Mechanisms Of Anti-tumor Effects Of Mibefradil And Resveratrol In Triple-negative Breast Cancer

Objective:Triple-negative breast cancer（TNBC）accounts for approximately 15%-20%of the breast cancer patients,and which is characterized by poor therapy efficacy and high risk of distant metastasis.Due to the specific molecular phenotype of TNBC,it cannot benefit from endocrine therapy or HER2 targeted therapy.Therefore,chemotherapy is still the recommended therapy in TNBC clinical treatment.However,due to the high rate of relapse and therapy resistance,the 5 year-mortality of TNBC patients is still 70%.Therefore,it is urgent to excavate more effective therapeutic strategies for TNBC.At present,there are still problems existed when developing new drugs for a certain disease,such as long work cycle,high cost,low success rate,and so on.Drug repurposing is one of the effective ways to solve these problems,and many researches have succeeded in reusing existing drugs for other applications by this method.Another important part of new drug development is to discover and optimize lead compounds from natural products.Many recent studies have focused on the development of pharmacologically safe and biologically active phytochemicals as preventive and therapeutic drugs for breast cancer.In this study,based on the concept of drug repurposing,we explored an anti-hypertensive drug Mibefradil,which could exert anti-tumor effect in TNBC.And based on the research of natural products,we found a natural active substance Resveratrol,which could improve the anti-tumor microenvironment in TNBC lung metastasis.This study explored the mechanisms of anti-tumor effects of two drugs,and may provide new strategies for TNBC therapy.Methods:1.The characteristic expression genes of TNBC was analyzed by using TCGA,METABRIC and GEO databases.2.Drug prediction was performed by using CMap database.3.Cell proliferative ability was detected by CCK-8 experiment.4.Cell apoptosis and cell cycle were detected by flow cytometry.5.Pharm Mapper and Swiss Target Prediction databases were used for drug target prediction.6.String database and Cytoscape software were used to construct protein interaction network.7.Autodock software was used for molecular docking analysis.8.The expression level of proteins were detected by Western blotting.9.Si RNA or c DNA plasmid were transfected to down-regulate or up-regulate the expression of the target protein.10.The xenograft model was constructed to explore the effect of Mibefradil in vivo.11.Immunohistochemical staining was used to detect the expression of proteins in subcutaneous tumor tissues.12.Hematoxylin-eosin staining was used to detect the effects of Mibefradil on the heart,liver and kidney of tumor-bearing mice.13.The experimental mouse model of TNBC lung metastasis was used to explore the effect of Resveratrol on TNBC lung metastasis.14.Hematoxylin-eosin staining was used to detect lung metastases in tumor-bearing mice.15.Immunohistochemical staining was used to detect the expression of proteins in lung tissue of tumor-bearing mice.16.The infiltration of immune cells in the lung tissue of tumor-bearing mice were detected by flow cytometry.17.The expression levels of m RNA were detected by real-time PCR.18.The concentration of cytokines were detected by ELISA.19.The proportion of target cell subsets were detected by flow cytometry.Results:1.Characteristic expression genes of TNBC were screened out by using public databases.By analyzing transcriptome data from TCGA,METABRIC and GSE76275 databases,167 up-regulated and 140 down-regulated characteristic expression genes of TNBC were screened out.2.Mibefradil is predicted to be the potential therapeutic drug for TNBC by CMap database.The characteristic expression genes of TNBC were uploaded to CMap database for drug prediction,and the T-type Ca²⁺channel blocker Mibefradil was screened out,which has potential anti-tumor effect on TNBC.3.Mibefradil inhibits the proliferation of TNBC cells.CCK8 assay showed that Mibefradil could decrease cell viability in TNBC cells MDA-MB-468 and MDA-MB-231.4.Mibefradil induces apoptosis of TNBC cells.Flow cytometry analysis showed that Mibefradil could induce apoptosis of TNBC cells in a dose-dependent manner.5.Mibefradil induces cell cycle arrest in TNBC cells.Flow cytometry analysis showed that Mibefradil could induced G1 phase arrest in TNBC cells in a dose-dependent manner.6.Predicting the potential targets of Mibefradil.A total of 185 potential human target genes of Mibefradil were predicted by using Pharm Mapper and Swiss Target Prediction databases.7.AURKA is the potential target of Mibefradil in TNBC.Via the construction of PPI network