptosis,and it can also promote cell survival by affecting signaling pathways.However,whether the death of colorectal cancer cells induced by 3-bromopyruvate has endoplasmic reticulum stress is not yet clear.ObjectivesTo clarify the effect of 3-bromopyruvate on apoptosis of colorectal cancer cells,to explore the role and mechanism of endoplasmic reticulum stress in3-bromopyruvate-induced apoptosis of colorectal cancer cells,combined with endoplasmic reticulum stress inhibitors.Provide a new combination of strategies for clinical treatment of colorectal cancer.Methods1.The expression of HK2 in colorectal cancer tissues and adjacent tissues was detected by immunohistochemistry.2.CCK8 method was used to detect the effects of different concentrations of 3-BP on the survival rate of colorectal cancer cells RKO and HCT116 and the most appropriate drug concentration was screened.3.Western blotting was used to detect the effect of different concentrations of 3-BP on the expression levels of Cleaved-caspase-3,a colorectal cancer cell RKO and HCT116apoptosis-related protein.Flow cytometry was used to detect the effect of different concentrations of 3-BP on the apoptosis of colorectal cancer cells.4.Western blotting to detect the effects of different concentrations of 3-BP on the expression levels of endoplasmic reticulum stress-related proteins BIP,eIf-2?,p-eIf-2?,and ATF4.5.Detect the effect of different concentrations of 3-BP on Ca²⁺concentration at different time points.6.Survival rate of CCK8:The effect of 3-BP combined with endoplasmic reticulum stress inhibitor on the survival rate of colorectal cancer cells RKO and HCT116.7.Western blotting was used to detect the effect of 3-BP combined with endoplasmic reticulum stress inhibitor on the expression of apoptosis-related protein Cleaved-caspase-3.Flow cytometry was used to detect the effect of 3-BP combined with endoplasmic reticulum stress on apoptosis Impact.Results1.Immunohistochemical results showed that compared with the tissue adjacent to colorectal cancer,the expression of HK2 in cancer tissue was significantly increased.2.CCK8 shows that with the increase of 3-BP drug concentration inhibited the survival of colorectal cancer cells RKO and HCT116.With the increase of drug concentration,the inhibitory effect on cell activity was significant,and the difference was statistically significant.?P<0.05?3.Western blotting results showed that 3-BP at different concentrations affected the expression of apoptosis-related protein Cleaved-caspase-3,and apoptosis increased significantly with increasing drug concentration.The results of flow cytometry detection corresponded to the former.The difference was statistically significant?P<0.05?.4.Western blotting results showed that the increase of 3-bromopyruvate significantly increased the expression of endoplasmic reticulum stress-related proteins BIP,eIf-2?,p-eIf-2?and ATF4,the difference was statistically significant?P<0.05?.5.Ca²⁺concentration test results show that 3-BP has significant time and concentration dependence on Ca²⁺concentration.6.CCK8 results showed that 3-BP combined with endoplasmic reticulum stress inhibitors significantly inhibited the survival rate of colorectal cancer cells RKO and HCT116,and the difference was statistically significant.?P<0.05?7.Western blotting results showed that 3-BP combined with endoplasmic reticulum stress inhibitors affected the expression of apoptosis-related protein Cleaved-caspase-3,and the apoptosis in the combined drug group was significantly increased.The results of flow cytometry detection corresponded to the former.The difference was statistically significant.?P<0.05?Conclusions3-BP can significantly inhibit the survival rate of colorectal cancer cells and induce the occurrence of tumor cell apoptosis and induce endoplasmic reticulum stress through the influence of calcium concentration;inhibition of endoplasmic reticulum stress can increase the cytotoxicity induced by 3-BP.