| Objectives: To investigate the effect of curcumin(Cur)on the proliferation of human ovarian cancer cell line SKOV3/CDDP in vitro and the expression of apoptosis related proteins Caspase-3,Bcl-2,Bax.Analyzing the possible mechanism of curcumin on SKOV3/CDDP cisplatin resistance reversal,so that we can provide theoretical basis for the application of traditional Chinese medicine in clinical use.Methods:1 SKOV3 cells and its cisplatin resistance cell SKOV3/CDDP were cultured in vitro,MTS assay tested the RI of the SKOV3/CDDP cells after the SKOV3 and SKOV3/CDDP cells was in different concentration of cisplatin(1μg/ml,2μg/ml,4μg/ml,8μg/ml,16μg/ml,32μg/ml,64μg/ml)for 24 h.2 MTS was used to measure the growth curve of the SKOV3/CDDP cells with different concentrations of Cur(0μmol/L,10μmol/L,20μmol/L,40μmol/L,60μmol/L,80μmol/L)at 24 h,48h,72 h.We also use MTS assay to test whether the low toxicity dose of Cur(10μmol/L)has a chemosensitizing effect on SKOV3/CDDP to cisplatin.3 The effect of Cur on apoptosis was measured by flow cytometry.The experiment was divided into the following four groups: 1)contorl group: SKOV3/CDDP cells cultured in DMEM/F12 cell culture medium which only containing 10% fetal bovine serum;2)Cur group: SKOV3/CDDP cells with 10μmol/L Cur;3)CDDP group: SKOV3/CDDP cells with 3.8μg/ml cisplatin;4)Cur+CDDP group: SKOV3/CDDP cells with combination of 10μmol/L Cur and 3.8μg/ml cisplatin.4 Western-Blotting method to detect the protein expression of apoptosis-related protein Caspase-3,Bcl-2,Bax after the deal with the above groups.5 The relative expression of Caspase-3,Bcl-2,Bax mRNA after the deal with the above groups are validated by RT-qPCR.Result:1 The result of SKOV3 and SKOV3/CDDP cells with different concentration of cisplatin 24 h shows that the cell viability of platinum-resistant cell line SKOV3/CDDP was significantly higher than its parental cell line SKOV3(P<0.001).The IC50 of SKOV3/CDDP and SKOV3 were 31.79 and 3.72 respectively.Resistance index(RI)= IC50(SKOV3/CDDP)/ IC50(SKOV3)= 8.54.2 MTS result showed that the inhibition rates of SKOV3/DDP with 0μmol/L、10μmol/L、20μmol/L、40μmol/L、60μmol/L、80μmol/L Cur 24 h were(0.00±6.17)%,(4.82±5.06)%,(23.15±2.56)%,(40.38±3.49)%,(57.88±1.27)%,(59.72±2.11)% respectively(F=242.67,P<0.001).Except for 10μmol/L and control group,60μmol/L and 80μmol/L group was compared no statistically significant,any the other two pairs were statistically significant.The inhibition rates after 48 h were(0.00±6.48)%,(8.56±3.48)%,(35.05±4.09)%,(55.27±4.00)%,(69.42±2.05)%,(80.33±0.95)% respectively(F = 347.66,P <0.001).Any two groups have statistical significance.The inhibition rates after 72 h were(0.00±6.19)%,(23.18±6.49)%,(45.20±3.62)%,(77.07±1.49)%,(79.79±1.52)%,(80.35±1.01)% respectively(F=349.96,P<0.001).In the 40μmol/L,60μmol/L,80μmol/L groups,there was no significant difference between any of the two,and the rest of any two groups both had statistically significant.The cell inhibition effect of curcumin was increased with the increase of drug dosage,and time.When the concentration of curcumin was ≤ 10μmol/L in any role for 24 h,48h,the cell viability rate was more than 90%.It can be considered that curcumin in this concentration has no toxic effect on SKOV3/CDDP cells,and regard the dose as the reversal dose.The MTS result showed that curcumin combination with cisplatin 48 h can significantly suppress the growth of SKOV3/CDDP cells,and enhance the toxic effect of cisplatin compared to use cisplatin alone,meanwhile,the IC50 of SKOV3/CDDP cells decreased from(1.77±0.03)μg/ml to(3.47±0.54)μg/ml(P<0.001).3 FCM showed that the apoptosis rate of SKOV3/CDDP cells in the blank control group,10μmol/L curcumin,3.8μg/ml cisplatin,and the combination group 48 h were(0.02±0.02)%,(4.45±0.83)%,(6.49±2.11)%,(21.31±3.60)%(P =0.01<0.05,P=0.03<0.05,P=0.009<0.01 compared with the control group).Compared with the control group 10μmol/L curcumin or in combination with 3.8μg/ml cisplatin could induce the increase of SKOV3/CDDP cell apoptosis.There was a different between the 10μmol/L curcumin and combination group,the combination group had a higher apoptosis rate(P=0.004<0.01).4 The relative expression of Caspase-3,Bax and Bcl-2 were detected by Western-Blotting method after 48 h of SKOV3/CDDP cells treated with 10 μmol/L Cur and 3.8μg/ml cisplatin alone or in combination.The results showed that the expression of Caspase-3 and Bax in the combination group was significantly higher than that in the control group(P<0.001,P<0.001),and the expression of anti-apoptotic protein Bcl-2 was decreased(P<0.001).Compared to 3.8μg/ml cisplatin group,the combination group decreased the expression of anti-apoptotic protein Bcl-2(P<0.01),and increased the expression of Caspase-3 and Bax(P<0.01,P<0.001).5 RT-qPCR analysis showed that SKOV3/CDDP cells treated with 10 μmol/L Cur and 3.8μg/ml cisplatin alone or in combination 48 h,the combination drug compared to control group can obviously up-regulate the transcriptional level of Caspase-3,Bax(P<0.001,P<0.01),simultaneously,down-regulate the transcriptional level of Bcl-2(P<0.001).The combination group can also increase the relative expression of Caspase-3,Bax mRNA(P<0.001,P<0.01),and decrease Bcl-2 mRNA(P<0.01).Conclusion:1 Curcumin has obvious inhibitory function on human cisplatin resistance ovarian caner cell lines SKOV3/CDDP,and its inhibition function has a dosage and time dependence.2 Curcumin in low cytotoxicity concentration can sensitize the chemotherapy of SKOV3/CDDP cells,and enhance the cytotoxicity of cisplatin.3 Curcumin has the ability to induce the apoptosis of SKOV3/CDDP cells,and it play a better results when combined with chemotherapeutic drug cisplatin.4 Curcumin in combination with cisplatin can significantly up-regulate the expression of apoptosis relative protein Caspase-3,Bax,meanwhile,down-regulate the expression of anti-apoptosis protein Bcl-2 both in mRNA and protein levels,so we think the mechanism of curcumin on antitumor and reversing drug-resistance may by means of inducing and enhancing the effection the apoptosis. |
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