Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells

Abstract

Since arsenic trioxide (As3+) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As4S4) in the treatment of APL, we investigated the effects of combining As4S4 and As3+ on the apoptosis and differentiation of NB4 and primary APL cells. As4S4, acting similarly to As3+, arrested the G1/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As4S4 (0.1-0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As4S4- or As3+-treated groups, the combination of As4S4 and As3+ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As4S4 acted synergistically with As3+ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As4S4 and As3+ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As4S4 and As3+ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.

Fig 1. The effects of combining As³⁺ and As₄S₄ on the growth of NB4 and primary APL cells.

(A) The effects of As³⁺ on cell viability. (B) The effects of As₄S₄ on cell viability. (C&D) The combined effects of As³⁺ and As₄S₄ on cell viability. (E) CI of concurrent treatment with As³⁺ and As₄S₄ in NB4 cells. (F) CI in primary APL cells. CI<1.0 indicated a synergistic effect. The viability of NB4 and primary APL cells were determined by WST-1 cell proliferation assay kit after 48 h of treatment. Error bars represent the S.D. from the mean of three separate experiments. **P<0.01 compared with the control. ^##P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 2. The effects of combining As³⁺ and As₄S₄ on the apoptosis of NB4 and primary APL cells.

(A) The apoptosis of NB4 cells. (B) The apoptosis of primary APL cells. (C) The percentage of apoptotic cells in NB4 and primary APL cells. After 48 h of treatment, the cells were stained with Annexin V-FITC and PI. Q₁ and Q_3, represent the dead cells and living cells, respectively. Q₂ and Q₄ were used to calculate the proportion of apoptotic cells. Figures show a representative experiment of three independent experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 3. The effects of combining As³⁺ and As₄S₄ on cell cycle distribution.

(A) Cell cycle distribution in NB4 cells. (B) Cell cycle distribution in primary APL cells. (C) The percentage of cell cycle distribution in each phase. After 48 h of treatment, NB4 and primary APL cells were stained with PI and analyzed by flow cytometry. Figures show a representative experiment of three independent experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 4. The effects of combining As³⁺ and As₄S₄ on cellular ROS accumulation.

(A) Cellular ROS were determined with DCFH-DA fluorescence probe in NB4 cells. (B) Cellular ROS in primary APL cells. (C) The effects of As₄S₄ and As³⁺ on HMOX1 expression. (D) The percentage of relative HMOX1 intensity obtained by RT-PCR. Error bars represent the S.D. from the mean of three independent experiments. *P<0.05 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 5. The effects of combining As₄S₄ and As³⁺ on mitochondria-mediated apoptosis.

(A) RT-PCR analysis of Bax, Bcl-2 and caspase-3 expression. (B) Western-blot analysis of Bax, Bcl-2 and caspase-3 expression. (C) Relative intensity of expression obtained by RT-PCR. (D) Relative intensity expression obtained by western-blot. Error bars represent the S.D. from the mean of three separate experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 6. The effects of combining As₄S₄ and As³⁺ on NFκB, caspase-12 and p53 expression.

(A) RT-PCR and Western-blot analysis of NFκB expression. (B) Relative NFκB intensity obtained by RT-PCR and western-blot. (C) The effects of As₄S₄ and As³⁺ on caspase-12 and p53 expression. (D) Relative intensities of caspase-12 and p53 obtained by western-blot. Error bars represent the S.D. from the mean of three separate experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Fig 7. As₄S₄ acts synergistically with As³⁺ to affect NB4 and primary APL cell differentiation.

(A) The effects of As₄S₄ on CD11b expression in NB4 cells. (B) The effects of As₄S₄ on As³⁺-induced differentiation in NB4 cells. (C) The effects of As₄S₄ on As³⁺-induced differentiation in primary APL cells. (D) The percentage of FITC-CD11b-positive primary APL cells. (E) Western-blot analysis of PML-RARα expression in NB4 and primary APL cells. The figures show a representative experiment of three independent experiments. **P<0.01 compared with 0.2 μM As³⁺ and 0.2 μM As₄S₄ combination treated cells.

Fig 8. Mechanism for the synergistic effects of As₄S₄ and As³⁺ on apoptosis and differentiation of acute promyelocytic leukemia cells.