| Chemotherapy play an important role in synthetic therapy of breast cancer.Resently new anticancer drugs has.greatly promoted the treating effect of many carcinoma including solid tumor.Multidrug resistance(MDR) remains to be a capital problem in chemotherapy.Once the tumor develop resistant to a certain anticancer drug,it will resist to drugs that are structurally and chemically unrelated.Many mechanisms are now know contribute to MDR,including changes①in drug transport such as Permeability-glycoprotein(P-gp),Multidrug Resistance Associated Protein(MRP),Lung Resistance-related Protein(LRP);②in metabolic detoxificationg such as Glutation(GSH);③in drug target,such as topoisoeraseⅡ(TopoⅡ),in DNA repair;④in apoptosis etc.Among these,the mechanism of P-gp/mdrl mediated was recognized earliestly and was researched most extensively.GSH and its enzyme systems not only play important roles in cellular drug detoxification and in the course of resisting oxidation,but also enhance the metabolism and transportation potency of anticancer drugs in cancer cells,the cancer cells then become drug resistance cell.Now the active drugs which can reverse multidrug-resistance are as follow:calcium channel blocker,immunomodulator,calmodulin inhibitor and so on.Because the curative effect is not good,target is single and toxic action is obvious,thy are not frequently applied in clinic.Chinese traditional drag possesses the good quality of high performance,multitarget and little toxic action,so it has the value of being investigated.So searching safe and active reversal drug for multidrug resistance from Chinese traditional drug is a hot spot now.Celastrusorbiculatus is the plant of celastrus,distribute generally,its rattan,root,leafage,fruit are all put in drug.The modern chemical pharmacy has confirmed that some component of Celastrus possess doughty antineoplasmic activity,inducing cell apoptosis is one of the pathway that kill and wound the tumor cell.1.Purpose:The purposes of this research was to study on reversal effect and possible mechanism of MDR in MCF-7 /ADM cell line by the total terpenes of Celastrus orbiculatus with methods of high performance liquid chroma-tography(HPLC),biochemical analyses,immunohisto chemical technique and so on to provide the theoretically and experimental bases for clinical use.2.Method:2.1 MTT methodApply MTT deoxidized method to measure direct cell toxicant effect on MCF-7(sensitive strain) and MCF-7/ADM(multidrug-resistant strain) by the total terpenes of Celastrus orbiculatus.To observe the reverse effect of the total terpenes of Celastrus orbiculatus which has screened for nontoxicant concentration to MCF-7/ ADM by MTT.To measure toxicant effect of ADM to both strains for resistant index and resistance fold by MTT;2.2 Hing performance liquid chromatography(HPLC)Apply Hing performance liquid chromatography to detect cellular ADM concentration in MCF-7,MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.2.3 Flow cytometryApply Flow cytometry to detect apoptosis cell population in MCF-7,MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.Apply Flow cytometry to survey P-gp proteinum transmutation in MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus through indirect fluorescent labeling methond.2.4 Biochemical analyseApply Biochemical analyse with DTNB to detect celluar GSH concentration of MCF-7,MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.2.5 Cell immunohistochemistryApply Cell immunohistochemistry S-P staining method to meaure P-gp proteinum masculine expressing rate in MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.3.Result3.1 There is no obvious cell toxicity of the total terpenes of Celastrus orbiculatus to MCF-7 and MCF-7/ADM cell when the concentration of the total terpenes of Celastrus orbiculatus is 10μg/ml below.The total terpenes of Celastrus orbiculatus(5μg/ml,10μg/ml) can increased cell toxic effect of ADM to MCF-7/ADM.Inhibitive concentration 50 of ADM to MCF-7 and MCF-7/ADM are1.09μg/ml and 62.62μg/ml respectively,resistance fold of MCF-7/ADM is57.45.3.2 The total terpenes of Celastrus orbiculatus(5μg/ml,10μg/ml)can increased MCF-7/ADM's accumulated concentration of ADM.3.3 MCF-7 and MCF-7/ADM are effected by different densities of the total terpenes of Celastrus orbiculatus,the rate of apoptisis cell step up obviously,and to offer dose dependent.Utilizing Flow cytometry immunofluorescence technic detect P-gp gene product which decreased along with the increase of dosage of the total terpenes of Celastrus orbiculatus.3.4 The total terpenes of Celastrus orbiculatus(10μg/ml) can decrease the celluar GSH concentration in MCF-7/ADM cell line,decreasing of celluar GSH concentration was one of the reversal mechanisms of MDR in MCF-7/ADM cell line by the total terpenes of Celastrus orbiculatus.3.5 After treated with the total terpenes of Celastrus orbiculatus(5μg/ml,10μg/ml), P-gp proteinum expression all have different degree decrease in MCF-7/ADM cell line.4.ConclusionThe total terpenes of Celastrus orbiculatus has certain breast tumout-resistant effect,it can increase MCF-7/ADM'S accumulated concentration of ADM,it can reduce MCF-7/ADM'S P-gp and GSH expression,it can increase the apoptosis cell population of MCF-7/ADM,the total terpenes of Celastrus orbiculatus can partly revese multidrug resistance of MCF-7/ADM in vitro.So Celastrus orbiculatus may be a kind of safe and efficient reversal agent for clinic.
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