| Objectives:Our previous study demonstrated that Polyphyllin I(PPI)obviously inhibited growth of MCF-7 and MCF-7/ADM cells.This study was designed to test the effect of PPI on reversing resistance of MCF-7/ADM cells and to explore the mechanism of action in vitro.Hemolytic activity of PPI was examined to understand its toxicity.Methods1.The modified MTT assay was used to detect the multidrug resistance of MCF-7/ADM cells to ADM,DDP and 5-FU.The low cytotoxic concentrations of PPI were used to reverse MCF-7/ADM cells to ADM,DDP and 5-FU.MAPK signaling pathway inhibitors were also examined for their reversal effect on MCF-7/ADM cells to ADM.2.Flow cytometry was used to detect the effect of PPI combined with ADM on apoptosis of MCF-7/ADM cells.3.The effect of PPI on ADM accumulation in MCF-7/ADM cells was detected by Fluorescence Spectrophotometry.The fluorescence intensity of ADM was observed by fluorescence microscope.4.In order to confirm the difference between MCF-7/ADM and MCF-7 cells at molecular level,the expression of ABCB1,ABCC1 and ABCG2 genes were detected by qPCR,and the expression of MAPK signal pathway-related proteins and P-gp protein were detected by western blot.5.Western blot method was used to detect the effect of PPI on the expression of MAPK signaling pathway proteins ERK,P-ERK,JNK,P-JNK,p38,P-p38,and on MCF-7/ADM cells treated for 24 and 48 h,and the effect of MAPK signaling pathway inhibitors on the expression of these proteins.6.The effect of PPI on the recovery rate of fluorescence bleaching on MCF-7/ADM cells was investigated by fluorescence bleaching recovery technology and laser confocal microscopy,in order to understand the effect of PPI on the fluidity of cell membrane.7.The hemolytic effect of PPI was determined by colorimetric method.Results1.MCF-7/ADM cells were resistant to ADM,DDP and 5-FU,with resistance index of 304.43,2.00 and 7.63,respectively.2.PPI 0.3,1,3 μM(low cytotoxic concentrations)and VER 10 μM combined with different concentrations of ADM,DDP and 5-FU,reversed resistance of MCF-7/ADM cells with RF of 4.67,2.30,32.73 and 29,83 to ADM;with RF of 1.42,2.32,4.66 and 2.29 to DDP;with RF of 2.23,2.69,121.32 and 1.25 to 5-FU.RF of p38 pathway inhibitor SB203580 10,20,40 and 80 μM to ADM was 1.96,3.51,4.64 and 4.77;RF of JNK pathway inhibitor SP600125 10,20 and 40 μM to ADM was 1.25,1.86 and 1.82;RF of ERK pathway inhibitor U0126 10,20,40 and 80 μM to ADM was 0.82,0.61,2.25 and 1.60.3.The apoptotic rate of MCF-7 and MCF-7/ADM cells treated with ADM for 48 hours was 85.13(P<0.01)and 9.40%,respectively.For MCF-7/ADM cells,the apoptotic rate increased to 18.17%(P<0.05)after treatment of PPI 3 μM，the apoptotic rate increased to 25.87%(P<0.01)when treated by PPI and ADM in combination,the apoptotic rate was 24.03%(P<0.01)when treated by VER and ADM in combination.4.After incubation with ADM for 3 h，the fluorescence intensity of ADM in MCF-7 cells was 359.63,which was significantly higher than that in MCF-7/ADM cells 56.34(P<0.01).After treatment with PPI 15 and 45 μM,ADM accumulation was increased in MCF-7/ADM cells with fluorescence intensity of 136.08(P<0.01)and 209.63(P<0.01),respectively.After treatment with VER 10 μM,the fluorescence intensity increased to 66.28(P<0.01).5.Compared with MCF-7 cells,ABCB1,ABCC1 and ABCG2 genes were highly expressed in MCF-7/ADM cells,with their relative expression levels of 4862.931(P<0.001),1.524(P<0.01)and 135.96(P<0.001)3 respectively.The relative expression levels of P-gp，P-p38 and P-ERK proteins in MCF-7 cells were 0.19,0.61 and 0.62,which were significantly lower than those in MCF-7/ADM cells:3.23(P<0.001),1.40(P<0.001)and 1.18(P<0.01).6.The relative expression of ERK protein was up-regulated from 1.53 to 2.40(P<0.05)by treatment of PPI 3μM for 24 h.Relative expression of P-JNK protein was down-regulated from 1.90 to 0.87(P<0.01).).After 24 and 48 h treatment of PPI combined with ADM,the expression of P-gp protein was down-regulated from 3.01 and 2.72 to 2.18(P<0.05)and 1.96(P<0.001),expression of P-ERK protein was down-regulated from 1.94 and 1.29 to 1.08 and 0.91(both P<0.05),expression of P-JNK protein was down-regulated from 1.95 and 1.68 to 1.37(P<0.05)and 0.7(P<0.001).After MAPK signaling pathway inhibitors treatment on MCF-7/ADM cells for 48 h,compared with control group,ERK pathway inhibitor U0126 40 μM decreased expression of P-ERK and P-gp protein from 1.56 and 2.61 to 1.17 and 2.15(both P<0.05);JNK pathway inhibitor SP600125 20 μM,suppressed expression of P-JNK and P-gp protein from 1.87 and 2.64 to 1.45 and 2.12(both P<0.05);p38 pathway inhibitor SB203580 20 μM down-regulated expression of P-p38 and P-gp protein from 1.27 and 2.91 to 0.84 and 2.46(both P<0.01).7.The recovery rate after fluorescent bleaching of MCF-7/ADM cells was 43.52%,which was significantly higher than that of MCF-7 cells(33.14%,P<0.05).After treatment of PPI 10 μM for 1 h,the fluorescence bleaching recovery rate of MCF-7/ADM cells was decreased to 26.80%(P<0.01),suggesting that membrane fluidity was decreased.8.PPI showed strong hemolytic activity,with ED50 of 4.26 μM.Conclusions1.MCF-7/ADM cells are resistant to chemotherapeutic drugs ADM,DDP and 5-FU,and are highly resistant to ADM,moderately resistant to 5-FU,and low-resistance to DDP.Mechanism of resistance may be:high expression of membranous transporter protein P-gp,low accumulation of drugs in cells,increased activation of p38MAPK and ERK signaling pathways,and elevated cell membranous fluidity.2.Low cytotoxicity of PPI(0.3,1,3 μM)reversed the resistance of MCF-7/ADM to three chemotherapeutic drugs.The reversal effect of 3 μM PPI was stronger than that of VER or comparable to VER.The reversal mechanism of PPI may be:increasing the accumulation of drugs in the cells,directly or indirectly down-regulating P-gp protein,inhibiting the activation of JNK,ERK signaling pathway,and reducing membranous fluidity of drug-resistant cells.3,PPI 3 μM combined with ADM induced apoptosis of MCF-7/ADM cells,but no obvious difference was observed compared with action of PPI alone,indicating that reversal mechanism of PPI may not be achieved by induction of apoptosis.4.PPI showed strong hemolytic activity in vitro. |
