As4S4 Exhibits Good Killing Effect on Multiple Myeloma Cells Via Repressing SOCS1 Methylation-Mediated JAK2/STAT3 Signaling Pathway

Abstract

Objective: This study aimed to investigate the effect of tetra-arsenic tetra-sulfide on treating multiple myeloma and its potential regulation on suppressor of cytokine signaling 1 methylation-mediated Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway.

Methods: Tetra-arsenic tetra-sulfide with different concentrations were used to treat U266 cells, and cell viability was measured at 12, 24, and 48 hours with 0 μM tetra-arsenic tetra-sulfide treatment as control by Cell Counting Kit-8 assay. Suppressor of cytokine signaling 1 methylation and expression were determined by methylation-specific polymerase chain reaction, quantitative polymerase chain reaction, and Western blot, respectively, in U266 cells and normal plasma cells and in U266 cells treated by tetra-arsenic tetra-sulfide. Then, rescue experiments were performed by transfecting suppressor of cytokine signaling 1 small interfering RNA into tetra-arsenic tetra-sulfide-treated U266 cells. Besides, phosphor-Janus kinase 2, Janus kinase 2, phospho-signal transducer and activator of transcription 3, and signal transducer and activator of transcription 3 expressions were determined by Western blot.

Results: Tetra-arsenic tetra-sulfide inhibited U266 cell viability efficiently in a dose- and time-dependent manner. Suppressor of cytokine signaling 1 methylation was higher while suppressor of cytokine signaling 1 expression was lower in U266 cells compared to normal plasma cells; when treated by tetra-arsenic tetra-sulfide, suppressor of cytokine signaling 1 methylation was decreased while suppressor of cytokine signaling 1 expression was increased in U266 cells, along with the reduced phospho-Janus kinase 2 and phospho-signal transducer and activator of transcription 3 expressions. Then, suppressor of cytokine signaling 1 small interfering RNA enhanced the cell viability and phospho-Janus kinase 2 as well as phospho-signal transducer and activator of transcription 3 expressions in both tetra-arsenic tetra-sulfide treatment-free and tetra-arsenic tetra-sulfide-treated U266 cells.

Conclusion: Tetra-arsenic tetra-sulfide exhibits good killing effect on multiple myeloma cells via repressing suppressor of cytokine signaling 1 methylation and downstream Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway, which might serve as a potential treatment option for multiple myeloma.

Keywords: As4S4; JAK2/STAT3; SOCS1 methylation; cell viability; multiple myeloma.

Figure 1.

Tetra-arsenic tetra-sulfide inhibited cell viability in U266 cells. Relative cell viability of U266 cells treated by As₄S₄ with different concentrations for 12, 24, and 48 hours (A). IC₅₀ value of As₄S₄ in killing U266 cells (B). Comparison among groups was determined by one-way analysis of variance. As₄S₄ indicates tetra-arsenic tetra-sulfide; IC₅₀, the concentration inhibiting 50% of in vitro cell growth; NS, no significance. P value < .05 was considered significant. ***P < .001; **P < .01; *P < .05.

Figure 2.

Comparison of SOCS1 methylation between U266 cells and control cells. Suppressor of cytokine signaling 1 methylation in U266 cells and control cells (A). Suppressor of cytokine signaling 1 mRNA expression in U266 cells and control cells (B). Suppressor of cytokine signaling 1 protein expression in U266 cells and controls (C and D). Comparison between groups was determined by unpaired t test. mRNA indicates messenger RNA; SOCS1, suppressor of cytokine signaling 1. P value < .05 was considered significant. **P < .01.

Figure 3.

Suppressor of cytokine signaling 1 methylation in U266 cells after treated by As₄S₄. Suppressor of cytokine signaling 1 methylation in U266 cells with As₄S₄ treatment (A). Suppressor of cytokine signaling 1 mRNA expression in U266 cells with As₄S₄ treatment (B). Suppressor of cytokine signaling 1 protein expression in U266 cells with As₄S₄ treatment (C and D). Protein expressions of pJAK2/JAK2 and pSTAT3/STAT3 in U266 cells with As₄S₄ treatment (E and F). Comparison between groups was determined by unpaired t test. As₄S₄ indicates tetra-arsenic tetra-sulfide; JAK2, Janus kinase 2; mRNA, messenger RNA; STAT3, signal transducer and activator of transcription 3. P value < .05 was considered significant. ***P < .001; **P < .01; *P < .05.

Figure 4.

Suppressor of cytokine signaling 1 expression, JAK2/STAT3 pathway, and cell viability in SOCS1 knockout U266 cells. Suppressor of cytokine signaling 1 mRNA expression in si-SOCS1 group and NC group (A). Suppressor of cytokine signaling 1 protein expression in si-SOCS1 group and NC group (B and C). JAK2/STAT3 protein expressions in si-SOCS1 group and NC group (D and E). Cell viability in si-SOCS1 group and NC group (F). Comparison between groups was determined by unpaired t test. JAK2/STAT3 indicates Janus kinase 2/signal transducer and activator of transcription 3; mRNA, messenger RNA; NC, negative control; SOCS1, suppressor of cytokine signaling 1. P value < .05 was considered significant. ***P < .001; **P < .01.

Figure 5.

Suppressor of cytokine signaling 1 expression, JAK2/STAT3 pathway, and cell viability in SOCS1 knockout U266 cells after As₄S₄ treatment. Suppressor of cytokine signaling 1 mRNA expression in As₄S₄ group and si-SOCS1 + As₄S₄ group (A); SOCS1 protein expression in As₄S₄ group and si-SOCS1 + As₄S₄ group (B and C). JAK2/STAT3 protein expressions in As₄S₄ group and si-SOCS1 + As₄S₄ group (D and E). Cell viability in As₄S₄ group and si-SOCS1 + As₄S₄ group (F). Comparison between groups was determined by unpaired t test. As₄S₄ indicates tetra-arsenic tetra-sulfide; JAK2/STAT3, Janus kinase 2/signal transducer and activator of transcription 3; mRNA, messenger RNA; SOCS1, suppressor of cytokine signaling 1. P value < .05 was considered significant. ***P < .001; **P < .01.