| Objective:Recently,the incidence of breast cancer presents persistent high growth,and it has became the highest incidence of women malignant tumor in the world.Surgical tumor removal,chemotherapy and radiation therapy have been the most important therapy means in breast cancer patients.With the progress of basic research and the development of various new drugs,chemotherapy plays an important role in the comprehensive treatment of breast cancer.Doxorubicin(Dox),a kind of anthracyclines,is often classified as the first-line drugs for breast cancer chemotherapy.However,with the prolongation of chemotherapy cycle,breast cancer cells are not only prone to resist to Dox,but also develop to cross-resistance to a variety of chemotherapeutic drugs with different structure and function,thereby resulting in breast cancer chemotherapy failure,relapse and metastasis.Therefore,how to effectively reverse the drug resistance of breast cancer has become one of the difficult problems of chemotherapeutic research.There are some different mechanisms associated with the development of drug resistance in breast cancer,mainly including efflux ATP-binding cassette(ABC)transporter proteins mediated,apoptosis regulatory genes mediated and tumor survival environment mediated,etc.With the deepening of understanding of the mechanism of drug resistance in breast cancer,a large number of reversal strategies for drug resistance in breast cancer have been involved in basic and clinical studies,among the total,chemosensitizer is the earliest and the most studied reversal strategy.However,current chemosensitizers commonly present the problem that the effective dose is similar to the poisoned dose,thus limiting the clinical application of these chemosensitizers.The ideal chemosensitizer should meet the following conditions:(1)It should be safe and less toxic to normal tissues;(2)It should have a certain antitumor activity;(3)It should be stable and have a long half-life.TanshinoneⅡA(TanⅡA)is one of the effective active ingredient of Salvia miltiorrhiza and commonly used to treat ischemic heart disease in clinic.Modern pharmacology studies show that TanⅡA has anti-tumor activity in addition to its cardiovascular protective effect.Although Dox plays an important role in the comprehensive treatment of breast cancer,its possible side effects such as hematopoietic depression,cardiac and renal toxicity and drug resistance sometimes lead to a reduction in the chemotherapeutic effect,even lead to the failure of chemotherapy.Since many toxic and side effects caused by Dox are the key points of tanshinoneⅡA in the treatment,meanwhile,also because TanⅡA has anti-tumor activity,therefore,TanⅡA meet the conditions as a chemosensitizer,however,few studied were done on TanⅡA as a chemosensitizer.In this study,by preparing human breast cancer MCF-7 cells,its corresponding Dox resistant MCF-7/dox cells and nude mice breast cancer model,here,we want to assess a new use of TanⅡA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms.The results of the study will provide valuable theoretical and experimental basises for developing a low toxicity and high efficient chemotherapeutic sensitizers which has no influence on the chemotherapy drugs in plasma dynamics.Methods:1.Effect of TanⅡA on the antitumor activity of Dox against breast cancer cellsMCF-7 and MCF-7/dox cells were exposed to Dox with or without TanⅡA intervention.Effect of TanⅡA on the inhibitory effect of Dox on the proliferation of breast cancer cells was assessed by MTS assay;Effects of TanⅡA on Dox inducing apoptosis and eliminating breast cancer stem cells(BCSCs)was analysed by flow cytometry;Effect of TanⅡA on the inhibitory effect of Dox on the invasion of breast cancer cells was detected by matrigel invasion assay;Effects of TanⅡA on intracellular Dox accumulation was analysed by flow cytometry.2.The mechanism of TanⅡA enhancing the antitumor activity of Dox against breast cancer cellsMCF-7 and MCF-7/dox cells were exposed to Dox,or combination of Dox and nontoxic dose of TanⅡA with or without bpV(HOpic)intervention.Meanwhile,untreated cells were employed for