Based On The GPER Approach To Explore The Effect Of Dihydrotanshinone I On Human Breast Cancer MCF-7 Cells And Its Molecular Mechanism

ObjectiveBreast cancer is the most commonly diagnosed cancer among women worldwide.Most patients diagnosed with breast cancer will continue to develop symptoms of post-traumatic stress disorder which usually last for at least one year.Among these symptoms,pain,fatigue,arm morbidity and postmenopausal symptoms are the most common ones.The identification and treatment of these symptoms is a big challenge and these symptoms could damage the patient's health-related quality of life.It might occur at any age,but most frequently in middle-aged women.Due to advances in early diagnosis and treatment,breast cancer mortality has declined in the past few years.Currently,drugs such as endocrine therapy are used to treat and prevent estrogen receptor positive breast cancer.Studies have shown that endocrine therapy is the most effective method treating breast cancer with less toxic.Though increased utilization of endocrine drugs has led to improved procedures for the prevention and curing of breast cancer,it has also brought new challenges.The main challenges it faces are late relapse,drug resistance,injection pain and lack of the risk of compliance.Therefore,looking for a more natural,effective,safe medicine which at the same time could improve the effects of the current endocrine method in treating breast cancer could provide support in breast cancer treatmentIn recent years,Chinese medicine has been widely applied in anti-cancer treatment.As a kind of traditional Chinese medicine in China,Danshen and its active ingredients including Tanshinone I and Tanshinone IIA have been proved to play anti-breast cancer role due to its anti-estrogenic effects.As another effective active ingredient of Salvia miltiorrhiza,Dihydrotanshinone I,has also been shown to have anticancer effect.But the molecular mechanism of its action remains unknownPhytoestrogen is a non-steroidal compound produced by plants and has a chemical structure similar to endogenous estrogen.Therefore,it can produce estrogen-like and anti-estrogen by activating or blocking the binding of estrogen to estrogen receptor sample effect.The estrogen receptor is a type of protein that can specifically bind to estrogen to form a hormone-receptor complex and exert biological effects.In addition to the classic nuclear receptors ER? and ER?,in recent years,researchers have targeted a new recogniazed type of estrogen binding protein as G protein-coupled estrogen receptor(GPER).GPER was shown the binding ability to estrogen and stimulate non-genomic signaling transduction including downstream signal molecules related to cell proliferation and cell apoptosis to further regulate cell proliferation and differentiation.The GPER mediated PI3K/AKT signaling pathway plays an important role in cell proliferation and differentiation.Studies have shown that excessive activation of PI3K/AKT has close relationship with tumorigenesis and development.It could also promote cell proliferation by regulating the activity of apoptosis-related proteins.Therefore,GPER/PI3K/AKT signaling pathway is a key point in breast cancer researchIn this research,human breast cancer MCF-7 cells has been selected as the research object.The effect of dihydrotanshinone I on proliferation and apoptosis of MCF-7 cells will be tested.And the intervention agent of GPER and the key factor in the related molecular pathway will be employed.to explore the role of GPER mediated PI3K/AKT pathway in anti-breast cancer MCF-7 effect of dihydrotanshinone I.The results will provide a reference to fully understanding of the anti-tumor effect and its mechanism of Salvia miltiorrhiza.Methods1.MTT cell proliferation experiment was used to observe the effect of dihydrotanshinone I on the proliferation inhibition rate of human breast cancer MCF-7 cells;2.Flow cytometry was used to detect the effect of dihydrotanshinone I on the proliferation cycle of human breast cancer MCF-7 cells;3.The Annexin V-FITC/PI double staining method was used to detect the apoptosis rate of hunan breast cancer MCF-7 cells induced by dihydrotanshinone I and the effect of the intervention agent on the apoptosis rate of human breast cancer MCF-7 cells treated with dihydrotanshinone I;4.Western Blot technic was used to detect the effect of dihydrotanshinone I on the expression of cell cycle-related and cell apoptosis-related proteins in GPER mediated PI3K/AKT signaling pathway in human breast cancer MCF-7 cells.The specific intervention agent including GPER agonist GI and PI3K specific inhibitor LY294002 was used to further observe the effect of GPER,PI3K