Arsenic Trioxide Induces Up-Regulation of BMF and NOXA and Overcomes an Anti-Oxidative Response To Induce Apoptosis in Myeloma Cells.

Arsenic Trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has shown promise as a therapeutic agent in multiple myeloma (MM). We have been characterizing the response of MM cell lines to ATO and have demonstrated that four human MM cell lines, including U266, MM.1s, 8226/S and KMS11 were sensitive to ATO. Affymetrix Hu133 2.0 Plus arrays were used for expression profiling at 0, 6, 24 and 48 h after ATO treatment. The pattern observed in all four cell lines was consistent with anti-oxidative responses and an imbalance of Bcl-2 family members favoring apoptosis. Most notably expression changes were associated with a response driven by the Nrf2-Keap1 pathway. Nrf2 is expressed at the mRNA level however the protein is not observed in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized within 6 h of ATO treatment. Importantly, this up-regulation is associated with Nrf2 accumulation in the nucleus where it functions. The activation of the Nrf2 is associated with the changes observed during ATO treatment, including up-regulation of the following genes: metallothionein-1 (MT-1), phase II detoxification enzymes (NQO-1), anti-oxidant enzymes [heme oxygenase-1 (HO-1), thioredoxin reductase, glutathione reductase and γ-glutamate cysteine ligase], NADPH-generating enzymes (malic enzyme), chaperone proteins (Hsp70, Hsp40, Hsp27KDa and Hsp105KDa) and the transcriptional repression of genes involved in the cholesterol/lipid biosynthesis pathway. HO-1 and MT-1 expression has been previously suggested to be associated with ATO-resistance in myeloma. However, the up-regulation of this gene (greater than 50 fold in MM.1s and KMS11 as early as 6 hrs) could not protect cells from ATO-induced apoptosis. Similarly up-regulation of MT-1 did not correlate with cell survival. Moreover, up-regulation of HO-1 or MT-1 prior to ATO treatment had no effect on ATO-induced apoptosis in U266 or MM.1s cells, consistent with each of these being components of a larger anti-oxidative response. Bcl-x, was down-regulated in all the four cell lines during ATO-induced apoptosis while one of its ligands, Bmf, a BH3-only pro-apoptotic Bcl-2 family member was up-regulated 1.5–3.0 fold at the mRNA level and correlated with response. An additional BH3-only protein Noxa was up-regulated in three out four cell lines at the mRNA and protein level. In U266, Noxa is expressed constitutively and not affected by ATO. Interestingly however, ATO induced a down-regulation of Mcl-1, the Bcl-2 anti-apoptotic partner of Noxa in U266 cells. Taken together the Bmf/Bcl-x and Noxa/Mcl-1 ratios favored an apoptotic response following addition of ATO. Additionally Noxa up-regulation was enhanced when glutathione was depleted with BSO and blocked by addition of N-acetylcysteine. Taken together these data indicate that while the cells initially respond to ATO treatment in a manner consistent with a strong anti-oxidative response, these protective mechanisms are not sufficient to block cell death induced by the up-regulation of two BH3-only proteins (Bmf and Noxa) and the down-regulation of Bcl-x and Mcl-1 resulting in the activation of the intrinsic apoptotic pathway as part of the ATO-induced apoptotic mechanism(s). New arsenical compounds or combined therapies, could avoid this protective anti-oxidative pathway activated by ATO increasing its activity.