control.Changes in proliferation inhibition rates of breast cancer cells after drug intervention were assessed by MTS assay;Changes in the apoptosis rates of breast cancer cells and the number of BCSCs were analysed by flow cytometry;Changes in the invasiveness of breast cancer cells were detected by matrigel invasion assay;Changes in the intracellular Dox accumulation of breast cancer cells were analysed by flow cytometry;Changes in the expressions of PTEN、p-AKT、P-gp、BCRP、MRP1 in breast cancer cells were determined by western blot.3.Study of the effect of TanⅡA on the antitumor activity of Dox against breast cancer cells in nude miceConstructing of breast cancer models in nude mice using MCF-7 cells.The mice were randomly divided into 4 groups when the tumor grew to about 0.7cm~3.The mice were administered with normal saline 0.2m L(control group),TanⅡA 10 mg/kg(TanⅡA group),Dox 5mg/kg(Dox group)and combinaton of Dox 5mg/kg and TanⅡA 10 mg/kg(combined drugs group)by intraperitoneal injection every other day.Changes in body weight and tumor volume of nude mice were assessed everyday;Changes in bone marrow hemopoietic level were determined by Wright’s staining;Changes in myocardial enzyme and renal function were detected by biochemical analyzer.Results:1.Effect of TanⅡA on the antitumor activity of Dox against breast cancer cellsTanⅡA could enhance the killing effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner;When combining treatment of Dox with different concentrations of TanⅡA,the killing effect of the combination on MCF-7 and MCF-7/dox cells increased with the increasing concentrations of TanⅡA;Even when combining treatment of Dox with nontoxic dose of TanⅡA,the inhibitory effects of Dox on cell proliferation and invasion and the effects of Dox on inducing cell apoptosis and killing BCSCs were significantly enhanced(P﹤0.05);Meanwhile,under the same concentration of Dox,Dox accumulation in breast cancer cells treated with Dox and nontoxic dose of TanⅡA was significantly higher than that of Dox treated alone(P﹤0.05).2.The mechanism of TanⅡA enhancing the antitumor activity of Dox against breast cancer cellsCompared to MCF-7 cells,the expression of PTEN in MCF-7/dox cells was decreased (P﹤0.05)while the expression of p-AKT in MCF-7/dox cells was increased(P﹤0.05);Meanwhile,the expression of MDR-related efflux ABC transporter proteins including P-gp,BCRP and MRP1 in MCF-7/dox cells was much higher than that of in MCF-7 cells(P﹤0.05);Compared to treatment with Dox alone,combined treatment of Dox and TanⅡA could significantly up-regulate the expression of PTEN and down-regulate the expression of p-AKT and efflux ABC transporter proteins including P-gp,BCRP and MRP1 in MCF-7 and MCF-7/dox cells(P﹤0.05);After the intervention of PTEN inhibitor bpV(HOpic),the enhanced cytotoxicity of Dox mediated by TanⅡA was greatly reduced;However,after the intervention of PTEN inhibitor bpV(HOpic),treatment of MCF-7 and MCF-7/dox cells with TanⅡA could result in decreased expression of PTEN(P﹤0.05)and the increased expression of p-AKT(P﹤0.05),but could not result in obvious changes in the expression of ABC transporter proteins including P-gp,BCRP and MRP1(P﹥0.05).3.Study of the effect of TanⅡA on the antitumor activity of Dox against breast cancer cells in nude miceWe successfully constructed breast cancer models in nude mice;Compared to single treatment with Dox or TanⅡA,combined treatment with Dox and TanⅡA could significantly inhibit tumor growth in nude mice(P﹤0.05);Meanwhile,combined treatment with Dox and TanⅡA could significantly attenuate the toxic side effects of Dox incuding weight loss,hematopoietic depression,cardiac and renal toxicity,etc.Conclusion:1.TanⅡA could obviously enhance the antitumor activity of Dox against breast cancer cells;2.TanⅡA enhanced the antitumor activity of Dox against breast cancer cells through inhibiting PI3K/AKT pathway and down-regulating the expression of efflux ABC transporters including P-gp,BCRP and MRP1.3.TanⅡA enhanced chemotherapeutic effect of Dox against human breast cancer cells while reducing toxic side effects of it. |
PDF Full Text Request