and AKT in the proliferation and apoptosis changes of MCF-7 cells induced by dihydrotanshinone IResults1.The effect of dihydrotanshinone I on the proliferation of human breast cancer MCF-7 cells1.1 The effect of dihydrotanshinone I on the proliferation inhibition rate of human breast cancer MCF-7 cells0.1-0.5 ?g/ml of dihydrotanshinone I could significantly inhibit the proliferation of MCF-7 cells after 48 hours(P?0.01)1.2 The effect of dihydrotanshinone I on the proliferation cycle of human breast cancer MCF-7 cellsThe results of flow cytometry showed that after 48h treating,0.1 ?g/ml of dihydrotanshinone I could arrest MCF-7 cells in G1 phase.Result of Western Blot test showed that 0.1 ?g/ml dihydrotanshinone I decreased the expression of cycle-related proteins cylinE,cyclinD1 and CDK2 to a certain extent after 48h of MCF-7 cells(P<0.01 or 0.05)But it has no obvious effect on cylinA2 protein expression2.The effect of dihydrotanshinone I on apoptosis of human breast cancer MCF-7 cells2.1 The effect of dihydrotanshinone I on the apoptosis rate of human breast cancer MCF-7 cellsThe results of Annexin V-FITC/P1 double staining showed that the effect of dihydrotanshinone I could increase the apoptosis rate of MCF-7 cells(P<0.01)2.2 The effect of dihydrotanshinone I on the expression of apoptosis-related proteins in human breast cancer MCF-7 cellsWestern Blot test results showed that 0.1 ?g/ml of dihydrotanshinone I increased the expression of apoptosis-related proteins caspase-3,caspase-9 and Bax after acting on MCF-7 cells for 48h(P<0.05).And the expression level of bcl-2 decreased(P<0.05).3.GPER mediated the role of dihydrotanshinone I in inhibiting proliferation and inducing apoptosis of human breast cancer MCF-7 cells3.1 G1 intervention on the effect of dihydrotanshinone I on MCF-7 cell apoptosis rateThe results of Annexin V-FITC/PI double staining showed that the apoptosis of MCF-7 cells caused by dihydrotanshinone I obviously decreased with G1 intervention(P<0.01)3.2 The effect of dihydrotanshinone I and the intervention agent G1 on the expression of GPER in MCF-7 cellsCompared with dihydrotanshinone I group,GPER protein expression further decreased(P<0.01)with GPER specific agonist G1 intervention3.3 The effect of G1 on the proliferation and apoptosis-related protein expression of MCF-7 cells treated with dihydrotanshinone IWestern Blot test showed that after adding GPER-specific agonist G1,compared with dihydrotanshinone I group,the expression of proliferation-related proteins including cyclinD1,cyclinE,CDK2 increased.The apoptosis-related proteins including caspase-3,caspase-9 and BAX expression decreased.At the meanwhile,bcl-2 expression obviously increased(P<0.01 or 0.05).4.Exploration of the molecular mechanism of dihydrotanshinone I inhibiting proliferation and inducing apoptosis of human breast cancer MCF-7 cells based on PI3K/AKT signaling pathway4.1 Effect of LY294002 on the apoptosis rate of MCF-7 cells treated with dihydrotanshinone IThe results of Annexin V-FITC/PI double staining showed that the apoptosis rate of MCF-7 cells further increased.(P<0.01)with LY294002 intervention4.2 Effect of dihydrotanshinone I and G1 on the expression of PI3K and AKT in MCF-7 cellsWestern Blot test results showed that after adding dihydrotanshinone I,compared with the control group,the expression of PI3K and AKT protein decreased(P<0.01 or 0.05).With 1?g/ml G1 intervention,PI3K and AKT protein expression further increased compared with the dihydrotanshinone I group(P<0.01)4.3 Effects of LY294002 on the proliferation of MCF-7 cells and the expression of apoptosis-related proteins treated with dihydrotanshinone IWestern Blot test results showed that after adding 1 ?g/ml P13K inhibitor LY294002,the expression of cycle-related proteins cyclinD1 and cyclinE in MCF-7 decreased(P<0.05)The expression of apoptosis-related protein caspase-3,caspase-9 and BAX increased(P<0.05)while the expression of bcl-2 decreased(P<0.05)Conclusion1.Dihydrotanshinone I could effectively inhibit the proliferation of human breast cancer MCF-7 cells and block its cell cycle in the G1 phase2.Dihydrotanshinone I could increase the apoptosis rate of MCF-7 cells and promote the expression of caspase-3,caspase-9 and Bax in MCF-7 cells that have a positive effect on the apoptosis process.At the meanwhile,the expression level of bcl-2 protein which hinders the process of apoptosis further decreased3.The effect of dihydrotanshinone I on proliferation inhibition and apoptosis induction of human breast cancer MCF-7 cells is mediated by GPER.4.Inhibition of GPER/P13K/AKT signaling pathway plays important role in the anti-breast cancer MCF-7 effect of dihydrotanshinone I.