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Inducing Apoptosis and Reversal of P-gp Mediated Multidrug Resistance of MCF-7/ADM by Ginsenoside Rh2

Author: ZhangZuo
Tutor: WuXianZhong;KongZuo;WangHuaQing
School: Tianjin Medical University
Course: Traditional Chinese Medicine
Keywords: Department of MCF7 cells Multidrug resistance MDR1 gene P-glycoprotein Ginsenoside Rh2 MCF-7 cell line Apoptosis Necrosis Cell cycle Reversal
CLC: R285.5
Type: PhD thesis
Year: 2007
Downloads: 320
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Abstract


SectionⅠ: THE ESTABLISHMENT OF MCF-7 DRUG RESISTANT CELL SUBLINES IN VITRO MODELS AND THE CORRELATED STUDYObjective: Multidrug resistance (MDR) is a phenomenon that cancercells develop a cross-resistant phenotype against several unrelated drugsthat differ widely with respect to molecular structure and target specificity.Overexpression of MDR1 is the most significant cause of MDR which hadcomplicated mechanism. In this study, MCF-7 drug-resistant cell sublineswas established to explore the developmental mechanism of the multidrugresistance which caused by P-gp, so as to establish in vitro models forexploring methods to overcome the drug-resistance in tumor.Methods: Adriamycin-resistant human mammary carcinoma cellsubline MCF-7/ADM was selected from MCF-7 by stepwise inducement:MCF-7 parental cells were exposed to progressively and intermittentlyincreasing concentration of Adriamycin (ADM) from 0.1mg/L to 0.5mg/Lfor 6 months. ADM IC50 of MCF-7 and MCF-7/ADM were compared. Thetransport activity of P-gp was determined by Rhodamine 123 assay.Comparison of mdr1 mRNA expression levels in drug-resistant cells andtheir parental cells was determined using reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry (FCM) was performed to assessthe expression of Permeability glycoprotein (P-gp).Results: Drug resistant cell sublines MCF-7/ADM were establishedsuccessfully. ADM IC50 of MCF-7/ADM is higher than MCF-7, (P<0.05).ADM IC50 of MCF-7/ADM is 65.43±3.94μM, ADM IC50 of MCF-7 is1.12±0.14μM. Rhodamine 123 tests showed that fluorescence intensity ofRhodamine 123 in MCF-7/ADM is lower than that in MCF-7, (P<0.05).fluorescence intensity of Rhodamine 123 in MCF-7/ADM and MCF-7 were(1.22±0.20)% and (97.67±1.01)% respectively. MDR1 mRNAexpression levels and P-gp expressions of MCF-7/ADM cells weresignificantly higher than that of parental cells.Conclusions: The typical multidrug resistant phenotypes showed inMCF-7/ADM cells, possibly mainly being caused by over expression ofMDR1 gene, which suggest that MCF-7/ADM cells might serve as an idealmodel for studying the mechanism of MDR and investigating the methodsto reverse it. SectionⅡ: STUDY OF INHIBITION BY GINSENOSIDE Rh2 IN MCF-7 AND IT’S DRUG-RESISTANT CELL SUBLINES MCF-7/ADMObjective: In recent years, we realized that inducing apoptosis oftumor cell are the targets of tumor therapy. Many studies showed thatChinese crude drug had effects in this area. Ginsenoside Rh2 had highsignificant anti-tumor effectiveness, which pharmacologic mechanism isworth to study deeply.Methods: Inhibition ratio of Ginsenoside Rh2 to MCF-7 andMCF-7/ADM were determined at 24h, 48h, and 72h. Flow cytometry wasperformed to compare apoptosis ratio, necrosis ratio and cell cycle changesof MCF-7 and MCF-7/ADM which explosure to Rh2. Effects of Rh2 tocaspase 3 activities in MCF-7 and MCF-7/ADM were detected too.Results: 5~80μmol/L Rh2 can inhibit proliferations of MCF-7 andMCF-7/ADM significantly with time and dose dependent, (P<0.05);>80μmol/L Rh2 had no dose dependent to MCF-7 and MCF-7/ADM; Rh2 hadstronger effects to MCF-7 than MCF-7/ADM. 40μmol/L Rh2 can lead tonecrosis in MCF-7 grossly, (P<0.05); but induce apoptosis in MCF-7/ADMmainly though caspase pathway, (P<0.05). The influence to cell cycle ofRh2 in MCF-7 and MCF-7/ADM were different too: Rh2 can lead MCF-7cell cycle blockage in S stage; but in MCF-7/ADM cells, S stage cellsdecrease, and G2/M stage cells increase.Conclusions: Rh2 inhibit proliferations of MCF-7 and MCF-7/ADMthrough different mechanism: Rh2 can lead to necrosis in MCF-7, butinduce apoptosis in MCF-7/ADM though caspase pathway. The finding ofinducing apoptosis in drug resistance cell sublines MCF-7/ADM showed individuality of Rh2 on anti-tumor effectiveness. SectionⅢ: REVERSAL OF P-gp MEDIATED MUTIDRUD RESISTANCE OF MCF-7/ADM BY GINSENOSIDE Rh2Objective: Multidrug resistance has played a major role in the failureof cancer chemotherapy. Overexpression of MDR1 is the most significantcause of MDR, P-gp is a member of the ABC (ATP-binding cassette)transporters, has been directly implicated in resistance to a broad spectrumof chemotherapeutic reagents. We need to find low toxicant, effectivemodulators of MDR. In this study, we want to investigate modulations ofP-gp-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2 invitro.Methods: In MTT test, Ginsenoside Rh2 and ADM were added toMCF-7 and MCF-7/ADM culture single or conjunct, IC50 were determined.In flow cytometric analysis using rhodamine 123 as an artificial substrate,ginsenoside Rh2 and verapamil were added to MCF-7 and MCF-7/ADMrespective, the transport activity of P-gp was determined by Rhodamine123 assay. Comparison of mdr1 mRNA expression levels in drug-resistantcells and their parental cells which exposure to Rh2 was determined usingreverse transcription polymerase chain reaction (RT-PCR). Flow cytometrywas performed to assess the expression of Permeability glycoprotein(P-gp).Results: Ginsenoside Rh2 can degrade ADM IC50 (P<0.05) andpromote accumulation of rhodamine 123 in drug-resistant cell sublineMCF-7/ADM in a dose-dependent manner, but it had no effect on MCF-7cells. Ginsenoside Rh2 had stronger effect than verapamil in inhibition ofrhdamine 123 efflux. RT-PCR and flow cytometric analysis showed that theexpression of MDR1 gone and P-gp were no difference of MCF-7/ADMcells which exposure to ginsenoside Rh2. Conclusion: Ginsenoside Rh2 can reverse drug resistance to ADM inMCF-7/ADM cells significantly in vitro.

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