| 【Purpose】The aim of the study was to investigate the effects of cisplatin combined with PD-0332991 on human lung cells A549 and the possibility of reversing the drug resistance,and to discuss the synergistic effect of the drug combination.【Method】1.Firstly,we detected the inhibitory effects of cisplatin and PD-0332991 on the proliferation of human lung cancer cell A549 and cisplatin-resistant cell A549/CDDP by CCK-8 assay.A459 and A459/CDDP cells were treated with cisplatin(0,1,2,4,8,16,32,64,128,256μg/ml)and PD-0332991(0,2.5,10,20,40,80,160μM/L)for 24 hours,then add 10%CCK-8 reagent,measure the absorbance and calculate the IC50 of each group.Then calculated the resistance index.2.Secondly,A549 cells and A549/CDDP cells were treated with different concentrations of cisplatin(0,1,2,4,8,16,32,64,128,256μg/ml)combined with PD-0332991 of 1/2*IC50 concentration for 24 hours,respectively.Calculated the inhibition rate and IC50 of cisplatin on the two kinds of cells in combination of the two drugs,obtain the cisplatin resistance index after adding PD-0332991.By comparing the changes of IC50 of drug-resistant cells before and after dosing PD-0332991.We can see whether PD-0332991 has reversal effect of drug resistance.Then calculate the combined effect index of cisplatin and PD-0332991 to determine whether the two drugs are synergistic.3.Cell flow assay single staining(PI)was used to detect the effect of PD-0332991 and cisplatin on the distribution of human lung adenocarcinoma cell cycle.Then,A549 and A549/CDDP cells were treated with 1/2*IC50 concentration of PD-0332991,1/2*IC50 concentration of cisplatin,drug combination and normal medium for 24 hours.The apoptosis of cells was detected by Annexin V/PI double staining.Observe whether the drug promotes apoptosis when used in combination.4.After the treatment of A549 cells with 1/2*IC50 concentration of PD-0332991,1/2*IC50 concentration of cisplatin,drug combination and normal medium for 24 hours.Western blot was used to detect cell cycle associated protein Rb,Phospho-Rb,CDK-4,CDK-6,E2F1,Cyclin D1 and the expression level of apoptosis-related protein Caspase-3 and bcl-2,and discuss the effects of combination therapy on cell cycle and apoptosis-related protein expression.【Result】PD-0332991 can reduce the activity of human lung cells and inhibit its proliferation;when combined with cisplatin,it can produce synergistic effects of drugs,and the inhibition of tumor cells is significantly enhanced.Acting on cisplatin-resistant cells can enhance the virulence of cells and reduce drug resistance to some extent.PD-0332991 combined with cisplatin treatment of lung adenocarcinoma cells,nuclear destruction and apoptosis increased,but as a cell cycle inhibitor,western blot result showed the apoptosis-related path Caspase was activated in drug-resistance cell,and the expression of anti-apoptosis protein Bcl-2 was inhibited.PD-0332991 inhibition of cell cycle was reduced after combined with cisplatin.Western blotting indicated that the expression of the protein in the Rb group was reduced after drug combination,but the expression of CDK-4/6 and Cyclin D1 was enhanced.【Conclusion】PD-0332991 can reduce the activity of human lung cancer cells,inhibit the cell cycle and arrest it in G1 phase;when combined with cisplatin,it can produce drug synergistic effect on lung adenocarcinoma cells A549 and A549/CDDP;and treated with PD-0332991 The drug resistance index of cisplatin-resistant cells was lower than before,and there may be some reversal of drug resistance.After PD-0332991 was combined with cisplatin,the proportion of apoptosis of lung adenocarcinoma cells increased.In addition,cisplatin may reduce the inhibition of PD-0332991 on the cell cycle and enhance the expression of some cyclins. |
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