The Molecular Mechanism Of 3-BrPA Targeted Inhibiting The Growth Of Breast Cancer Cells With Overexpression Of ErbB2

The energy metabolism of tumor cells was significantly higher than that of normal cells in order to maintain their high growth rate,proliferation and invasion.The ErbB2 gene is overexpressed in many malignant tumors,and the high expression of ErbB2 gene in breast cancer is as high as20%-30%.High expression of ErbB2 leads to an increase in glucose metabolism and uptake,an increase in lactate and a decrease in oxygen consumption.Breast cancer cells with high expression of ErbB2 are more likely to be resistant to ErbB2 targeted drug herceptin.It may be a new target for breast cancer therapy to inhibit the apoptosis of breast cancer cells by inhibiting the expression of ErbB2 in breast cancer cells.Purpose:Three breast cancer cell lines with high expression and low expression of ErbB2 were established.In order to study the ability of ErbB2 gene regulating activity of breast cancer cells after 3-BrPA treatment,expression of activity and mitochondrial distribution of HK? protein,Western blot method,mitochondrial separation,JC-1 mitochondrial membrane potential detection,xenograft in vivo experiments and other methods were used.To investigate the mechanism of 3-BrPA induced apoptosis of breast cancer cells with high expression of ErbB2 and the inhibition of xenograft breast cancer,the dynamic changes of breast cancer cells with high expression of ErbB2 was observed after treatment with HK? inhibitor 3-BrPA.Materials and Methods:1.Three types of breast cancer cell lines with high expression and low expression of ErbB2 were successfully established by transfection of wild type ErbB2 and ErbB2 siRNA.Detection of ErbB2 expression in three breast cancer cell lines by Western blot,The activity of the cells was measured by 48 hours after 3-BrPA treatment.The morphology and apoptosis of the cells were observed by fluorescence microscope.2.Western blot method was used to analyze the expression of HK?in three cell lines and the changes of mitochondria and cytoplasm.3.Western blot method was used to analyze the changes of HK ?expression in the mitochondria and cytoplasm of breast cancer cells with high expression of ErbB2,and to observe the effect of 3-BrPA on the dissociation of HK?.4.Mitochondrial membrane potential was detected by JC-1fluorescent probe.Western blot method was used to analyze the distribution of cytochrome C in mitochondria and cytoplasm and the cleavage of PARP protein.To observe the effect of 3-BrPA treatment on the apoptosis of breast cancer cells with high expression of ErbB2.5.In the xenograft experiment,231 V and 231ErbB2 two kinds of breast cancer cells were inoculated into nude mice to observe the anti-tumor effect of 3-BrPA treatment on breast cancer bearing nude mice with high expression of ErbB2.Result:1.Western blot method was used to detect the expression of ErbB2 in three breast cancer cell lines with high expression and low expression of ErbB2.The activity of the cells in the 48 hours groups after 3-BrPA treatment was determined,and the activity of the cells with high expression of ErbB2 was significantly decreased obviously.2.Western blot analysis showed that the expression of HK? in the cells with high expression of ErbB2 was significantly increased and most of them were distributed in mitochondria,but there was no significant change in the cells with low ErbB2 expression.3.Western blot analysis showed that the expression of HK? in breast cancer cells with high expression of ErbB2 was significantly reduced after treatment with 3-BrPA,but increased significantly in the cytoplasm,indicating that HK? was dissociated from mitochondria.4.JC-1 mitochondrial membrane potential detection after treatment of3-BrPA high expression of mitochondrial membrane potential of ErbB2 breast cancer cells were significantly decreased and the analysis of the content of cytochrome C in mitochondria decreased significantly increased in the cytoplasm of Western blot method,PARP lysis increased significantly,indicating the apoptosis of 3-BrPA induced by the high expression of ErbB2 in breast cancer cells.5.Xenograft experiments on the nude mice bearing breast cancerbearing nude mice after 3-BrPA treatment can significantly inhibit the expression of tumor cells with high expression of ErbB2.Conclusion and Innovation:1.Breast cancer cells with high expression of ErbB2 were more sensitive to 3-BrPA treatment.2.HK ? was increased in mitochondria by the high expression of ErbB2.3.3-BrPA treatment promoted the dissociation of HK ? from mitochondria of breast cancer cells with high expression of ErbB2.4.While reducing the mitochondrial membrane potential and the release of cytochrome C from mitochondria to the cytosol and the cleavage of PARP,mitochondrial mediated apoptosis induced by 3-BrPA treatment.5.The 3-BrPA treatment to the nude mice bearing tumor of breast cancer cells with high expression of ErbB2 is found tumor inhibition.