| Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among wemon worldwide.The number of new cases continues to grow,and the increasing trend is observed in younger women.Triple-negative breast cancer,in particular,has a poor prognosis due to high rate of metastasis and lack of clear therapeutic targets.Chemotherapy is one of the main treatments for breast cancer.However,the common chemotherapeutic drugs still have problems with severe side effects and drug resistance.So there is an urgent need to develop new drugs to treat breast cancer.Ethoxysanguinarine（ESG）,a kind of benzophenanthridine alkaloid,is extracted from traditional Chinese herbal medicine Boluohui with anti-viral,anti-bacterial,anti-inflammatory and anti-cancer biological activities.However,the role and molecular mechanism of ESG in breast cancer have not been clearly elucidated.This thesis mainly studies the anti-breast cancer activity of ESG and its underlying molecular mechanism,especially in triple negative breast cancer,and identifies the interaction network of Hakai protein involved in the anti-breast cancer activity of ESG using proteomics technology.The main research contents include the following three parts:（1）Effects of ESG on the proliferation and apoptosis of breast cancer cells.MTT assay showed that ESG inhibited the proliferation of breast cancer MCF7 cells and the highly aggressive triple negative breast cancer cells MDA-MB-231 in a dose-dependent manner.The IC50 values of ESG at 48 hr in MCF7 and MDA-MB-231 were2.008±0.424μM and 1.233±0.119μM,respectively.Flow cytometry results showed that ESG induced apoptosis in breast cancer cells.Western blot results further suggested that apoptosis-related pathway proteins Caspase-8,Caspase-9 and PARP were significantly cleaved,suggesting that ESG could activate both exogenous and endogenous apoptotic pathways in breast cancer.The combined use of ESG and Docetaxel（Doc）,a common drug used in clinical treatment of triple negative breast cancer,could synergistically inhibit the proliferation of MDA-MB-231 cells via interfering with CIP2A/PP2A/p AKT pathway.（2）The effect of ESG on breast cancer cell migration.Scratch assay and transwell assay showed that ESG significantly inhibited the migration of MDA-MB-231 cells.Molecular mechanism studies suggested that ESG could upregulate the epithelial marker E-cadherin and downregulate the mesenchymal marker N-cadherin,and the up-regulation of E-cadherin might be related to ESG-triggered inhibition of its E3 ubiquitin ligase Hakai,which suggested that ESG inhibited the epithelial mesenchymal transition（EMT）in breast cancer.Moreover,the migration of MDA-MB-231 cells were synergistically inhibited under combination treatment of low-dose ESG and Doc,and ESG-triggered down-regulation of Hakai protein might be involved in this synergistic effect.The above results suggest that Hakai protein plays an important role in ESG-induced anti-breast cancer activity.（3）Studies on the interactive network of Hakai protein.Given that the protein interaction network of Hakai in breast cancer has not been clearly elucidated,the Proximity-dependent Biotin Identification Technology（Bio ID）was used to systematically analyze the interactive network of Hakai at protome level.First,the Bio ID-only vector expressing biotin ligase and Hakai-Bio ID vector expressing biotin ligase and Hakai fusion protein were successfully constructed and subsequently transfected into MDA-MB-231 cells by cationic liposome.Immunofluorescence co-localization assay suggested that these two vectors could be expressed and localized correctly in MDA-MB-231 cells.Furthermore,western blot results suggested that Bio ID-only and Hakai-Bio ID fusion proteins could be efficiently experessed with the function of biotin labeling.Finally,the biotinylated proteins were purified from the Bio ID-only and Hakai-Bio ID transfected cells using streptavidin-modified magnetic beads,and identified by mass spectrometry followed by data analysis.A total of 85potential Hakai interactive proteins were found.Enrichment analysis showed that these proteins were involved in the regulation of various pathways such as nucleosome binding,RNA binding,adhesion protein binding and ER binding,including various proteins playing important roles in cancer,such as EGFR,RAB11A and KRAS.In summary,ESG inhibits breast cancer cell proliferation,migration and induces apoptosis via regulating CIP2A/PP2A/p AKT and Hakai/E-cadherin pathways,and can sensitize the anti-cancer effect of Doc,which lays foundation for development of ESG-based anti-breast cancer drugs.In addition,using Bio ID technology,85 candidates of Hakai-interacting proteins have been identified,many of which are closely related to the occurrence,development and treatment of cancer.The following study of Hakai interactome will facilitate to elucidate the molecular pathogenesis of breast cancer. |
