Arsenic trioxide induces apoptosis in B-cell chronic lymphocytic leukemic cells through down-regulation of survivin via the p53-dependent signaling pathway

Abstract

Arsenic trioxide (As2O3) can induce apoptosis in many tumors. However, the associated mechanisms are not clearly understood. We found that As2O3 significantly inhibited the proliferation of WSU-CLL cells and induced apoptosis in dose- and time-dependent manners. WSU-CLL cells treated with 2μM As2O3 showed survivin down-regulation and p53 up-regulation. Survivin siRNA combined with As2O3 further inhibited the proliferation of WSU-CLL cells. p53 inhibition by siRNA prevented the down-regulation of survivin by As2O3 and prevented the As2O3-induced cytotoxicity of WSU-CLL cells. These results suggest that As2O3 may be of therapeutic value for chronic lymphocytic leukemia.

Keywords: Apoptosis; Arsenic trioxide; P53 and CLL; Survivin; WSU-CLL.

Fig. 1

Cytotoxicity of As₂O₃ to WSU-CLL cells. WSU-CLL cells were cultured without or with different concentrations of As₂O₃ for 24, 48, 72 and 96 h. Cell viability was determined based on trypan blue exclusion. Control cell viability was set at 100%, and the survival relative to the control is presented. Each point represents the mean ± SD of three experiments with nine replicates per dose.

Fig. 2

Apoptosis of WSU-CLL cells after As₂O₃ treatment. (A–D) Apoptosis of WSU-CLL cells after treatment with different concentrations of As₂O₃ for 48 h. (A) 0 μM, (B) 1 μM, (C) 2 μM, (D) 4 μM and (E) WSU-CLL cells were treated with 2 μM As₂O₃ for 0, 24, 48, 72 and 96 h. Cell apoptosis was evaluated by flow cytometry after Annexin V and PI staining. The data are presented as the mean ± SD of three independent experiments.

Fig. 3

Analyses of survivin protein and mRNA levels. WSU-CLL cells were exposed to 2.0 μM As₂O₃ for 0, 24, 48 and 72 h. Survivin was analyzed by Western blotting. (A) Representative immunoblots of the expression level of survivin in cells treated with 2.0 μM As₂O₃ for 0, 24, 48 and 72 h. (B) β-actin expression was used as a loading control, and the relative survivin protein levels (means ± SD, n = 3) were determined. Survivin mRNA levels were measured by RT-PCR. (C) RT-PCR images of survivin in WSU-CLL cells treated with As₂O₃ for different lengths of time. (D) Quantification of the survivin mRNA level in WSU-CLL cells treated with As₂O₃ for different lengths of time in three separate experiments. As₂O₃ reduced survivin transcriptional activity.

Fig. 4

Involvement of survivin in As₂O₃-induced cytotoxicity. WSU-CLL cells were transfected with myc-survivin or pcDNA (vector) for 20 h and then incubated with or without 2 μM As₂O₃ for 48 h. (A) Survivin protein expression was analyzed by Western blotting. Survivin protein was overexpressed in WSU-CLL cells transfected with survivin. (B) Cell viability was determined by trypan blue exclusion. Survivin overexpression reduced As₂O₃-induced cytotoxicity. The viability of control cells was set at 100%, and the viability relative to the control is shown.

Fig. 5

Survivin inhibition by siRNA sensitizes WSU-CLL cells to As₂O₃-induced cell death. WSU-CLL cells were transfected with survivin or nonspecific siRNA for 20 h and then treated with or without 2 μM As₂O₃ for 48 h. (A) Cells that were pre-treated with survivin siRNA (survivin) showed a significant decrease in survivin expression compared with the control group (C) and the nonspecific siRNA group (NS). (B) Cell viability was determined by trypan blue exclusion. Control cell viability was set at 100%, and the viability relative to the control is shown.

Fig. 6

Effect of As₂O₃ on p53 protein expression in WSU-CLL cells. WSU-CLL cells were exposed to 2.0 μM As₂O₃ for 0, 24, 48, and 72 h. Western blot analyses were performed. (A) Representative immunoblots of the p53 expression level following treatment with 2 μM As₂O₃ for 0, 24, 48 and 72 h. (B) β-actin expression was used as a loading control. Relative p53 protein levels (means ± SD, n = 3) were determined.

Fig. 7

Knockdown of p53 with siRNA targeting p53 can prevent cell death due to As₂O₃. WSU-CLL cells were transfected with p53 siRNA or nonspecific siRNA for 20 h and then incubated with or without 2 μM As₂O₃ for 48 h. Western blot and cell viability analyses were performed. (A) Representative immunoblots of p53 and survivin. The p53 siRNAs were highly effective at decreasing the level of p53 protein and simultaneously increased survivin protein expression. (B) The relative protein expression of p53 and survivin (means ± SD, n = 3) was determined. p53 siRNA decreased p53 protein expression, which was up-regulated by As₂O₃. The decrease in the survivin protein level caused by As₂O₃ was partially rescued by p53 siRNA. (C) Cell viability was determined by trypan blue exclusion. Control cell viability was set at 100%, and the viability relative to the control is shown. The knockdown of p53 using siRNA resulted in the partial rescue of cell viability.