Dose-dependent effects of selenite (Se(4+)) on arsenite (As(3+))-induced apoptosis and differentiation in acute promyelocytic leukemia cells

Abstract

To enhance the therapeutic effects and decrease the adverse effects of arsenic on the treatment of acute promyelocytic leukemia, we investigated the co-effects of selenite (Se(4+)) and arsenite (As(3+)) on the apoptosis and differentiation of NB4 cells and primary APL cells. A 1.0-μM concentration of Se(4+) prevented the cells from undergoing As(3+)-induced apoptosis by inhibiting As(3+) uptake, eliminating As(3+)-generated reactive oxygen species, and repressing the mitochondria-mediated intrinsic apoptosis pathway. However, 4.0 μM Se(4+) exerted synergistic effects with As(3+) on cell apoptosis by promoting As(3+) uptake, downregulating nuclear factor-кB, and activating caspase-3. In addition to apoptosis, 1.0 and 3.2 μM Se(4+) showed contrasting effects on As(3+)-induced differentiation in NB4 cells and primary APL cells. The 3.2 μM Se(4+) enhanced As(3+)-induced differentiation by promoting the degradation of promyelocytic leukemia protein-retinoic acid receptor-α (PML-RARα) oncoprotein, but 1.0 μM Se(4+) did not have this effect. Based on mechanistic studies, Se(4+), which is similar to As(3+), might bind directly to Zn(2+)-binding sites of the PML RING domain, thus controlling the fate of PML-RARα oncoprotein.

Figure 1

Effects of Se⁴⁺ on As³⁺-induced cell death. (a) The viability of NB4 cells was determined with the WST-1 cell proliferation assay after 48 h of treatment. (b) The viability of primary APL cells. Error bars represent S.D. from the mean of three separate experiments. *P<0.05 and ^**P<0.01 compared with As³⁺-treated cells

Figure 2

Effects of As³⁺ and Se⁴⁺ on cell apoptosis. (a) Cell apoptosis in NB4 cells measured by Annexin V-FITC and PI double staining. (b) Cell apoptosis in primary APL cells. Q₁ and Q₃ respectively represent the proportions of dead cells and living cells, and Q₂ and Q₄ were used to calculate apoptotic cells. Figures show a representative experiment from three independent experiments

Figure 3

Cell cycle distribution profiles of NB4 cells. (a) Cell cycle distribution of NB4 cells analyzed by flow cytometry. (b) Se⁴⁺ at 1.0 μM and (c) 4.0 μM arrested the G₁/S transition. (d) Effects of As³⁺ on the cell cycle. (e) Effects of 1.0 μM Se⁴⁺ combined with 2.0 μM As³⁺ on the cell cycle distribution. (f) Effects of 4.0 μM Se⁴⁺ and 2.0 μM As³⁺ on the cell cycle distribution. SubG₁ represents the apoptotic cells. Figures show a representative experiment from three independent experiments

Figure 4

Effects of Se⁴⁺ on NB4 cell uptake of arsenic. Error bars represent S.D. from the mean of three independent experiments. *P<0.05 and ^**P<0.01 compared with As³⁺-treated groups at corresponding concentrations

Figure 5

Effects of Se⁴⁺ on As³⁺-induced accumulation of ROS in NB4 cells. (a) Effects of Se⁴⁺ on ROS analyzed by flow cytometry. (b) Effects of Se⁴⁺ on As³⁺-induced accumulation of ROS in NB4 cells. (c) RT-PCR analysis of the expression of HMOX1 induced by Se⁴⁺, As³⁺, or their combination. (d) Percentage of relative intensity obtained from the corresponding RT-PCR. Error bars represent S.D. from the mean of three independent experiments. *P<0.05 compared with control, ^#P<0.05 and ^##P<0.01 compared with As³⁺-treated cells

Figure 6

The effects of Se⁴⁺ and As³⁺ on apoptotic factors in NB4 cells. (a) Effects of As³⁺, Se⁴⁺, and their combination on the expression of Bax, Bcl-2, NF-кB, and Caspase-3 were analyzed by RT-PCR. (b) Effects of drugs on the expression of apoptosis factors were analyzed by western blots. (c) Relative intensity expression obtained from the corresponding RT-PCR and (d) western blots. Error bars represent S.D. from the mean of three separate experiments. *P<0.05 and ^**P<0.01 compared with control, ^#P<0.05 and ^##P<0.01 compared with As³⁺-treated cells

Figure 7

Cell differentiation and the fate of PML–RARα oncoprotein. (a) Effects of Se⁴⁺ on the differentiation of NB4 cells were analyzed using FITC anti-human CD11b antibody with flow cytometry. (b) Effects of Se⁴⁺ and As³⁺ on the differentiation of NB4 cells. (c) Proportions of FITC-CD11b-positive NB4 cells. (d) Effects of Se⁴⁺ on the differentiation of primary APL cells. (e) Effects of combined Se⁴⁺ and As³⁺ on the differentiation of primary APL cells. (f) Proportions of FITC-CD11b-positive primary APL cells. (g) Expression of PML–RARα fusion protein analyzed by western blot. (h) Relative intensity expression obtained from corresponding western blot. Error bars represent S.D. from the mean of three separate experiments. *P<0.05 and ^**P<0.01 compared with control, ^#P<0.05 and ^##P<0.01 compared with As³⁺-treated cells

Figure 8

Interactions between Se⁴⁺ and PML-R. (a) UV spectra for the effects of Zn²⁺, As³⁺, and Se⁴⁺ on the absorbance of PML-R at 280 nm. (b) CD spectra for the effects of Zn²⁺, As³⁺, and Se⁴⁺ on the conformation of PML-R. (c) Changes of β-pleated sheet calculated according to CD spectra, ^**P<0.01 compared with the other two. (d) Effects of Zn²⁺, As³⁺, and Se⁴⁺ on the R_H of PML-R measured by DLS, ^**P<0.01 compared with Zn²⁺-treated group. (e) Synchronous fluorescence spectra (Δλ=60 nm) for the interactions between metal/semimetal ion and PML-R at 285 nm. (f) Thiol groups of PML-R determined by Ellman's test, *P<0.05 and ^**P<0.01 compared with control. (g) Peaks corresponding to Cys9 and Cys12 were detected in PML-R by MALDI-TOF-MS. (h) Effects of Se⁴⁺, (i) As³⁺, and (j) Zn²⁺on Cys residues 9 and 12. *Represents IA-modified fragments. The molar ratio of metal ion to protein is 2 : 1. Figures show a representative experiment from three independent experiments

Figure 9

Mechanism for the effects of Se⁴⁺ (1.0 and 4.0 μM) on As³⁺-induced apoptosis and differentiation in NB4 cells and primary APL cells. Se⁴⁺ promotes the degradation of PML–RARα fusion protein by directly binding to the PML-R-ZFs. The decomposition of PML–RARα oncoprotein contributes to the differentiation of NB4 cells and primary APL cells. On one hand, Se⁴⁺ (4.0 μM) prevents the cells from undergoing As³⁺-induced apoptosis by eliminating ROS, downregulating the Bax pro-apoptotic factor, and upregulating the Bcl-2 anti-apoptotic factor. On the other hand, Se⁴⁺ enhances As³⁺-induced apoptosis by downregulating NF-ĸB and activating caspase-3