| Objective This present research aims to study the inhibition of proliferation, invasion, migration and induction of apoptosis in nasopharyngeal carcinoma cells by aqueous extract of Yiqi Jiedu formula combined with Salinomycin, and explore the might mechanism of this proliferation inhibition.Methods 1. MTT assay was employed to investigate the viability of nasopharyngeal carcinoma CNE-2, 5-8F, 6-10 B cells, which is treated by aqueous extract of Yiqijiedu formula(YQ) and salinomycin(SAL) in different concentrations alone and in company for 72 hours. The morphological changes of different treated groupswere also observed under the microscope.2. The cell colony formation assay was used to detect the cloning efficiency of nasopharyngeal carcinoma cell lines CNE-2, 5-8F and 6-10 B, which were treated with different concentrations of YQ and SAL alone and in company for 14 days.3. Use the mitochondrial membrane potential assay kit(JC-1) to study the change of mitochondrial membrane potential of nasopharyngeal carcinoma cell lines CNE-2 and 5-8F, which weretreated with different concentrations of YQ and SAL alone and in company for 48 hours respectively, by detecting and the red/green cell fluorescence ratio, and observe the effects of apoptosis induction of medicines.4. The wound healing assay and vitro Transwell migration assay was employed to detect the migration capacity of nasopharyngeal carcinoma cell lines CNE-2 and 5-8F, which were treated bydifferent concentrations of YQ and SAL alone and in company for 24 hours.Use the vitro Transwell invasion assayto study the effects of different concentrations of YQ and SALalone and in company on the invasion ability of nasopharyngeal carcinoma cell lines CNE-2 and 5-8F, 24 hoursafterintervention,by counting the number of cells which has penetrated the membrane matrixand invade in the under chamber side of bottom membrane of the Tanswell.5. Flow cytometrywas used to analyze cell cycle of nasopharyngeal carcinoma cell lines CNE-2 and 5-8F which were treated by different concentrations of YQ and SAL alone and in company for 48 hours.6. Western Blot test was employed to detect the expression of cycle related proteins such as Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6, P21 and P15 in the CNE-2 and 5-8F cell linestreated withdifferent concentrations of YQ and SAL alone and in companyfor 48 hours.Results 1. YQ in combination with SAL inhibits proliferation and induces apoptosis of nasopharyngeal carcinoma cells.(1)Compared with the solvent control group, the cell viability of both YQalone and SAL alone treated group were significantly decreased, the differences were statistically significant(P<0.01). YQ(0.625mg/m L, 1.25mg/m L, 2.5mg/m L) were combined with 1.25?mol/L salinomycin(the cell viability were 37.1%, 16.4% and 12.8% respectively), 2.5?mol/L salinomycin(the cell viability were 38.7 %, 23.9% and 17.7% respectively) and 5.0?mol / L salinomycin(the cell viability were 27.3%, 14.5%, 13.5% respectively), the cell viability decreased significantly, the differences among them were statistically significant(P<0.01).(2) The number of cloning colonies and colony formation rate were significantly reduced after treated with 0.5?mol/L SAL alone in CNE-2, 5-8F and 6-10 B cells(colony formation rate were 70%,60% and 50%)compared with solvent control group(all P<0.01). Compared with the solvent control group, CNE-2 cells(colony formation rate: 80.6% and 62%) intervened by 0.05mg/m L,0.1mg/m L YQ alone, 5-8F cells(colony formation rate: 56% and 15%) and 6-10 B cells(colony formation rate: 80% and 25%) intervened by 0.025mg/m L,0.05mg/m L YQ alone, the colony formation rate decreased, the differences among them were statistically significant(all P<0.01). Compared with the solvent control group, YQ and SAL alone group, the cell colony formation rate of CNE-2(48% and 35%), 5-8F(5% and 2%) and 6-10B(3% and 2%) treated with 0.5?mol/L SAL combined with YQ(CNE-2 cells were treated with 0.05mg/m L,0.1mg/m L YQ, 5-8F and 6-10 D cells were treated with 0.025mg/m L, 0.05mg/m L YQ) were significantly reduced, the differences among them were statistically significant(all P< 0.01).