Yi Huang1, Yu Sun1, Wei-Wei Wang1, Lei Zhang1. 1. Department of Anus and Intestine Surgery, The Affiliated Zhongshan Hospital of Dalian University Dalian 116001, Liaoning, China.
Abstract
BACKGROUND: Epidermal growth factor receptors (EGFR) are identified to be favorable targets for cancer treatment. In present work, we showed that Boeravinone B, a Rotenoid from natural origin has significant anticancer activity via internalization of ErbB2 and EGFR, and thereby resulting in destruction of the receptors. METHODS: For cell viability and apoptosis were done by MTT assay. Annexin V-FITC staining was done for determining the extent of apoptosis. Immunoblotting for expression of proteins in HT-29 cell lysates after exposing them to Boeravinone G. Immunofluorescence and Confocal microscopic analysis was done for HT-29 cells incubated with anti-EGFR or anti-ErbB2 antibodies. Surface biotinylation assay was done followed by western blot analysis for expression of proteins using antibodies against transferrin receptor, ErbB2 and EGFR. RESULTS: Exposure of HT-29 cells with Boeravinone B suppressed constitutive as well as ligand mediated phosphorylation of ErbB2, ErbB3 and EGFR. The treatment also inhibited the activation of mitogen-activated protein kinase (MAPK), Akt and Erk1/2 which are downstream signaling molecules. The treatment also bought about internalization of ErbB2 and EGFR causing destruction of receptors, Boeravinone B also caused apoptosis in HT-29 cells. Boeravinone B mediated degradation was halted by Chloroquine (lysosomal inhibitor). Boeravinone B caused nuclear translocation of apoptosis-inducing factor (AIF) and caused proteolytic processing of PARP along with caspase-3, confirming Boeravinone B may induce caspase-independent apoptosis in HT-29 cells. CONCLUSION: The findings of present study provide first ever evidences for Boeravinone B suggesting anticancer activity via internalization and destruction of EGFR family receptors i.e. ErbB2 and EGFR in HT-29 cell lines.
BACKGROUND:Epidermal growth factor receptors (EGFR) are identified to be favorable targets for cancer treatment. In present work, we showed that Boeravinone B, a Rotenoid from natural origin has significant anticancer activity via internalization of ErbB2 and EGFR, and thereby resulting in destruction of the receptors. METHODS: For cell viability and apoptosis were done by MTT assay. Annexin V-FITC staining was done for determining the extent of apoptosis. Immunoblotting for expression of proteins in HT-29 cell lysates after exposing them to Boeravinone G. Immunofluorescence and Confocal microscopic analysis was done for HT-29 cells incubated with anti-EGFR or anti-ErbB2 antibodies. Surface biotinylation assay was done followed by western blot analysis for expression of proteins using antibodies against transferrin receptor, ErbB2 and EGFR. RESULTS: Exposure of HT-29 cells with Boeravinone B suppressed constitutive as well as ligand mediated phosphorylation of ErbB2, ErbB3 and EGFR. The treatment also inhibited the activation of mitogen-activated protein kinase (MAPK), Akt and Erk1/2 which are downstream signaling molecules. The treatment also bought about internalization of ErbB2 and EGFR causing destruction of receptors, Boeravinone B also caused apoptosis in HT-29 cells. Boeravinone B mediated degradation was halted by Chloroquine (lysosomal inhibitor). Boeravinone B caused nuclear translocation of apoptosis-inducing factor (AIF) and caused proteolytic processing of PARP along with caspase-3, confirming Boeravinone B may induce caspase-independent apoptosis in HT-29 cells. CONCLUSION: The findings of present study provide first ever evidences for Boeravinone B suggesting anticancer activity via internalization and destruction of EGFR family receptors i.e. ErbB2 and EGFR in HT-29 cell lines.