| Objectives:Our previous study found that Polyphyllin I(PP?)had a significant inhibitory effect on MCF-7 and MCF-7/ADM cells.Low cytotoxic concentration of PP? may reverse drug resistance of MCF-7/ADM cells by inhibiting the expression of P-gp and phosphorylation of ERK and JNK.ERK and JNK signaling pathway can modulate cell cycle,which may be one of the mechanisms of PP? reversing drug resistance in breast cancer.The purpose of this study is to further investigate the effect of PP? with low cytotoxic concentrations on the P-gp function and cell cycle of MCF-7/ADM cells.Methods:1.FCM was used to detect the effect of PP? at low cytotoxic concentrations(0.3,1 and 3?mol/L)on the accumulation of ADM and Rh123 in MCF-7 and MCF-7/ADM cells after 48h treatment.The fluorescence intensity changes were also observed under fluorescence microscope.2.By counting cell numbers with automatic cell counter,the growth curves of MCF-7 and MCF-7/ADM cells were drawn and the doubling time of both cell lines was calculated.3.FCM was used to detect the distribution of cell cycle phase of MCF-7 and MCF-7/ADM cells treated by the effect of 1 and 3 ?mol/L PP? combined with ADM.4.Western blot was used to detect the expression of CyclinDl,CDK4,CDK6,p16,CyclinEl,CDK2,p53,p21,p27,Rb and P-Rb proteins in MCF-7/ADM cells treated with 1 and 3?mol/L PP? combined with ADM for 24 hours.Results:1.The MFI of ADM and Rh 123 in MCF-7 cells was significantly higher than that of MCF-7/ADM cells.1,3 ?mol/L PP? and 20?mol/L VER increased the MFI of ADM in MCF-7/ADM cells in a concentration dependent manner,with the increase folds of 2.00(P<0.05),2.90(P<0.01)and 4.18(P<0.001).Similarly,PP? and VER increased MFI of Rh123 in MCF-7/ADM cells for 1.84(P<0.01),4.04(P<0.001),and 4.92 folds(P<0.001).Notably,the MFI of ADM and Rh123 was significantly weak in MCF-7 cells after PP? treatment.' Under the fluorescence microscope,similar results were observed in MCF-7 and MCF-7/ADM cells.2.Doubling time of MCF-7 cells was about 64 hours,and that of MCF-7/ADM cells was about 47 hours.Compared with MCF-7 cells,MCF-7/ADM cells G0/G1 phase decreased from 73.37%to 67.87%(P<0.05),G2/M phase increased from 18.07%to 22.13%(P<0.05),The proliferation of MCF-7/ADM cells was more vigorous.3.In MCF-7 cells,the proportion of cells in G0/G1 phase,S phase and G2/M phase of control group was73.37%,8.13%and 18.07%,respectively.1?mol/L ADM increased S and G2/M proportion to 39.13%and 31.37%(P<0.001).The ratio of G0/G1 phase by ADM treatment was 28.23%,it was increased to 39.87%(P<0.05)and 48.87%(P<0.001)after PP?(1,3/Pmol/L)combined with ADM.VER combined with ADM increased G0/G1 phase to 46.23%(P<0.01).For MCF-7/ADM cells,the proportion of cells in G0/G1 phase,S phase and G2/M phase in the control group were 67.87%,9.60%and 22.13%,respectively.G0/G1 phase was increased to 73.63%(P<0.05),77.77%(P<0.01)after treatment of 1 and 3?mol/L PP?.The proportion of cells in G0/G1 phase,S phase and G2/M phase of ADM group was 65.80%?1.53%and 22.13%,respectively.1 and 3 ?mol/L PP? combined with ADM increased G0/G1 phase to 75.63%(P<0.05),78.90%(P<0.001),respectively.VER combined with ADM increased S and G2/M phase to 14.07%(P<0.05),and 35.93%(P<0.01).4.Compared with MCF-7 cells,CyclinDl,CDK4,CDK6,p16,CyclinEl and p53 proteins were highly expressed in MCF-7/ADM cells,up-regulation folds were 1.55(P<0.01),1.46(P<0.001),1.42(P<0.01),2.24(P<0.001),2.29(P<0.001)and 9.31(P<0.001).The expression of CDK2,p21,p27 and Rb proteins in MCF-7/ADM cells were all lower than that in MCF-7 cells,the down-regulation folds were 1.73(P<0.01),4.11(P<0.001),6.88(P<0.001)and 2.91(P<0.001),respectively.5.In MCF-7/ADM cells,compared with the control group,expression of CyclinD 1,CDK4,CyclinEl,p16,p53,Rb,P-Rb were decreased with 3?mol/L PP? treatment for 1.33(P<0.01),1.39(P<0.05),1.70(P<0.001),1.51(P<0.01),2.33(P<0.001),14.46(P<0.001)and 4.30 folds(P<0.001),respectively.On the other hand,3 ? mol/L PP? increased the level of p21 and p27 proteins expression to 2.39 times(P<0.001)and 5.80 times(P<0.001).Compared with ADM treatment,when 1,3?mol/L PP? combined with ADM,CyclinD1,CDK4,p16 and P-Rb were down regulated in a concentration dependent manner,and the down-regulated folds of 3?mol/L PP? were 1.25(P<0.05),1.78(P<0.01),1.57(P<0.001)and 9.63(P<0.001),respectively.Simultaneously,p21 and p27 were increased to 3.11 times(P<0.001)and 3.07 times(P<0.001).VER combined with ADM significantly decreased the expression ofp16,p21,p27 and P-Rb,the down-regulation folds were 1.71(P<0.001),2.41(P<0.05),1.85(P<0.05)and 2.14(P<0.01),respectively,however,p53 was slightly up regulated 1.13 times(P<0.05).Conclusions:1.Drug resistance of MCF-7/ADM cells may be caused by the enhancement of P-gp efflux function which decrease ADM accumulation in cells.On the other hand,it may be caused by higher expression of CyclinD1,CDK4,CDK6,p16,CyclinE1 and p53,and lower expression of CDK2,p21,p27 and Rb in MCF-7/ADM cells.These G1/S checkpoint regulators were involved in the regulation of cell cycle,their coordinate effect promote the process of cell cycle,which shortened the doubling time in MCF-7/ADM cells with elevated proportion of G2/M phase and the more vigorous proliferation.2.Low cytotoxic concentration of PP? may increase the intracellular accumulation of chemotherapeutic drugs by inhibiting the efflux function of P-gp,which reversed the chemoresistance of MCF-7/ADM cells.3.Low cytotoxic concentration of PP? alone or in combination with ADM can block MCF-7/ADM cells in the G0/G1 phase,and its internal mechanism may be a result of the suppression of CyclinD 1-CDK4/Rb signal transduction,as well as up regulation of CKI:p21 and p27,which in turn prevented MCF-7/ADM cells crossing G1/S checkpoint.4.ADM inhibited the proliferation of MCF-7 cells by blocking cells in S and G2/M phase,however,because of drug resistance,this was not displayed in MCF-7/ADM cells.PP? combined with ADM exhibited G0/G1 phase blocking effect,which maybe a result of synergism with ADM,nevertheless,the blocking effect of PP? in G0/G1 phase may mask the blocking effect of ADM in S and G2/M phase. |
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