Effect Of Trastuzumab On NOTCH-1 Signaling Pathway In Breast Cancer Cells SK-BR3

Objective: To investigate the effects and mechanisms of trastuzumab on Notch-1 signaling pathway and the relationship between Notch-1 and HER proteins in HER2 overexpression breast cancer cells, recognizing the significance of Notch-1 signaling pathway in trastuzumab resistance of breast cancer cells.Methods: HER2 Overexpression breast cancer cells SK-BR3 and HER2 non-overexpression breast cancer cells MDA-MB-231 were selected in this study. Grown curves of the two cell lines were detected by trypan blue exclusion method and cell counting. Immnocytochemistry and Western-blotting were used to detect the expression of Notch-1 and activated molecule of Notch-1 (NICD-1) proteins in SK-BR3 and MDA-MB-231 cells, respectively. SK-BR3 cells were treated with trastuzumab, Western-blotting was used to detect the expression of NICD-1 and HER2 proteins, RT-PCR assayed the expression of HES-1 (hairy/enhancer of split) mRNA, the target gene of Notch-1. Double immunofluorescence staining and immunoprecipitations were used to analyze the relationship of Notch-1 and HER2 proteins in SK-BR3 cells. Results: The expression of Notch-1 could be showed in both cell lines, but the nuclear accumulation of Notch-1 in SK-BR3 cells were significantly lower than in MDA-MB-231 cells. The results of Western-blotting showed that the expression of activated Notch-1 protein in HER2 overexpression cells SK-BR3 was obviously lower than in HER2 non-overexpression cells MDA-MB-231 (P <0.01). The expression of activated Notch-1 and HES-1 mRNA were obviously increased after trastuzumab treatment (P <0.01), and HER2 protein did not change significantly for trastuzumab treating (P > 0.05). Moreover, Notch-1 was discovered to co-localize and interact with HER2 in SK-BR3 cells by double immunofluorescence staining and immunoprecipitations.Conclusion: Overexpression of HER2 decreased Notch-1 activity by the formation of a HER2-Notch1 complex in HER2 overexpression breast cancer cells, and trastuzumab can restore the activity of Notch-1 signaling pathway, which could be associated with cells resistance to trasuzumab.