奥普佐米协同三氧化二砷抑制急性早幼粒白血病细胞增殖的分子机制

Molecular mechanism of oprozomib synergistic with arsenic trioxide in inhibiting proliferation of acute promyeloid leukemia cells

目的探讨奥普佐米协同三氧化二砷抑制急性早幼粒白血病细胞增殖分子机制。方法 MTT法检测不同联合用药对急性早幼粒白血病NB4、HL-60细胞存活率的影响:(1)梯度浓度三氧化二砷(NB4细胞:0、0.1、0.2、0.3、0.4、0.5μmol/L;HL-60细胞:0、0.4、0.8、1.2、1.6、2.0μmol/L)单用或与固定浓度奥普佐米(NB4细胞:40 nmol/L;HL-60细胞:160 nmol/L)联用;(2)梯度浓度奥普佐米(NB4细胞:0、5、10、20、40 nmol/L;HL-60细胞:0、40、80、120、160、200 nmol/L)单用或与固定浓度三氧化二砷(NB4细胞:0.4μmol/L;HL-60细胞:1.2μmol/L)联用;(3)单独或联合用药作用不同时间(12、24、48 h);(4)相同浓度的硼替佐米或奥普佐米分别与梯度浓度三氧化二砷联用。单独用药或联合用药或用NAC预处理后,流式细胞仪检测细胞凋亡及ROS生成量;Western blot检测凋亡相关蛋白cleaved-Caspase 3、cleaved-Caspase 9、PARP-1及融合基因蛋白PML-RARA的表达。结果与单用组比较,奥普佐米与三氧化二砷联用后,NB4、HL-60细胞存活率显著降低(P<0.01),呈剂量和时间依赖性,并具有明显的协同作用,协同系数小于1。联合用药可使约80%的NB4细胞ROS水平明显升高,融合蛋白PML-RARA表达降低,细胞凋亡率增加约70%(P<0.01);与联合用药组比较,ROS抑制剂NAC预处理后,细胞凋亡减少约41%(P<0.01),PML-RARA的表达升高。结论奥普佐米协同三氧化二砷诱导急性早幼粒白血病细胞ROS生成,并导致PML-RARA蛋白表达降低,促进细胞凋亡。

Objective To investigate the molecular mechanism of oprozomib synergistically acting with arsenic trioxide in inhibiting the proliferation of acute promyeloid leukemia cells. Methods MTT assay was used to observe the effects of different medication strategies on the survival rate of NB4 and HL-60 cells respectively:(1)gradient concentration of arsenic trioxide was adopted alone( for NB4 cells: 0, 0.1, 0.2, 0.3, 0.4, 0.5 μmol/L;for Hl-60 cells: 0, 0.4, 0.8, 1.2, 1.6, 2.0 μmol/L) or combined with fixed concentration of oprozomib(for NB4 cells: 40 nmol/L;for Hl-60 cells: 160 nmol/L);(2)gradient concentration of oprozomib was applied alone(for NB4 cells: 0, 5, 10, 20, 40 nmol/L;for Hl-60 cells: 0, 40, 80, 120, 160, 200 nmol/L) or combined with fixed concentration of arsenic trioxide(for NB4 cells: 0.4 μmol/L;for Hl-60 cells: 1.2 μmol/L);(3) the treatment results of single drug or combination were observed for 12, 24 and 48 h respectively;(4)the effects of bortezomib or oprozomib(both at the same concentration) were compared when in combination with arsenic trioxide(at gradient concentration). In addition, flow cytometry was used to detect cell apoptosis and reactive oxygen species(ROS) production, and Western blotting was performed to determine the expression of apoptosis related proteins cleaved-Caspase 3, cleaved-Caspase 9, PARP-1 and the fusion gene protein PML-RARA, in the presence or absence of N-acetylcysteine(NAC, ROS inhibitor, 5 mmol/L). Results As compared with the single drug group, the combination of oprozomib and arsenic trioxide resulted in a significant decline of cell viability of NB4 and HL-60 cells(P<0.01) in a dose-and time-dependent manner, showing an obvious synergistic effect, and the synergistic coefficient was less than 1. The combined medication strategy also increased the ROS production by about 80%, improved the apoptotic rate by 70%, and reduced the expression of PML-RARA in NB4 cells(P<0.01). However, inhibiting ROS production by NAC pretreatment decreased the apoptotic rate by about 41%(P<0.01), along with elevated expression of PML-RARA. Conclusion The combination of oprozomib and arsenic trioxide promotes apoptosis by inducing ROS generation and reducing the expression of PML-RARA protein in acute promyelocytic leukemia cells.