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Objective: Tetrandrine(Tet), a bisbenzylisoquinoline albaloid isolated from the Chinese herb“Hanfangji”(Radix Stephania Tetrandra), could effectively reverse P-glycoprotein mediated multidrug resistance. This study to evalue the revese effect of Tet on cisplanin resistance hunman ovarian cell line SKOV3/DDP in vitro, and to study the possible reverse mechanism by detecting the expression of the multidrug-resistance genes--MDR-1 and LRP, then to provide the theory evidence for Tet in clinical therapy.Method:1 SKOV3 and SKOV3/DDP cells were incubated in culture medium in vitro, treated with DDP at different concentrations and detected cell’s proliferation inhibitory rate by MTT assays,in order to calculated the 50% inhibitory rate(IC50) of the two cell lines and then calculate the resistance index of SKOV3/DDP.2 The cytotoxic effect of Tet on SKOV3 /DDP cells was examined by MTT assays and the non-cytoxic dose(cell’s proliferation inhibitory rate<10%) of Tet was identified.3 The changes of DDP resistance was observed after SKOV3/DDP cells were treated with non-cytoxic dose of Tet by MTT assays,then calculated the reverse fold.4 The mRNA expression of MDR-1 and LRP were determined by reverse transcription Polymerase chain reaction (RT-PCR) at 0h, 24h, 48h, 72h after non-cytoxic dose of Tet intervention.5 The expression of P-gp and LRP protein were examined by FCM at 0h, 24h, 48h, 72h after non-cytoxic dose of Tet intervention. 6 Apoptosis and distribution of cell cycle were observed by flow cytometry(FCM) after SKOV3/DDP cells was treated with DDP combinated with or without non-cytoxic dose of Tet.Results:1 MTT assay showed that the IC50 of DDP in SKOV3 cells was2.37μg/ml and in SKOV3/DDP cells was 6.24μg/ml. The resistance index was 2.63.2 2μmol/L Tet or even below than 2μmol/L dose of Tet showed not evident inhibitory effect on the growth of SKOV3/DDP cells(cell’s proliferation inhibitory rate<10%), and 2μmol/L was chosen for the non-cytoxic dose of Tet.3 MTT assay showed that IC50 of DDP in SKOV3/DDP cells decreased from 6.24μg/ml to 3.89μg/ml after 2μmol/L Tet intervention for 48h, reverse fold was 1.60.4 RT-PCR assay showed that after added 2μmol/L Tet for 24h, 48h, 72h, the relative expressions of MDR-1 mRNA in SKOV3/DDP cells were 0.221±0.018, 0.074±0.013, 0.073±0.011, There was statistically significant difference compared with contrl group(0.778±0.017), respectively(P<0.05). but there was not statistically significant difference when 72h group compared with 48h group(P>0.05). After added 2μmol/L Tet for 24h, 48h, 72h, the relative expressions of LRP mRNA were 0.789±0.012, 0.680±0.014, 0.544±0.025, there was statistically significant difference when 48h group and 72h group compared with contrl group(0.777±0.016), respectively(P<0.05). But there was not statistically significant difference when 24h group compared with control group(P>0.05).5 After added 2μmol/L Tet for 24h, 48h, 72h, the fluorescence intensity of P-gp in SKOV3/DDP cells were 0.843±0.012, 0.811±0.008, 0.806±0.010, there was statistically significant difference when 48h group and 72h group compared with 24h group(P<0.05), but there was not statistically significant difference when 72h group compared with 48h group(P>0.05). After added 2μmol/L Tet for 24h, 48h, 72h, the fluorescence intensity of LRP in SKOV3/DDP cells were 0.992±0.003, 0.964±0.002, 0.955±0.002, there was statistically significant difference between each two groups(P<0.05).6 Cell apoptosis was observed by flow cytometry: The apoptotic percentage(%) in DDP group,Tet group,Tet +DDP group was 24.607±1.201、0.963±0.495、32.660±1.247, respectively. There was statistically significant difference in DDP group and Tet+DDP group compared with control group(0.511±0.353), and between each other(P<0.05). There was not statistically significant difference when Tet group compared with control group(P>0.05).7 Distribution of cell cycle was observed by flow cytometry: In control group, DDP group and Tet+ DDP group,the number of cells in G0/G1 phase were 58.175±0.982, 58.400±0.637, 12.663±0.978, the number of cells in S phase were 21.088±1.722, 28.950±1.334, 86.426±0.972, the number of cells in G2/M phase were 20.738±1.380, 12.650±1.097, 0.912±0.082. In DDP group, the number of cells increased in S phase while decreased in G2/M phase compared with the control group, there was statistically significant difference(P<0.05).In Tet+DDP group,the number of cells increased in S phase while decreased in G2/M and G0/G1 phase comparede with DDP group, there was statistically significant difference(P<0.01). In Tet group, there was not statistically significant difference compared with control group(P>0.05).Conclusion:1 Tet in certain range of concentration could inhibit SKOV3/DDP cells’growth in vitro,and the inhibitory rate increased as the concentration of Tet increased.2 The mechanism of non-cytoxicty Tet partly reverse SKOV3/DDP cells’drug resistance may be related with downning regulation of MDR-1 and LPR’s expession.3 Non-cytotoxic concentration of Tet could enhanced the effect of DDP on SKOV3/DDP cells, it induced an apoptosis and an arrest of cell cycle in S phase to inhibit the cells’proliferation. 4 Tet could as an effectual MDR reverse agent for ovarian cancer’s treatment, but it still need further study to interpret the reverse mechanism.
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