The Inhibitory Effect Of Cepharanthine On Breast Cancer And Its Molecular Mechanism Of Reversing Breast Cancer Endocrine Resistant

Background and aims:Breast cancer is one of the most common malignant tumors of the serious threat to women’s health.On a global scale in recent years,breast cancer has become the second malignant tumor that leads the deaths of women.Our country,according to the epidemiological study of breast cancer incidence and mortality are increased year by year.In 2015,about 4 million Chinese women diagnosed with breast cancer,about 2 million people died of breast cancer.The standardized comprehensive therapies of breast cancer include surgery,chemotherapy,radiotherapy,endocrine therapy,targeted therapy,and other supplementary treatment.Traditional Chinese medicine as an important regime in adjuvant treatment of breast cancer has more and more be taken seriously.Cepharanthine is a kind of have anti-cancer effect of traditional Chinese medicine,it has shown a variety of antitumor effects in previous study.But its antitumor mechanism is still unclear in breast cancer.In this study,we explore the mechanism of Cepharanthine in breast cancer through the application of molecular biology technology research,provides the basis for seeking new treatment options for patients with breast cancer.Methods and ResultsIn this study,we intend to explore the mechanism of Cepharanthine in breast cancer;specific research includes the following two parts:Part I Cepharanthine induces autophagy,apoptosis and cell cycle arrest in breast cancer cellsMethods:In order to detect inhibitory effect of Cepharanthine on breast cancer,we used MTT,clone formation,the in vitro scratch,invasion and migration assays to validate.We conducted further research to learn more about specific mechanism of Cepharanthine in inhibiting cell proliferation of breast cancer.In order to learn more about the effect of Cepharanthine on cell apoptosis and cell cycle G0/G1 arrest,we used Flow cytometry and Western blot were used to detect.Acridine orange staining,MTT and flow cytometry assays were used to detect the effect of Cepharanthine on cell autophagy.Then,we further investigate the effect of Cepharanthine in regulating AKT/mTOR pathway through the plasmid construction and Western blot assays.Results:As measured by MTT,clone formation assays,Cepharanthine showed the inhibitory effect of cell proliferation on MCF-7 and MDA-MB-231 cells;The in vitro scratch,invasion and migration assays showed Cepharanthine could obviously inhibit motility of breast cancer cell;Flow cytometry and Western blot assays demonstrated that Cepharanthine could induce apoptosis and cell cycle G0/G1 arrest in breast cancer cells.As measured by acridine orange staining,flow cytometry,Western blot and MTT assays,Cepharanthine could induce autophagy in breast cancer cells;Then,we transfected AKT overexpressing vector(pCDNA3.1-AKT)into the breast cancer cells in vitro,and analyzed by flow cytometry and Western blot analysis showed that Cepharanthine inhibited proliferation through regulating AKT/mTOR signaling pathway in breast cancer cells.Part II The mechanism of Cepharanthine reversed endocrine resistant in breast cancer cellsMethods:Our team using 10 μM tamoxifen treated MCF-7 in vitro for one year,and established tamoxifen-resistant cell lines(M7-TR).MTT assay was used to detect the sensitivity of tamoxifen resistant cell line(M7-TR)and parental MCF-7 to tamoxifen.Transwell assay was used to detect the invasion and metastasis of M7-TR.The changes of EMT-related markers in M7-TR cells measured by qRT-PCR and Western blot assays.In addition,the inhibitory effect of Cepharanthine on M7-TR cells was detected by MTT.The inhibitory effect of Cepharanthine on the invasion and migration of M7-TR cells was detected by in vitro scratch and Transwell assays.Then,qRT-PCR and Western blot were used to detect the changes of EMT-related markers in M7-TR cells after treatment with Cepharanthine.Then,we carried out P65-siRNA transfection in M7-TR cells and treated with or without Cepharanthine,the changes of NF-κB pathway and migration ability of M7-TR cells detected through MTT,Western blot and Transwell assays.Results:MTT assay showed that M7-TR was less sensitive to tamoxifen than that of parental MCF-7 cells.Transwell assay showed that M7-TR increased the invasion and migration ability.Meanwhile,cell morphological changes,qRT-PCR and Western blot revealed that M7-TR exhibited typical EMT-like changes.MTT assay revealed that Cepharanthine had inhibitory effect on M7-TR.In vitro scratch,Transwell assays showed that Cepharanthine could inhibit the invasion and metastasis ability of M7-TR cells.The expression of EMT markers of M7-TR cells after treated with Cepharanthine was detected by RT-PCR and Western blot.Furthermore,we transfected P65 siRNA into M7-TR cells,then cells were treated with or without Cepharanthine.After that,MTT,Western blot and Transwell assays were used to detect the inhibition of invasion and migration via regulating NF-κB pathway in M7-TR cells.Conclusions:1.Cepharanthine could induce apoptosis,autophagy and cell cycle G0/G1 phase arrest in breast cancer cells.2.Cepharanthine regulated the proliferation of breast cancer cells through regulating AKT/mTOR signal pathway.3.Cepharanthine inhibiting invasion and metastasis caused by breast cancer endocrine resistant via inhibiting EMT.4.Cepharanthine reversed breast cancer endocrine resistant via suppressing NF-κB pathway.Significances1.Reveals the effect of Cepharanthine on inhibiting cell proliferation of breast cancer.2.Clarify the effect of Cepharanthine on reversing breast cancer endocrine resistant.3.Confirmed the anti-tumor effect of Cepharanthine on breast cancer,and provided the theoretical basis for the clinical adjuvant therapy of breast cancer.