and the method of molecular docking,AURKA was screened out to be the poteintial target for Mibefradil judged by its strongest binding affinity.8.Mibefradil inhibits the expression and phosphorylation of AURKA protein.Western blot results showed that Mibefradil could dose-dependently downregulate the protein level and phosphorylation level of AURKA in TNBC cells.9.Mibefradil regulates apoptosis and cell cycle signal pathway of TNBC cells.Western blot results showed that Mibefradil treatment significantly increased the expression of pro-apoptosis protein cleaved-PARP,and decreased the expression of anti-apoptosis protein BCL2 in TNBC cells.Meanwhile,it could also decrease the expression of cell cycle-related proteins Cyclin D1 and Cyclin B1.10.Mibefradil regulates apoptosis of TNBC cells via targeting AURKA.Flow cytometry analysis showed that knockdown of AURKA attenuated the apoptosis induced by Mibefradil,whereas overexpression of AURKA enhanced Mibefradil-induced apoptosis in TNBC cells.11.Mibefradil regulates the expression of apoptosis-related proteins by targeting AURKA.Western blot results showed that knockdown of AURKA attenuated the Mibefradil-induced increase of cleaved-PARP and the decrease of BCL2,while overexpression of AURKA significantly enhanced the increase of cleaved-PARP and the decrease of BCL2 induced by Mibefradil.12.Mibefradil inhibits TNBC growth in vivo.The xenograft model results showed that the tumor weight and the tumor size of Mibefradil‐treated group were significantly decreased comparing with the vehicle group.Simultaneously,the immunohistochemistry results showed that the expression of Ki67 decreased,the expression of TUNEL increased,and the expression of its target gene AURKA and p-AURKA decreased in the Mibefradil‐treated tumor tissues.13.Mibefradil has no obvious side effects in vivo.Hematoxylin-eosin results showed that no toxicity-related pathological changes were observed in vital organs such as heart,liver and kidney in Mibefradil‐treated group.14.Resveratrol inhibits TNBC lung metastasis and its growth in lung.TNBC lung metastatic experimental mouse model results showed that intraperitoneal injection of Resveratrol could significantly inhibit TNBC lung metastases.Immunohistochemistry results showed that the expression of Ki67 decreased,the expression of TUNEL increased in Resveratrol-treated lung metastasis lesion.15.Resveratrol promotes the infiltration of T cells into lung tissue.Flow cytometry analysis showed that Resveratrol treatment could significantly increase both the proportion and the absolute number of CD8⁺T cells,and increase the number of CD4⁺T cells in the lungs of tumor bearing mice.16.Resveratrol affects the frequency and M1polarization of macrophages in lung.Flow cytometry analysis showed that Resveratrol treatment significantly increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages,which results in a significant increase in the ratio of M1/M2 macrophages in tumor bearing lungs.17.Resveratrol has no effect on myeloid-derived suppressor cells（MDSCs）.Flow cytometry analysis showed that Resveratrol had no effect on the proportion and the number of MDSCs in the lungs of tumor bearing mice.18.Resveratrol promotes Th1 immune response in TNBC lung metastasis.Resveratrol treatment could significantly increase the m RNA and protein levels of Th1 cytokines（IL-2 and IFN-γ）,while decreased Th2 cytokine（IL-6）in lung tissues.19.Resveratrol elevates the anti-tumor activity of CD8⁺T cells.Real-time PCR results showed that Resveratrol significantly increased the expression levels of IFN-γ,perforin and granzyme B m RNA in CD8⁺T cells of lung tissues.Moreover,flow cytometry analysis further verified that Resveratrol increased the number of CD8⁺T cells producing IFN-γ,perforin and granzyme B in the lung tissue of tumor-bearing mice.20.Resveratrol down-regulates the expression of PD-1 in CD8⁺T cells.Real-time PCR results showed that Resveratrol down-regulated the expression of PD-1 m RNA in CD8⁺T cells in lung tissues.Simultaneously,flow cytometry analysis showed that Resveratrol down-regulated the proportion and the number of CD8⁺T cells expressing PD-1 in lung tissues of tumor-bearing mice.Conclusion:1.Mibefradil inhibits the growth of TNBC,and regulates apoptosis-related signal pathways by inhibiting the expression of AURKA and its phosphorylated protein to exert anti-tumor effects.2.Resveratrol inhibits the lung metastasis of TNBC,and affects the infiltration of immune cells in the microenvironment of lung tissue.Resveratrol improves Th1 immune response,and down-regulates the expression of PD-1in CD8⁺T cells to enhances the anti-tumor immune response mediated by CD8⁺T cells.