Mechanism Of α-mangostin Targeting RXRa And Regulating Apoptosis,migration And Invasion Of Breast Cancer Cells

Background:Breast Cancer is one ofmalignant tumors with high case-fatality rate,high morbidity and poor prognosis.Currently,there are several difficult problems for clinical treatment,such as obvious side effects of the first-line drug like Paclitaxel,the problem of the drug resistance and recurrence due to potential invasion and high frequent metastasis.The research work on targeting therapeutic agent has been make some progress,which have more the patients who survive longer with their breast cancer.But there are big issues focus on low efficiency of single drug treatment,high treatment cost,and tending to recrudesce.So looking for new targets and developing new target drugs are becoming a necessity.Truncated RXRα(tRXRα)clearly present in some cancer cells specifically,which be hydrolyzed by a certain protease in the cytoplasm.TRXRa may affect the tumorigenic process via PBK/Akt signal pathway.We find that tRXRαis closely related to highly invasive and metastatic features in breast cancer.So RXRα/tRXRα is a rather plausible target for anti-breast cancer drugs.Objective:On the drug therapy aspect,some natural drugs have carried out on clinical stage for some of the virtues of higher activity,little side-effect,wide raw material resources and long role time,et al.Natural drugs are the good source of a candidate for potential antitumor agents.They treat some of intractable diseases with great potential and vast development prospects.With the development finding and screening natural target drugs is becoming a hot topic in anti-cancer research in recent years.In this study,we will screen active pharmaceutical compound of Natural drugs for breast cancer,which based on targeting RXRa/tRXRa.We try to explore their possible molecular mechanisms,and with this study we can lay the foundation for futher development of anti-breast cancer.Methods:We performed virtual screening of drug targets against RXRa with docking procedures.And tested the effect on transcriptional activity of RXRa of the human embryonic kidney 293T cells by dual-luciferase reporter gene assay,which be treated by a-Mangostin.SPR analysis of the interaction of a-Mangostin with RXRa was performed with biacore system.MTT method was used to check the effect of breast cancer cells proliferation treated by Mangostin.Then the growth changes of breast cancer cells were observed by colony formation assay.Annexin V-FITC/PI double dye method and Flow cytometry were used to detect breast cancer cells cycle and cell apoptosis after treatment.Western blot(WB)were used to detect the expression of apoptosis related proteins or invasion-associated proteins after treatment.Cell migration was measured by wound healing assay.Cell invasion was evaluated by Transwell assay.The expression of mmp2 and mmp9 in MDA-MB-231 cells after treatment was detected by qRT-PCR.In our study,the expression of cyclin D1 in the clinical tissue specimens was assayed by immunohistochemical staining.In addition,to verify cyclin D1 be located in the cytoplasm and nucleus,the methods of subcellular fraction and immunofluorescence stain were used.Results:1,the energy Values of docking with Mangostin and RXRaare relatively smal The compounds showed strong binding ability to RXRa.Among them,αMangostin is the most prominent representative.The values of KD of a-Mangostin is 2.99uM.And the IC50 values of a-Mangostin suppressed the growth of breast cancer cells are 3.693uM(MCF-7),2.372uM(SKBR-3)and 5.464uM(MDA-MB-231).2,aMangostin could inhibit the proliferation of human breast cancer cells,trigger PARP cleavage,induce apoptosis,and hinder MDB-MB-231 cells cycle in concentrationdependent manners.3,the results shown that a-Mangostin may induce upregulation of bax expression in cells,in contrast α-Mangostin down-regulated expression of Bad/cleaved caspase 3/RXRa/tRXR/p-Akt,IKKβ/cyclin D1/etc in cells.4,the changes of p-Akt/cleaved caspase 3 after a-Mangostin treatment were similar with the circumstances that RXRa was knocked down in MDB-MB-231 cells.5,the changes of p-Akt/cleaved caspase 3 after a-Mangostin treatment were similar with the circumstances that LY294002 was mixed in MDB-MB-231 cells,too.6,a-Mangostin could can impede wound healing in the MDA-MB-231 cells experiments and repress invasive ability in vitro of MDA-MB-231 cells,and knocking down of cyclin D1 had similar results.7,it shown that α-Mangostin may induce upregulation of mmp2 and mmp9 expression in MDA-MB-231 cells,this is analogous to knock RXRa down or mix LY294002 solution.8,the abnormal high expressions of cyclin Dl were found in tissue samples of triple-negative breast cancer patients.It investigated that localization of cyclin D1 not only in nucleus but also in cytoplasm.Conclusions:1,according to the results of docking analysis and reporter gene assay,α-Mangostin show strong antagonistic effect on transcriptional activity of RXRα.The further experimental results indicated that a-Mangostin has high anti-breast cancer activity.2,the molecular mechanism of anti-breast cancer of α-Mangostin are fairly complicated,which involve in many interaction between the molecules in cells.It was discovered that a-Mangostin regulate downstream effectors via PI3K/Akt signaling pathway by degrading RXRa/tRXR.3,NF-kB signal pathway implicated in the regulation of anti-breast cancer activity of a-Mangostin.4,cyclin D1 involves in mediating apoptosis,invasion and migration of MDA-MB-231 cells.Therefore,it has important academic value.5,the functions of cyclin D1 in cytoplasm of MDA-MB-231 cells are the main emphasis of the next stage of the research.