Reactive oxygen species are not required for an arsenic trioxide-induced antioxidant response or apoptosis

Abstract

Arsenicals are both environmental carcinogens as well as therapeutic agents for the treatment of trypanosomiasis and more recently cancer. Arsenic trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has activity in multiple myeloma (MM). While signaling events associated with carcinogenesis have been well studied, it still remains to be determined which of these events are involved in anti-cancer signaling. To better define this response, gene expression profiling following ATO treatment of four MM cell lines was performed. The pattern was consistent with a strong antioxidative response, particularly of genes activated by Nrf2. While Nrf2 is expressed constitutively at the mRNA level, the protein is not detected in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized and present in the nucleus within 6 h of ATO treatment. Despite the activation of this antioxidative response, ROS may not be important in ATO-induced death. Inhibition of ATO-induced ROS with butylated hydroxyanisole (BHA) does not affect Nrf2 activation or cell death. Moreover, silencing Nrf2 had no effect on ATO-induced apoptosis. Together these data suggest that ROS is not important in the induction of the antioxidative response or cellular death by ATO.

FIGURE 1.

ATO-induced apoptosis in four human myeloma cell lines. U266, MM.1s, 8226/S, and KMS11 were treated for 6, 24, and 48 h with 2μm ATO. Viability was measured by Annexin-V-FITC/PI staining. Percent (%) of control viability, as percent of Annexin-V-negative cells, was plotted versus time (h). The data are presented as the mean ± S.D. of eight independent experiments.

FIGURE 2.

Induction of HO-1 is not protective against ATO-induced apoptosis in MM cell lines. U266, MM.1s, 8226/S and KMS11 were treated for 0, 6, 24, and 48 h with 2 μm ATO. Total protein lysates were obtained and NQO1 (A) and HO-1 (B) protein expression was determined by Western blot. KMS11 treated with ATO for 24 h was used as a positive control for the U266 and MM.1s NQO1 blot. C, U266 cell line was pretreated for 24 h with 50, 100, and 200 μm of hemin and HO-1 protein up-regulation was demonstrated by Western blot. Cells treated with ATO for 24 h were used as a positive control. Hemin-pretreated U266 cells were treated with 2 μm ATO for 24 h and viability was evaluated by Annexin-V-FITC/PI staining. Data are presented as mean ± S.D. of three independent experiments. D, U266 cell line was stably transfected with pcDNA3-HO-1 or an empty vector control (Neo). Expression of HO-1 was confirmed by Western blot. U266, U266-Neo, and U266-HO-1 were treated with 2 μm ATO for the indicated time and viability was evaluated by annexin-V-FITC/PI staining. Percent (%) of control viability was plotted versus time (h). Data are presented as mean ± S.D. of three independent experiments.

FIGURE 3.

MT induction does not protect cells from ATO-induced cell death. A, U266, MM.1s, and 8226/S were treated for 0, 6, 24, and 48 h with 2 μm ATO, and total RNA was obtained. RT-PCR for MT1 and GAPDH was performed as described under “Materials and Methods.” B, U266 was treated with 25, 50, and 100 μm of ZnCl₂, and MT1H and MT2A gene expression was measured by real-time PCR. C, untreated and ZnCl₂-pretreated U266 cells were treated with 2 μm ATO for 24 h, and viability was evaluated by annexin-V-FITC/PI staining. Data are presented as mean ± S.D. of three independent experiments.

FIGURE 4.

ATO induced activation of the Nrf2-Keap1 pathway. Genes related to the Nrf2-Keap1 signaling pathway, included in the array, were clustered using Cluster and TreeView. The clustering of these genes was supervised to maintain the time course of the arrays. Ratios versus baseline expression at 6, 24, and 48 h were included in the analysis for U266, MM.1s, 8226/S, and KMS11. The scale represents the magnitude of indicated changes. A black square indicates no change in expression at that time point compared with control, while a gray box indicates that no expression was observed in that cell line.

FIGURE 5.

ATO induces up-regulation of Nrf2 protein, translocation to the nucleus and target gene expression. A, Western blot analysis of Nrf2 and Keap1 protein expression at 0, 6, 24, and 48 h after 2 μm ATO treatment, for U266 and MM.1s cell lines. B, cells were treated with 40 nm of Bortezomib and/or 2 μm ATO for 6 h, lysed, and subjected to Western blot analysis for HO-1, Nrf2, Keap1, and actin protein expression. C, U266 and D, MM.1s cells were treated as in B and fractionated into cytosolic and nuclear protein fractions. Nrf2 and Keap1 protein expression was analyzed by Western blot. Actin and Lamin A/C were used as cytosolic and nuclear loading controls, respectively.

FIGURE 6.

Activation of Nrf2 and Noxa up-regulation by ATO are not mediated by ATO-induced ROS. A, KMS11 was incubated with 100 μm BHA for 16 h. Control and BHA-treated cells were then treated with 1 μm ATO for 6 h. DHE was used for ROS determination. The shaded histogram represents untreated cells while the bold-lined open histograms represent the indicated treatment. B, KMS11 was treated as in A and protein lysates were subjected to Western blot analysis of HO-1, NQO1, Nrf2, Keap1, Noxa, and actin. C, KMS11 was treated as in A, and cell viability was measured by Annexin-V-FITC/PI staining after 48 h of ATO treatment. Percent (%) of control viability was calculated, and data are presented as mean ± S.D. of four independent experiments.

FIGURE 7.

Nrf2 signaling is not protective during ATO-induced apoptosis. A, MM.1s and KMS11 were electroporated with si(-) (negative control), siNrf2, and siKeap1. After 16 h, cells were treated with 2 μm ATO. Silencing of Nrf2 and Keap1 proteins was determined by Western blot at 6 and 24 h after ATO treatment. B, viability was evaluated by Annexin-V-FITC/PI staining. Percent (%) of control (untreated, transfected, UT) viability was plotted versus time (h). Student's t test was used to compare differences between samples, si(-) and experimental samples unless otherwise indicated, with confidence intervals of 95%. ND, no difference; ^*, p < 0.05.