Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells

Abstract

Arsenic has played a key medicinal role against a variety of ailments for several millennia, but during the past century its prominence has been displaced by modern therapeutics. Recently, attention has been drawn to arsenic by its dramatic clinical efficacy against acute promyelocytic leukemia. Although toxic reactive oxygen species (ROS) induced in cancer cells exposed to arsenic could mediate cancer cell death, how arsenic induces ROS remains undefined. Through the use of gene expression profiling, interference RNA, and genetically engineered cells, we report here that NADPH oxidase, an enzyme complex required for the normal antibacterial function of white blood cells, is the main target of arsenic-induced ROS production. Because NADPH oxidase enzyme activity can also be stimulated by phorbol myristate acetate, a synergism between arsenic and the clinically used phorbol myristate acetate analog, bryostatin 1, through enhanced ROS production can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can effectively kill leukemic cells. Our findings pinpoint the arsenic target of ROS production and provide a conceptual basis for an anticancer regimen.

Fig. 1.

Microarray analysis of NB4 cells before and after arsenic treatment. Expression profiles of the 24 genes (with the lower bound fold change of the 90% confidence intervals ≥5) were shown across the five samples in Eisen's heat map. No As, untreated NB4 cells; As, arsenic-treated NB4 cells. Red and green colors represent high and low expression levels, respectively. The corresponding gene symbols, fold changes, and the lower bound fold change of the 90% confidence intervals are also listed. –, down-regulated gene expression. Those genes related to ROS production were marked with an asterisk. The genes are ordered from the most down-regulated genes to the highest up-regulated genes, based on the lower bound fold change.

Fig. 2.

Up-regulation of NADPH oxidase and eosinophil peroxidase expression by arsenic. (A) Absent immunohistochemical staining in control cells (Left) and intense staining of eosinophil peroxidase in arsenic-treated NB4 cells (Right). The staining in arsenic-treated cells depended on the primary antibody (data not shown). As, arsenic. (B) Immunoblotting of p47^PHOX and p67^PHOX shows dramatic increases in the protein levels in NB4 cells after arsenic treatment. α-Tubulin was used as a loading control. (C) Arsenic increased mRNA of all of the NADPH oxidase subunits in NB4 cells as measured by SYBR green quantitative real-time PCR.

Fig. 3.

Induction of ROS formation from NADPH oxidase by arsenic. Chemiluminescence measured with luminol plus HRP (A), lucigenin (B), and flow cytometry (C) by using dihydrorhodamine 123 showed significant ROS induction in NB4 cells treated with arsenic. (D and E) Luminol plus HRP (D) and cytochrome c reduction (E) showed that induction of ROS was further dramatically enhanced by the addition of 50 nM PMA. Incubation with DPI completely blunted the baseline, arsenic-induced, or PMA-stimulated chemiluminescence and cytochrome c reduction. SOD, superoxide dismutase.

Fig. 4.

NADPH oxidase is the main source of arsenic-induced ROS. (A) p47^PHOX protein was induced by arsenic in cells transfected with scrambled (Scram.) siRNA, but this induction is diminished in cells transfected with p47^PHOX siRNA (Inset). The lucigenin chemiluminescence induced by arsenic in p47^PHOX siRNA-transfected cells was significantly diminished compared with those transfected with scrambled siRNA. As, arsenic. (B) Lucigenin chemiluminescence was absent in X-CGD cells before and after arsenic treatment. In contrast, the baseline chemiluminescence of the parental cells, PLB-985, was enhanced by arsenic treatment. (C) Cytochrome c reduction assay showed PMA-enhanced superoxide production in arsenic-treated parental cells PLB-985 but not in X-CGD cells. (D) mRNAs of most NADPH oxidase subunits in PLB-985 or X-CGD cells were increased by arsenic, as determined by real-time PCR.

Fig. 5.

Synergistic cytotoxicity between arsenic and either PMA or bryostatin 1 (Bryo). (A) PMA or byrostatin 1 at the doses used did not prevent cell proliferation. Without arsenic, the cells continued proliferation in the presence of PMA or bryostatin 1 in absence (Left) or presence (Right) of NAC. NAC had mild toxicity to NB4 cells. (B Left) Synergistic toxicity is evident in cells pretreated with arsenic and then exposed to PMA or byrostatin 1, as compared with cells pretreated with arsenic only. (Right) NAC blocks the synergistic toxicity. (C) NAC decreased arsenic-induced ROS signals. After the addition of 1 nM bryostatin, the arsenic-treated cells showed significant induction of chemiluminescence of luminol plus HRP than those cells coincubated with NAC.