Dose-dependent effects of selenite (Se4+) on arsenite (As3+)-induced apoptosis and differentiation in acute promyelocytic leukemia cells

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 (Se⁴⁺) and arsenite (As³⁺) on the apoptosis and differentiation of NB4 cells and primary APL cells. A 1.0-μM concentration of Se⁴⁺ prevented the cells from undergoing As³⁺-induced apoptosis by inhibiting As³⁺ uptake, eliminating As³⁺-generated reactive oxygen species, and repressing the mitochondria-mediated intrinsic apoptosis pathway. However, 4.0 μM Se⁴⁺ exerted synergistic effects with As³⁺ on cell apoptosis by promoting As³⁺ uptake, downregulating nuclear factor-кB, and activating caspase-3. In addition to apoptosis, 1.0 and 3.2 μM Se⁴⁺ showed contrasting effects on As³⁺-induced differentiation in NB4 cells and primary APL cells. The 3.2 μM Se⁴⁺ enhanced As³⁺-induced differentiation by promoting the degradation of promyelocytic leukemia protein–retinoic acid receptor-α (PML–RARα) oncoprotein, but 1.0 μM Se⁴⁺ did not have this effect. Based on mechanistic studies, Se⁴⁺, which is similar to As³⁺, might bind directly to Zn²⁺-binding sites of the PML RING domain, thus controlling the fate of PML–RARα oncoprotein.Acute promyelocytic leukemia (APL) is a subtype of human acute myeloid leukemia.¹ The promyelocytic leukemia protein–retinoic acid receptor-α (PML–RARα) fusion protein, which is generated from a specific chromosome translocation t(15;17)(q22;q21), is the key driver of APL leukemogenesis.² Arsenic trioxide (ATO), which has been successfully used in the treatment of APL, induces the catabolism of PML–RARα oncoprotein.³ ATO is one of the primary therapeutic agents for APL, but organ toxicity, especially for the liver and kidney, causes excessive pain for patients.^{4, 5} Studies on the toxicity of arsenic suggest that ATO metabolism increases its toxicity because of oxidative damage and generation of more toxic metabolites, including monomethylarsonous acid and dimethylarsinous acid.^{6, 7, 8, 9} Thus, identifying new therapeutics to decrease the adverse effects of ATO is necessary.ATO induces both apoptosis and differentiation in human APL cells.¹⁰ Apoptosis is an ordered cascade of enzymatic events.¹¹ Studies on the mechanism of ATO-induced apoptosis in APL cells suggest that ATO promotes apoptosis through the mitochondria-mediated intrinsic pathway that is induced by oxidative stress and regulated by Bcl-2 family members.^{10, 12, 13} ATO can also induce apoptosis by inhibiting the nuclear factor-кB (NF-кB) pathway that regulates the expression of various survival proteins.^{14, 15} In addition to apoptosis, ATO can induce the differentiation of APL cells by degrading the PML–RARα fusion protein and activating the retinoic acid signaling pathway.^{10, 16} Zhang et al.¹⁶ reported that ATO induced the degradations of PML and PML–RARα oncoprotein by directly binding to PML. PML is a zinc-finger protein with a Cys-rich motif that contains a RING domain. The PML RING domain (PML-R) contains two Zn²⁺-binding sites (ZFs) and requires Zn²⁺ for autonomous folding.¹⁷ The conserved Cys12, Cys29, and Cys32 residues in PML-R-ZF1, and Cys24, Cys40, and Cys43 residues in PML-R-ZF2 are the binding sites for trivalent arsenic.¹⁶Selenium is an essential nutrient element that shows chemopreventive effect and anticancer potential.¹⁸ Li et al.¹⁹ suggested that high dose (5.0–20 μM) of selenite (Se⁴⁺) could induce the accumulation of reactive oxygen species (ROS) and the apoptosis of NB4 cells. Subsequently, Zuo et al.²⁰ and Guan et al.²¹ confirmed that high concentrations of Se⁴⁺ induced the apoptosis of NB4 cells through an ROS-mediated pathway. However, the accumulation of ROS could induce adverse effects to noncancer tissues by causing oxidative damages.²² For cancer treatment, we attempt to increase the anticancer efficacy while decreasing the adverse effects. Thus far, few studies have investigated the effects of 2.0–4.0 μM Se⁴⁺ on the apoptosis and differentiation of human APL cells. Selenium exerts its biological functions dose-dependently.²² In addition, selenium has chemical properties and metabolic fates similar to those of arsenic. In consideration of the typical characteristics of ATO in the treatment of APL, we hypothesized that 2.0–4.0 μM Se⁴⁺ might induce some interesting changes in APL cells, such as differentiation and the degradation of PML–RARα.Combination therapy is widely used in cancer treatment. The relationship between selenium and arsenic is complex. Selenium and arsenic act as metabolic and toxic antagonists.²³ Combining a low concentration of Se⁴⁺ with ATO might decrease the toxicity and increase the curative potency of ATO in the treatment of APL. Thus, it is of great significance to evaluate the effects of combining selenium with arsenic on the apoptosis and differentiation of human APL cells.In this study, we found dose-dependent contrasting effects of Se⁴⁺ on arsenite (As³⁺)-induced apoptosis and differentiation in NB4 cells and primary APL cells. A 4.0-μM concentration of Se⁴⁺ enhanced As³⁺-induced apoptosis through downregulation of NF-кB and activation of caspase-3, but 1.0 μM Se⁴⁺ failed to elicit these effects. At 2.0–4.0 μM, Se⁴⁺ induced cell differentiation and synergistically promoted As³⁺-induced cell differentiation. Mechanistic studies suggested that Se⁴⁺ might bind directly to PML-R in the form of divalent selenium (Se²⁺) to promote the degradation of PML–RARα oncoprotein.