The Effects Of Bufadienolides On HER2 Overexpressing Breast Cancer Cells

Objective: HER2(Human epidermal growth factor receptor 2),a member of human epidermal growth factor family(EGFR family),is a proto-oncogene frequently amplified in human breast cancer.HER2 overexpression is correlated with rapid tumor growth,highly aggressive ability,early metastasis and frequent spread to the central nervous system,patients with HER2 overexpression usually face a relatively poor prognosis.The typical therapy for HER2 overexpression breast cancer patients is therapeutic HER2 signaling inhibition together with chemotherapy.Approximately half of HER2 positive breast tumor express estrogen receptor(ER),and the treatment for this tumor type also includes endocrine therapy of which the most standard medication is ER antagonist tamoxifen.Membrane receptor HER2 is also a tyrosine kinase(TK)receptor,and activation of HER2 after homo-or heterodimerization with other EGFR family member could lead to an initiation of downstream signaling pathway including the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(m TOR)and mitogen activated protein kinase(MAPK).This activated downstream kinase further modify and phosphorylated transcription factors and other components of the transcriptional and cell cycle machineries.This modification leads to alteration of gene expression and acceleration of tumor cell survival and proliferation as well as promotion of metastatic and angiogenic abilities.The typical targeted therapy for HER2 overexpression breast cancer is trastuzumab(also known as Herceptin),which belongs to humanized HER2 monoclonal antibody.Trastuzumab could block the dimerization of HER2 by binding to extracellular domain of HER2,and show obvious effect on HER2 positive breast tumor.However,drug resistance would rapidly occur to most patients.Preclinical and neoadjuvant trials indicate that ER could act as an escape pathway to bypass HER2 inhibition.It is reported that targeted therapy of lapatinib leads to restoration of extra survival pathways including ER,and the possible mechanism is enhanced ER expression caused by extended inhibition of PI3K/AKT pathway and consequent upregulation of transcription factors.HER2 overexpression is a possible mechanism of tamoxifen resistance under endocrine therapy,which lead to an attention to HER2 and ER positive breast cancer.As a traditional Chinese medicine,toad venom is capable of cardiotonic and anti-inflammatory effect.Arenobufagin and bufalin are two homogeneous bufadienolides with cardiac glycoside structure,and it has been reported that cardiac glycosides could inhibit tumor cell proliferation.Recent studies demonstrated that arenobufagin repress the adhesion,migration and invasion of hepatoma carcinoma cells,as well as inducing cell apoptosis and autophagy and blocking angiogenesis mediated by vascular endothelial growth factor.Multiple studies also indicated suggested that bufalin could induce cell cycle arrest and tumor cell apoptosis in leukemia and numerous solid tumor including hepatoma carcinoma,breast cancer and melanoma.In this study,we aimed at investigating whether arenobufagin and bufalin could be used as neoadjuvant drug for breast cancer.Methods:(1)To investigate the effect of arenobufagin and bufalin on HER2 overexpressing breast cancer,we used T47 D,MCF-7 cell lines and HER2 gene modified T47D/HER2 and MCF-7/HER2 cell lines were used in this study.(2)MTT,colony formation and wound healing assay were used to testify cell survival,proliferation and migration under treatment of arenobufagin and bufalin.(3)The important signaling pathways and key proteins associated with cell survival and proliferation were detected by western blot assay.(4)We further used flow cytometry to determine whether cell cycle changed when cells were treated with arenobufagin and bufalin.Results:(1)Results of MTT and colony formation assay demonstrated that arenobufagin and bufalin could significantly inhibit the cell survival and proliferation of T47 D and T47D/HER2 breast cancer cells in a time-and dose-dependent manner,and those two drugs could still function at a low dose.Combination of arenobufagin or bufalin with tamoxifen could obviously enhance the ability of tamoxifen to block proliferation of HER2 overexpressing cell.(2)Western blot assay indicated that arenobufagin and bufalin could decrease the level of of HER2 protein,inhibit the phosphorylation of AKT and ERK,down-regulate the expression of nuclear receptor coactivator SRC-1,SRC-3 and transcriptional factor E2F1,as well as induce fragmentation of apoptosis related protein PARP.These two drugs could also induce apoptosis and down-regulate the level of SRC-1 and SRC-3 in triple negative breast cancer cell line MDA-MB-231.(3)The result of flow cytometry assay suggested that arenobufagin and bufalin could induce cell cycle G2 M phase arrest in T47 D,MCF-7,T47D/HER2 and MCF-7/HER2 cell lines.Conclusions:Our results showed that arenobufagin and bufalin could significantly inhibit the proliferation and survival of HER2 overexpressing breast cancer cells.And the combination of each bufadienolide in low dose with tamoxifen could significantly enhance the inhibitory effect of tamoxifen on HER2 overexpressing breast cancer cells.Further study on HER2 related signaling pathways along demonstrated the declination of SRC-1,SRC-3,nuclear transcription factor E2F1,phosphorylated AKT and ERK,as well as cell cycle arrest and apoptosis.It is also indicated that arenobufagin and bufalin could affect the survival of All above suggest that arenobufagin and bufalin may be potential therapy adjuvants for HER2 overexpressing breast cancer therapy.