| Objective: To observe the effect of curcumin(CUR)and cisplatin(DDP)on the proliferation,apoptosis,migration,and invasion of neuroblastoma SK-N-SH cells,and preliminarily explore the possible mechanisms with the expression of apoptosis-related genes and patterns under the treatments of CUR and(or)DDP.Methods:(1)According to the protocol,this experiment was divided into 5 groups:Blank control group(Only cells without treatment factors),Solvent control group(0.1% DMSO),CUR group(5?mol/L?10?mol/L?15?mol/L?20?mol/L?25?mol/L?30?mol/L?35?mol/L),DDP group(5?mol/L?7.5?mol/L?10?mol/L),combing group(DDP5?mol/L+CUR 30?mol/L;DDP7.5?mol/L+CUR30?mol/L;DDP10?mol/L+CUR30?mol/ L).When SK-N-SH cells were cultured with different treatment factors for 24 hours,the proliferation of SK-N-SH cells was detected by CCK-8 assay and screened the best joint that was used to next experiment.(2)Experiment was four groups :solvent control group(0.1% DMSO),CUR group(30?mol/L),DDP group(7.5?mol/L),and combing group(DDP7.5?mol/L+CUR30?mol/L).Cell apoptosis were determined by flow cytometry.Real time-PCR was used to detect the Bcl-2 and Caspase-3 mRNA expression,Western blot tested the Bcl-2 and Caspase-3 protein expression.Wound healing assay and transwell invasion assay explored migration and invasion of SK-N-SH cell.Results:(1)The result of CCK-8-8 demonstrated that CUR and DDP were both able to inhibit the proliferation of SK-N-SH cells respectively,and both of CUR and DDP showed a concentration dependence on inhibiting the SK-N-SH cells proliferation.The two drugs combined effect were stronger than single agent.Jin Zheng jun method analyzed that the q values of three groups were from 0.85 to 1.15,which implied only an additive effect in these two drugs.Besides,the best joint was CUR30?mol/L and DDP 7.5 ?mol/L.(2)Flow cytometry detection showed that the apoptosis rates of SK-N-SH cells in the CUR group and the DDP group are higher than solvent control group.Apoptosis rate of combing group was higher than in single agent group.The apoptosis rates of the solvent control group,CUR group,DDP group and combing group were 3.11±0.14%?14.25±0.29%?8.89±0.28%?19.13±0.40%.(3)Real-timePCR result indicated that CUR and DDP can both down-regulate the expression of Bcl-2 mRNA and up-regulate the expression of caspase-3 mRNA.Compareing with single agent groups,combing group was more obvious on regulating these apoptosis-related genes.(4)Western blot demonstrated that the expression of Caspase-3 protein in SK-N-SH cells treated with CUR or DDP up-regulating significantly,while Bcl-2 protein was reduced.Moreover,When CUR combined with DDP,the change of Bcl-2 and Caspase-3 protein expression level were greater.(5)Transwell invasion assay and wound healing assay results showed that CUR and DDP can reduce neuroblastoma SK-N-SH cell migration and invasion.In the wound healing assay,the number of neuroblastoma cell migration distance in solvent control group was significantly greater than other groups.In the transwell invasion experiments,CUR or DDP decreased SK-N-SH cells moving to bottom hole than solvent control group,a more significant effect of two drugs combination than single agent group.Conclusions:(1)CUR and DDP both have the inhibitory effect with concentration dependent to the SK-N-SH cell.And they had additive effect on reducing the proliferation of SK-N-SH cell when they are used together.(2)CUR and DDP can promote SK-N-SH cell apoptosis,and inhibit the proliferation.Two drug combination effect is more obvious.The effect of CUR and DDP to induce the apoptosis of SK-N-SH possibly caused by adjusting the expression of the apoptosis-related genes,such as Bcl-2 and Caspase-3.(3)CUR and DDP can inhibit SK-N-SH cell migration and invasion,combing group than single-agent using even more significant... |
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