Molecular Mechanism Of Cryptotanshinone Affecting Breast Cancer Cell Proliferation And Apoptosis Based On GPER Pathwa

ObjectiveBreast cancer is the most frequently diagnosed cancer among women worldwide and it mostly affects urban women aged from 40 to 55.Recently,with the acceleration of urbanization in China,the incidence of breast cancer is increasing constantly so that its prevention and treatments has became a social problem.As the target organ responded the estrogen,breast is regulated by estrogen.Breast diseases,especially the occurrence and development of breast cancer,are inseparable from the estrogen receptor.In addition to the classical nuclear estrogen receptor(nER α and β),which plays an estrogen-mediated role in breast cancer cells,there is still a new type of estrogen receptor,G protein-coupled estrogen receptor(GPER),which was discovered in the end of the 20th century.GPER has been shown to bind estrogen at the cell membrane,triggering rapid non-genomic effects and rapidly activating downstream signal pathways within seconds to minutes to achieve regulatory effects on target organs or cells.Studies indicated that GPER inhibits the proliferation of breast cancer cells,but the specific mechanism is still unclear.It is of great importance to elucidate the molecular mechanism of the role of GPER in breast cancer cells for the study of molecular targeted drugs based on GPER.PI3K/AKT signaling transduction is a key pathway of cellular proliferation and apoptosis regulation,which could be regulated by GPER.The up-regulation of the activity of this pathway can significantly promote the proliferation of breast cancer cells.Therefore,PI3K/AKT signaling pathway has become a popular topic in the research of to prevent from breast cancer.GPER’s regulation of the PI3K/AKT signaling pathway can rapidly activate PI3K by binding estrogen,causing phosphorylation of downstream core factor AKT,promoting the proliferation of target cells and inhibiting the occurrence of apoptosis.Phytoestrogens(PEs)are a class of active substances with a structure similar to endogenous estrogens.Resent studies indicated that estrogen works in both directions by binding toestrogen receptors,Cryptotanshinone(CPT)is the active component of salvia miltiorrhiza,which is the effect in activating blood circulation and removing blood stasis.Preliminary experimental study confirmed that salvia miltiorrhiza the other two kinds of active ingredients of Tanshinone I and Tanshinone IIA can through combination with estrogen receptor,give play to the role of phytoestrogens sample,achieve the goal that inhibit breast cancer cell proliferation,but the implicit whether tanshinone estrogen receptors in breast cancer cells by effect is still not clear,reveal the implicit function targets and the way of tanshinone will and provide a new experiment for the prevention and treatment of breast cancer cells.This study will mainly choose two kinds of breast cancer cells which express the different estrogen recepors.SKBR-3 cells perform the GPER positive and nER negative and in MCF-7 cells,both GPER and nER are positive expression.For reaserching the curative effect of the targetd medicine to provide new experimental basis,the molecular mechanism will be reaserched through testing the cell proliferation,cell cycle,cell apoptosis rate,GPER mediating role in PI3K/AKT signaling pathway and some other effects after treatment with CPT.Methods1,MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)cell viability assay was performed to detect the cell proliferation both on SKBR-3 and MCF-7 after treating with CPT for 24H and 48H.2.Flow cytometry was used to detect the cell cycle distribution affected by CPT on SKBR-3 and MCF-7 cell.3.Annexin V-FITC/PI double staining was performed to detect the cell apoptosis rate on SKBR-3 and MCF-7 after treatment with CPT.4.Small interfering RNA(siRNA)transfection was used to make a cell model of low-expression of GPER.And then,use these cell model to detect the proliferation by MTT assay.5.Western Blot was used to research the expression of cell cycle related protein(cyclins),cell apoptosis related protein(caspases),and key protein of PI3K/AKT signaling pathway.Furthermore,the specific GPER agonist G1,antagonist G15 and the inhibitor of PI3K-LY294002 were used respectively to explain the affect of CPT mediated through GPER/PI3K/AKT signaling pathway.Results1.Effect of CPT on proliferation of breast cancer cells1.1 Effect of CPT on proliferation rate of breast cancer cellsMTT cell proliferation assay showed that 1-10μM CPT inhibited SKBR-3 and MCF-7 cells(P<0.01)in a time-dose dependent manner.1.2 Effect of CPT on the cell cycle of breast cancer cellsFlow cytometry showed that 5,10μM of CPT induced an cell cycle arrest at G0/G1 phase in SKBR-3 cells after treatment with CPT for 48H(P<0.05),while the same concentration of CPT induced a cell cycle arrest at G2/M phase in MCF-7 cells(P<0.05).Western Blot results showed that 5,10μM CPT inhibited the expression of cyclin A,cyclin B,cyclin D and CDK2 in SKBR-3 and MCF-7 cells to varying degrees(P<0.05).In addition,the results indicated that CPT performed a more significant effect on SKBR-3 cells.2.Effect of CPT on cell apoptosis of breast cancer cells2.1 Effect of CPT on cell apoptosis rate of breast cancer cellsAnnexin V-FITC/PI double staining results showed that CPT significantly increased the apoptosis rate of SKBR-3 and MCF-7 cells(P<0.05).However,SKBR-3 cells were more sensitive to the effects of CPT compared with the MCF-7 cells.2.2 Effects of CPT on the expression of caspase-3 in breast cancer cellsWestern blot results showed that CPT could increase the expression of caspase-3 both in SKBR-3 and MCF-7 cells(P<0.05).3.CPT inhibits SKBR-3 cells viability through a GPER-mediated mannerThe results of MTT cell proliferation assay showed that the inhibitory effect of cryptotanshinone on SKBR-3 and MCF-7 cells with low expression of GPER established after transfection with GPER siRNA was weakened to different degrees(P<0.05).The results of Western Blot showed that the combined effect of cryptotanshinone and GPER agonist G1 promoted its inhibitory effect on the proliferation of breast cancer cells(P<0.05),on the contrary,its combined effect with GPER antagonist G15 weakened its inhibitory effect on the proliferation of breast cancer cells(P<0.05).4.Explore the molecular mechanism of cryptotanshinone inhibiting breast cancer cell proliferation through PI3K/AKT signaling pathwayResults indicated that CPT had inhibitory effects on the expression of PI3K,AKT,and p-AKT both in the two types of breast cancer cells(P<0.05),especially the decreasing of p-AKT(P<0.01).Compared with the two types of breast cancer cells,SKBR-3 cells were more sensitive to CPT.Secondly,the combined effect of G1 and CPT enhanced the inhibitory effect(P<0.05),while G15 could weaken the inhibitory effect(P<0.05).Finally,the intervention of LY294002 significantly inhibited expression of cyclins both in SKBR-3 and MCF-7 cells(P<0.05).Conclusion1.CPT could effectively inhibit the proliferation of SKBR-3 and MCF-7 cells,and block their cell cycle in G0/G1 phase and G2/M phase respectively to delay the process of cell proliferation cycle;2.CPT could significantly increase the apoptosis rate of SKBR-3 and MCF-7 cells,and increase the expression of caspase-3,the apoptotic effector protein in cells.3.CPT inhibits SKBR-3 cells viability through a GPER-mediated manner.4.Regulation of CPT on PI3K,p-AKT and cell cycle-associated proteins expression in SKBR-3 cells is realized by a GPER mediated manner.