Inhibition of Autophagy Gene Beclin1 Enhances the Drug Sensitivity of Human Myeloid Leukemia Resistant Cell K562/IMA to Imatinib / 中国药学杂志

ABSTRACT

OBJECTIVE: To investigate the effect of autophagy gene Beclin1 silencing on enhancing the drug sensitivity of imatinib-resistant human myeloid leukemia cell line K562/IMA. METHODS: Imatinib-resistant human myeloid leukemia cell line K562/IMA was constructed by the combination of the first dose of high-dose shock therapy and gradual increase of dose. Imatinib interfered with K562 and K562/IMA cell lines. Cell viability was detected by CCK-8. Apoptosis rate was detected by flow cytometry. Western-blot assay was used to determine the drug resistance level. The expression of Beclin1 in drug resistant strains was detected. Small interfering RNA (siRNA) transfected Beclin1 siRNA-Beclin1 was used to silence the Beclin1 gene of K562/IMA. Control group (control), siRNA negative control group (siRNA-NC) and Beclin1 siRNA group (siRNA-Beclin1) were set up. RT-QPCR and Western-blot were used to identify the expression level of Beclin1 protein and mRNA in each group after silencing. CCK-8 method was used to detect the sensitivity of each group of cells to imatinib, and the half inhibitory concentration IC50 was calculated. The apoptosis rate of each group was detected by flow cytometry. The expression levels of Beclin 1, Bcl-2, Bax, cleaved-caspase 3, caspase-3 and cytochrome C(cyto-C) were detected by Western-blot assay, and the metastatic and invasive ability of cells was detected by Transwell chamber. RESULTS: K562 / IMA cell lines were resistant to imatinib at 10 μmol•L-1, and the expression of Beclin1 was higher than that of K562 cell lines. The cell viability of K562/IMA was significantly higher than that of K562 cell lines in the gradient concentration range, while the apoptosis rate was lower than that of K562 cell lines. After siRNA silencing Beclin-1, the cell viability and apoptosis rate of siRNA-Beclin 1 group were significantly lower than those of control group, and the IC50 value of half inhibitory concentration was significantly lower than that of control group. The levels of Bax, cleaved-caspase 3, caspase-3 and cyto-C in the cells were significantly higher than those in the control group, while the levels of Bcl-2 were significantly lower than those in the control group. CONCLUSION: Beclin1 plays an important role in imatinib resistance in human myeloid leukemia, and autophagy may be involved in the drug resistance process. After silencing Beclin1, cell resistance level can be reduced and drug sensitivity improved.