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

Abstract

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: ①gradient 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); ②gradient 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; ④the 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.

Keywords

• oprozomib
• acute promyelocytic leukemia
• pml-rara
• reactive oxygen species
• apoptosis