(3)Apoptosis was detected that: Compared to the solvent control group, YQ and SAL alone can significantly reduce the CNE-2 and 5-8F mitochondrial membrane potential, the differences were statistically significant(P<0.01). The combination treatment of YQ(1.25, 2.5mg/ml) and SAL(1.25?mol/L) can significantly reduce the mitochondrial membrane potential, compared to the solvent control(4.92) and SAL alone treated group(3.59) in CNE-2(ratio of red/green fluorescence reduced to 0.68 and 0.47). The combination treatment of YQ(1.0, 2.0mg/ml) and SAL(1.25?mol/L) can significantly reduce the mitochondrial membrane potential, compared to the solvent control(4.92) and SAL alone treated group(3.59) in 5-8F cells(ratio of red/green fluorescence reduced to 0.58 and 0.36). There were significantly different(P< 0.01).2. YQ in combination with SAL inhibits nasopharyngeal carcinoma cells from migration and invasion.(1)24 hours later after scratched, CNE-2 and 5-8F cells could migrate rapidly, especially the CNE-2 cells, of which the scratch area were almost filled 24 hours later. In contrast to the solvent control group, both single YQ and SALtreated group can significantly inhibited cell migration(the cell migration rate were significantly reduced), the difference was statistically significant(P<0.01). The cell migration rate of CNE-2(16.9% and 11.9%) and 5-8F(16.5% and 6.6%) in YQ(1.25mg/m L,2.5mg/m L to CNE-2, 1.0mg/m L,2.0mg/m L to 5-8F) in combination with 1.25?mol/L SAL treated group were significantly reduced than those of the single SAL group(CNE-2: 31.8%, 5-8F: 35.2%) and the solvent control group(P<0.01). Moreover, it's easily to found that, the higher the concentration of YQ, the stronger the migration inhibition effect.(2)The cell migration rate and number of migrated CNE-2 and 5-8F cells relative to the control group, were significantly decreased, both in the SAL and YQ single treated groups. The differences were statistically significant(P<0.01). Combination treatment of 1.25?mol/L SAL and YQ(1.25mg/m L, 2.5mg/m L to CNE-2, 1.0mg/m L, 2.0mg/m L to 5-8F) high concentration and low concentration significantly reduced cell migration rate of CNE-2(22.1% and 12.7%) and 5-8F(19.5% and 11.7%), in contrast with single SAL group(CNE-2: 45.6%, 5-8F: 50.8%) and the solvent control group. And with the YQ concentration increased, the migration inhibition strengthened, indicates a significant a concentration-dependent manner.(3)The cell invasion rate and number of invaded CNE-2 cells, relative to the control group, were significantly decreased, both in the SAL and YQ single treated groups. The differences were statistically significant(P<0.01); Combination treatment of 1.25?mol/L SAL and YQ(1.25mg/m L, 2.5mg/m L to CNE-2, 1.0mg/m L, 2.0mg/m L to 5-8F) significantly reduced invasion rate of CNE-2(21.9%), in comparison with the single SAL group(46.6%)and single YQ(43.8%) group(P< 0.01).3. The mechanism of combination treatment of YQ and SAL inhibits proliferation in nasopharyngeal carcinoma cells.(1)Flow cytometry results showed that with YQ concentrations increased, there was an decrease in the number of cells in G0/G1 phase, as well as an increase in the number of cells in S and G2 phase in the group treated with YQ alone. However, the cell cycle of SAL single treated group did not change much. In the groupstreated by YQ combined with 1.25?mol/L SAL, the cells in G0/G1 phase were decreased, while the cells in S phase, G2 and M phase were increased.(2)Several cell cycle regulatory related protein were analyzed in CNE-2 and 5-8F cells by western blot, the result shows that the expression of protein Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6 and P21 were reduced slightly in SAL single treated group. YQ individual treated group, the expression of Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6 and P21 were down-regulated, while the expression of P15 were increased, and both of them are in a significant concentration-dependent manner. Moreover, the combination treatment of SAL and YQ had down-regulated the expression of Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6, P21 and up-regulated the expression P15 more significantly.Conclusions Aqueous extract of Yiqi Jiedu formula combined with Salinomycinco-inhibits proliferation, migration and induce apoptosis of nasopharyngeal carcinoma cells. Aqueous extract of Yiqi Jiedu formula combined with Salinomycininhibit cell proliferation of nasopharyngeal carcinoma cells through affecting the expression of cell cycle related proteins such as Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6, P21, P15, and then blocking cell cycle and inducing apoptosis. |
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