As2S2 Mediates the ROS/P38 MAPK Signaling Pathway to Induce Apoptosis and S-Phase Arrest in Myelodysplastic Syndrome Cells

Abstract

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid clonal disorder that represents a significant threat to human health. As₂S₂, a natural compound, has been shown to exert therapeutic effects on various malignant tumors, including acute myeloid leukemia (AML), breast cancer, and osteosarcoma, based on extensive clinical experience. In this study, we investigated the mechanism by which As₂S₂ inhibits the proliferation of the myelodysplastic syndrome (MDS) SKM-1 cell line. Our findings revealed that As₂S₂ inhibited the proliferation of SKM-1 cells in a time- and dose-dependent manner. Flow cytometry, protein immunoblotting, and real-time fluorescence quantitative PCR analyses demonstrated that As₂S₂ promotes the phosphorylation of P38 MAPK, thereby activating the MAPK signaling pathway. Additionally, it promotes apoptosis by increasing the BAX/Bcl-2 ratio and induces S-phase arrest through the downregulation of the cell cycle-related protein cyclin A2. Further studies demonstrated that As₂S₂-treated cells exhibited ROS accumulation under fluorescence microscopy, along with activation of the P38 MAPK signaling pathway, increased apoptosis, and S-phase arrest in the cell cycle. This process could be partially reversed by the ROS inhibitor N-acetylcysteine. Therefore, the results of the present study suggest that As₂S₂ induces ROS accumulation in SKM-1 cells, which contributes to the activation of the P38 MAPK signaling pathway, promoting apoptosis and S-phase arrest in the cell cycle. Additionally, As₂S₂ may serve as a potent therapeutic agent for the treatment of myelodysplastic syndromes, with ROS acting as one of the key therapeutic targets.

Keywords: As2S2; P38 MAPK; ROS; apoptosis; myelodysplastic syndrome.

Figure 1

Inhibitory effect of As₂S₂ on SKM-1 cells. The inhibitory effect of As₂S₂ on SKM-1 cells was time- and dose-dependent, with IC₅₀ values of 22.33, 15.16, and 3.973 μM/L at 12, 24, and 48 h, respectively ($\overline{\mathrm{X}}$ ± s; n = 3).

Figure 2

Effects of co-culture of As₂S₂ and inhibitors SB203580 (10 μM/L) and NAC (300 mM/L) on apoptosis and cell cycle of SKM-1 cells. (A) Effect of co-culture of As₂S₂ and the inhibitor SB203580 on apoptosis levels in SKM-1 cells. (B) Flow cytometric fluorescence scatter plots of SKM-1 cell apoptosis ((B(1)): control group; (B(2)): As₂S₂ group; (B(3)): As₂S₂ and SB203580 co-culture group; (B(4)): As₂S₂ and NAC co-culture group). (C) Effect of co-culture of As₂S₂ and inhibitors SB203580 and NAC on the cell cycle. (D) Histograms of flow cytometry cell cycle distribution ((D(1)): control group, (D(2)): As₂S₂ group, (D(3)): co-culture of As₂S₂ and inhibitor SB203580, (D(4)): co-culture of As₂S₂ and inhibitor NAC). $\overline{\mathrm{X}}$ ± s; n = 3; ** p < 0.01 vs. control group; ^▲ p < 0.01 vs. As₂S₂ group; ^▲▲ p < 0.01 vs. As₂S₂ group.

Figure 3

Effect of co-culture of As₂S₂ and inhibitors SB203580 (10 μM/L) and NAC (300 mM/L) on apoptosis and cell cycle-regulated proteins in SKM-1 cells. (A) Effect of co-culture of the As₂S₂ group and inhibitor on the transcript levels of P38 MAPK, BAX, BCL-2, and cyclin A2 mRNA in SKM-1 cells. (B,C) Effect of co-culture of As₂S₂ and inhibitor SB203580 on protein expression levels and quantitative analysis in SKM-1 cells. ($\overline{\mathrm{X}}$ ± s; n = 3) ** p < 0.01 vs. control group; ^▲▲ p < 0.01 vs. As₂S₂ group.

Figure 4

Effect of co-culture of As₂S₂ and ROS inhibitor NAC on SKM-1 cells. (A) Levels of ROS in SKM-1 cells as measured by flow cytometry. (B) Flow fluorescence histogram of ROS. (C) The expression level of ROS in SKM-1 cells observed under a fluorescence microscope (100×, (C(1)): control group, (C(2)): As₂S₂ group, (C(3)): As₂S₂ + NAC group). Arrows indicate cells exhibiting pronounced DCF fluorescence, signifying elevated reactive oxygen species (ROS) levels. (D) Effects of co-culture of As₂S₂ with SB203580 (P38 inhibitor, 10 μM/L) and NAC (ROS inhibitor, 300 mM/L) on cell viability of SKM-1 cells for 48 h. (E,F) Effect of co-culture of As₂S₂ and ROS inhibitor NAC on protein expression levels and quantitative analysis. ($\overline{\mathrm{X}}$ ± s; n = 3) ** p < 0.01 vs. control group; ^▲ p < 0.01 vs. As₂S₂ group; ^▲▲ p < 0.01 vs. As₂S₂ group.

Figure 4

Effect of co-culture of As₂S₂ and ROS inhibitor NAC on SKM-1 cells. (A) Levels of ROS in SKM-1 cells as measured by flow cytometry. (B) Flow fluorescence histogram of ROS. (C) The expression level of ROS in SKM-1 cells observed under a fluorescence microscope (100×, (C(1)): control group, (C(2)): As₂S₂ group, (C(3)): As₂S₂ + NAC group). Arrows indicate cells exhibiting pronounced DCF fluorescence, signifying elevated reactive oxygen species (ROS) levels. (D) Effects of co-culture of As₂S₂ with SB203580 (P38 inhibitor, 10 μM/L) and NAC (ROS inhibitor, 300 mM/L) on cell viability of SKM-1 cells for 48 h. (E,F) Effect of co-culture of As₂S₂ and ROS inhibitor NAC on protein expression levels and quantitative analysis. ($\overline{\mathrm{X}}$ ± s; n = 3) ** p < 0.01 vs. control group; ^▲ p < 0.01 vs. As₂S₂ group; ^▲▲ p < 0.01 vs. As₂S₂ group.