Gene Profile during Arsenic Trioxide-Induced Apoptosis in Multiple Myeloma: Strong Anti-Oxidantive Response and Pro-Apoptotic Imbalance.

Arsenic Trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has promising activity in multiple myeloma (MM). The purpose of the present study was to evaluate the changes in the gene expression profile in four human MM cell lines (U266, MM.1S, RPMI 8226 and KMS11) following exposure to ATO in a 48H time-course to gain further insight into the mechanism of action in MM. U266 and 8226 show some resistance to ATO, while MM.1S and KMS11 are more sensitive to ATO-induced apoptosis. Two μM ATO resulted in 45.3, 74.2, 48.3 and 76.4 percent apoptosis for U266, MM.1S, 8226 and KMS11, at 48H respectively. Affymetrix Hu133 2.0 Plus Chips containing 54,675 probe sets were used for the mRNA expression profiling at 0, 6, 24 and 48H after ATO treatment. A total of 654, 478 and 600 common up or down-regulated genes showed a clustered expression pattern in response to ATO at 6, 24 and 48H, respectively. The pattern was consistent with strong anti-oxidative and heavy metal responses and pro-apoptotic imbalance. Altered cell processes included: protein folding, response to metal ion, cation and amino acids transport, heme metabolism, cell proliferation, cysteine metabolism, fatty acid metabolism, cell proliferation and apoptosis. Common up-regulated genes including clusterin (apolipoprotein J), ferritin, biliverdin reductase B, glutamate-cysteine ligase, heme oxygenase (decycling) 1, metallothionein 1, NAD(P)H dehydrogenase quinone 1, and solute carrier family 7 imply a clear anti-oxidative response. Heme oxygenase-1 (HO-1) and metallothioneins (MTs) expression has been suggested to be associated with ATO-resistance. HO-1 baseline expression is 5 fold higher in U266 and 8226 cell lines compared to MM.1S and KMS11, also suggesting a possible role of HO-1 in ATO-resistance. However, the strong up-regulation of this protein (greater than 50 fold in MM.1S and KMS11 as early as 6H) could not protect cells from ATO-induced apoptosis. Similarly the strong up-regulation of MT-1 did not correlate with cell survival. Moreover, up-regulation of HO-1 and MT-1 prior to ATO treatment (using Hemin and ZnCl2, respectively) only marginally and transiently protected MM.1S from ATO-induced apoptosis. Common down-regulated genes such as bcl-x, survivin and insulin induced gene 1 suggest a pro-apoptotic imbalance in the cell fate. 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 significantly (2.5 and 3.0 fold) in MM.1S and KMS11 while to a lesser extent in U266 and 8226 (1.5 and 1.6 fold). Additionally, Bmf baseline expression is higher in MM.1S and KMS11 compared to U266 and 8226, while Bim is highly expressed in the four cell lines and did not show any transcriptional regulation during ATO-induced apoptosis. Bim and Bmf are induced following JNK activation. JNK1 is phosphorylated in all cell lines in response to ATO. Taken together these data indicate that while the cells initially respond to ATO treatment in a manner consistent with oxidative stress, the compensatory mechanisms are not sufficient to block cell death. The mechanism(s) of cell death are not completely clear although changes in the expression of Bcl-2 family members suggest that activation of the intrinsic apoptotic pathway is likely to be important.