Molecular mechanism for apoptosis of leukemia cells induced by chromosome region maintenance 1 selective inhibitor KPT-330

Abstract

Objective To investigate the induction effect and molecular mechanisms of chromosome region maintenance 1(CRM1) selective inhibitor KPT-330 on apoptosis of human leukemia cells. Methods Human acute promyelocytic leukemia cells(NB4), human chronic myelogenous leukemia cells (K562) and human promyelocytic leukemia cells(HL-60) were divided into a control group(without KPT-330 treatment) and KPT-330 treatment group(NB4, K562 and HL-60 were treated with 0.01, 0.10, 0.20, 0.50, 1.00, 5.00, 10.00 μmol/L KPT-330, respectively). Cell counting kit-8 assay was used to quantify the growth inhibition of cells after exposure to KPT-330.Fluorescence microscope was used to observe the morphological change in leukemia cells when treated with KPT-330.Annexin V/propidium iodide staining followed by flow cytometry was used to detect the apoptosis of leukemia cells.Then, Western blot was used to detect the expression level of CRM1 and the activation of apoptosis marker cysteinyl aspartate specific proteinase-9(Caspase-9), cysteinyl aspartate specific proteinase-3(Caspase-3) and poly ADP-ribose polymerase(PARP). Long non-coding RNA(lncRNA) microarray was used to analyze the differentially expressed genes differentially by the cluster analysis. Results The inhibition of cell proliferation of leukemia cells was in a dose-dependent manner with KPT-330 treatment.Expression of CRM1 was also down-regulated when leukemia cells were treated with KPT-330.Compared with the control group, abnormal cells were observed under fluorescence microscopy.Much more cells showed apoptotic feature after being treated with 0.20 μmol/L and 1.00 μmol/L KPT-330, the apoptosis rate of NB4, K562 and HL-60 were respectively(4.95±0.21)%, (14.05±0.07)%, (4.95±0.63)%(0.20 μmol/L 24 h); (6.10±0.56)%, (20.75±0.21)%, (5.85±0.07)% (0.20 μmol/L 48 h); (41.15±0.21)%, (35.45±0.35)%, (15.65±0.07)% (1.00 μmol/L 24 h); (52.45±0.35)%, (47.45±2.47)%, (16.80±1.98)% (1.00 μmol/L 48 h), respectively, and the differences were statistically significant compared to the control group[24 h NB4, K562, HL-60: (3.30±0.28)%, (9.50±0.56)%, (2.00±0.14)%; 48 h NB4, K562, HL-60: (3.70±0.14)%, (3.50±0.28)%, (2.15±0.21)%](all P<0.05). G2 phase cells obviously reduced and subG1 phase cells increased.The expression level of cleaved Caspase-3, cleaved Caspase-9 and cleaved PARP increased.Molecular function analysis showed that apoptosis induced by KPT-330 in leukemia cells was partially rela-ted to Glutathione metabolism and Toll-like receptor signaling pathway. Conclusions KPT-330 can effectively inhibit proliferation and induce apoptosis of leukemia cells.KPT-330-induced apoptosis in leukemia cells is partially related to Glutathione metabolism and Toll-like receptor signaling pathway. Key words: KPT-330; Chromosome region maintenance 1; Leukemia cell; Apoptosis; Long non-coding RNA