| Objective:Acute lymphoblastic leukemia（ALL）is the most common malignant tumor in children,accounting for almost one-third of paediatric malignancies.Glucocorticoid（GC）resistance is still a challenge for children with ALL.Traditional Chinese medicine has the effects of anti-tumor cell proliferation and promoting apoptosis,and can be used as an auxiliary drug in chemotherapy.Salidroside（SAL）has anti-tumor and enhanced tumor chemotherapy sensitivity,but there is no report on the anti-leukemia effect of SAL.Therefore,this experiment aims to explore the effects of SAL on the proliferation,apoptosis and autophagy of human T-ALL cell lines CEM-C7 and CEM-C1 cells and whether it can enhance GC-resistant CEM-C1 cells against Dexamethasone（DEX）sensitivity provides a new treatment strategy for overcoming the drug resistance of leukemia cells.Methods:1.Cell culture:Human T-ALL cell line CEM-C7 and CEM-C1 cells are cultured in vitro.2.Experimental grouping:（1）CEM-C7 and CEM-C1 cells were divided into 5 groups according to the intervention concentration of SAL（5.0 mg/ml,7.5 mg/ml,10.0 mg/ml,12.5 mg/ml and 15.0 mg/ml）;（2）CEM-C7 and CEM-C1 cells were divided into 5 groups according to the intervention concentration of DEX（25μg/ml,50μg/ml,100μg/ml,150μg/ml and 200μg/ml）;and DEX at different concentrations（25μg/ml,50μg/ml,100μg/ml,150μg/ml and 200μg/ml）combined with SAL（1.5 mg/ml）to divide CEM-C7 and CEM-C1 cells into 5 groups;（3）According to whether T-ALL cell line CEM-C7 and drug-resistant cell line CEM-C1 had been treated with SAL optimized concentration（0 mg/ml,5.0 mg/ml,7.5 mg/ml,10.0 mg/ml）,they were divided into 4 groups;（4）CEM-C1 cells are divided into control group,SAL group（1.5 mg/ml）,DEX group（100μg/ml）and combination group（DEX100μg/ml+SAL 1.5mg/ml）according to whether SAL is combined with DEX.3.Cell counting kit-8（CCK-8）:（1）To detect the changes in the proliferation of CEM-C7 and CEM-C1 cells after 24h,48h,72h treatment with different concentrations of SAL;（2）Detect the changes in the proliferation of cells in each group after treatment with different concentrations of DEX and combined with SAL;（3）Simultaneously determine the intervention concentration and intervention time of SAL and the IC50 for CEM-C1 and CEM-C7 cells,and calculate the drug resistance multiple,reversal multiple and relative reversal rate.4.Cell apoptosis:Flow cytometry was used to detect the apoptosis of the CEM-C1 and CEM-C7 at different concentrations（0 mg/ml,5.0 mg/ml,7.5mg/ml,10.0 mg/ml）SAL intervention for 48 hours;and whether SAL combined with DEX treatment of CEM-C1 cell apoptosis in each group.5.Cell cycle:The cell cycle changes of CEM-C7 and CEM-C1 were detected by flow cytometry at different concentrations（0 mg/ml,5.0 mg/ml,7.5mg/ml,10.0 mg/ml）SAL intervention for 48 hours.6.Quantitative real-time PCR:The expression of C-MYC and LC3 m RNA in CEM-C7 and CEM-C1 cells at different concentrations（0 mg/ml,5.0 mg/ml,7.5 mg/ml,10.0 mg/ml）was detected after 48 hours of SAL intervention.7.Acridine Orange staining:To detect the autophagy of CEM-C7 and CEM-C1 cells at different concentrations（0 mg/ml,5.0 mg/ml,7.5 mg/ml,10.0mg/ml）SAL intervention for 48 hours.8.Western Blot:（1）Detection of C-MYC protein expression in CEM-C7and CEM-C1 cells;（2）Detect the expression of CEM-C7 and CEM-C1 cell apoptosis-related proteins,autophagy-related proteins and C-MYC proteins at different concentrations（0 mg/ml,5.0 mg/ml,7.5 mg/ml,10.0 mg/ml）SAL intervention for 48 hours;（3）Detect the expression of apoptosis proteins,autophagy protein and C-MYC protein in each group after SAL combined with DEX treatment of CEM-C1 cells.Results:1.SAL inhibited the proliferation of T-ALL cells:（1）SAL inhibited the proliferation of CEM-C7 and CEM-C71 cells in a dose-dependent manner;（2）The 48h IC50 of SAL on CEM-C7 and CEM-C1 cells were 8.03 mg/ml and 6.69 mg/ml,respectively.2.SAL promoted apoptosis of T-ALL cells:（1）Flow cytometry showed that with the increase of SAL concentration,the apoptosis rate increased significantly（P<0.001）;（2）SAL could induce cell apoptosis,accompanied by down-regulation of BCL-2 protein and up-regulation of Cleaved-PARP and Bax protein（P<0.01）;（3）Compared with the DEX group alone,the combination group（SAL+DEX）could more significantly induce GC-resistant CEM-C1 cell apoptosis（P<0.01）.3.SAL blocked CEM-C7 cells in S phase（P<0.01）,but had no effect on CEM-C1 cell cycle（P>0.05）.4.SAL promoted autophagy in T-ALL cells:（1）Acridine orange staining detected that SAL promoted autophagy in ALL cells;（2）SAL could promote autophagy in CEM-C7 and CEM-C1 cells,accompanied by increased LC3 gene and protein levels（P<0.001）;（3）Compared with the DEX alone group,the combination group（SAL+DEX）could more significantly induce autophagy in GC-resistant CEM-C1 cells（P<0.001）.5.The reversal effect of SAL on CEM-C1 cell resistance:（1）After 48 hours of intervention with different concentrations of DEX,the IC50 of CEM-C1 cells was（111.83±2.87）μg/ml,the IC50 of CEM-C7 cells was（0.67±0.02）μg/ml,and the resistance index was 166.92 times;（2）After SAL combined with DEX,the IC50 of CEM-C1 cells to DEX dropped to（35.59±3.73）μg/ml,the reversal fold was 3.14（t=16.21,P=0.000）,and the relative reverse rate was 0.69.It showed that the resistance of SAL（1.5mg/ml）to CEM-C1 was partially reversed.（3）Compared with the DEX group,the combined group（SAL+DEX）had a significant increase in the inhibition rate of CEM-C1 cells（P<0.001）,and the coefficient of drug interactionwas<l.6.SAL down-regulated C-MYC m RNA and protein expression in CEM-C1 resistant cells:（1）Compared with CEM-C7 cells,the expression of C-MYC protein in CEM-C1 cells increased significantly（t=12.75,P=0.000）;（2）SAL down-regulated C-MYC gene and protein levels in a dose-dependent manner（P=0.000）;（3）Compared with the DEX alone group,the combination group（SAL+DEX）could down-regulate the expression of C-MYC in GC-resistant CEM-C1 cells more significantly（P<0.01）.Conclusion:1.SAL can significantly inhibit the proliferation of CEM-C7 and CEM-C1cells,promote leukemia cells apoptosis,and induce autophagy.2.SAL can enhance the sensitivity of CEM-C1 cells to DEX and partially reverse the resistance of CEM-C1 cells to GC.The mechanism may be related to the down-regulation of C-MYC. |
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