Original Article
Effect of Trastuzumab on Notch-1 Signaling Pathway in Breast Cancer SK-BR3 Cells
Abstract
Objective: To investigate the effects and mechanisms of trastuzumab on Notch-1 pathway in breast cancer cells, recognizing the significance of Notch-1 signaling pathway in trastuzumab resistance.
Methods: Immunocytochemistry staining and Western blotting were employed to justify the expression of Notch-1 protein in HER2-overexpressing SK-BR3 cells and HER2-non-overexpressing breast cancer MDA-MB-231 cells. Western blotting and reverse transcription PCR (RT-PCR) were used to detect the activated Notch-1 and Notch-1 target gene HES-1 mRNA expression after SK-BR3 cells were treated with trastuzumab. Double immunofluorescence staining and co-immunoprecipitation were used to analyze the relationship of Notch-1 and HER2 proteins.
Results: The level of Notch-1 nuclear localization and activated Notch-1 proteins in HER2-overexpressing cells were significantly lower than in HER2-non-overexpressing cells (P<0.01), and the expressions of activated Notch-1 and HES-1 mRNA were obviously increased after trastuzumab treatment (P<0.05), but HER2 expression 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.
Conclusion: Overexpression of HER2 decreased Notch-1 activity by the formation of a HER2-Notch1 complex, and trastuzumab can restore the activity of Notch-1 signaling pathway, which could be associated with cell resistance to trastuzumab.
Methods: Immunocytochemistry staining and Western blotting were employed to justify the expression of Notch-1 protein in HER2-overexpressing SK-BR3 cells and HER2-non-overexpressing breast cancer MDA-MB-231 cells. Western blotting and reverse transcription PCR (RT-PCR) were used to detect the activated Notch-1 and Notch-1 target gene HES-1 mRNA expression after SK-BR3 cells were treated with trastuzumab. Double immunofluorescence staining and co-immunoprecipitation were used to analyze the relationship of Notch-1 and HER2 proteins.
Results: The level of Notch-1 nuclear localization and activated Notch-1 proteins in HER2-overexpressing cells were significantly lower than in HER2-non-overexpressing cells (P<0.01), and the expressions of activated Notch-1 and HES-1 mRNA were obviously increased after trastuzumab treatment (P<0.05), but HER2 expression 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.
Conclusion: Overexpression of HER2 decreased Notch-1 activity by the formation of a HER2-Notch1 complex, and trastuzumab can restore the activity of Notch-1 signaling pathway, which could be associated with cell resistance to trastuzumab.
