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.A lthough arsenic has played a significant therapeutic role in various diseases for Ͼ2,000 years (1, 2), it was not used clinically for decades, until recently when clinical trials worldwide confirmed its dramatic therapeutic effects in acute promyelocytic leukemia (APL) (3, 4). APL is a subtype of acute myelocytic leukemia with most cases carrying the characteristic chromosomal translocation t(15, 17) that results in the PML-RAR␣ fusion protein (5). Although APL is highly responsive to arsenic, the presence of PML-RAR␣ fusion protein is neither absolutely necessary nor sufficient for sensitivity to arsenic (3, 6, 7). The mechanism by which arsenic is effective against APL remains elusive, despite studies suggesting that arsenic can promote degradation of the oncogenic PML-RAR␣ fusion protein (8, 9). Paradoxically, arsenic is also an established human carcinogen that can induce reactive oxygen species (ROS), leading to DNA damage or cell death (10-13).Some previous mechanistic studies (14, 15) were limited to exposure of cells other than myeloid cells, or to arsenite rather than arsenic trioxide for brief periods, and hence do not reflect the clinical setting for cytotoxic effects of arsenic on APL cells. To explore the molecular mechanisms of arsenic's therapeutic effects in the treatment of APL patients with daily continuous infusion of arsenic trioxide, we treated a human APL cell line, NB4, for Ͼ1 week with arsenic trioxide at a dose lower than the plasma trough level achieved in APL patients. We reported previously that arsenic at this dose was able to down-regulate human telomerase hTERT transcription (16). In this report, we determined changes in gene expression profiles by using oligonucleotide microarrays, and we found that NADPH oxidase components were dramatically up-regulated within days in m...

Inorganic Arsenic Activates Reduced NADPH Oxidase in Human Primary Macrophages through a Rho Kinase/p38 Kinase Pathway

“…Chou et al (15) have shown that prolonged treatment of NB4 cells (9 days) with 0.75 M As 2 O 3 markedly up-regulate mRNA levels and membrane expression of p47 phox . In contrast, our results show that p47 phox protein levels were similar in whole cell lysates of untreated and As 2 O 3 -treated cells; consequently, they suggest that iAs does not alter global NCF1 gene expression in macrophages.…”

“…Besides phagocytic NADPH oxidase, iAs can also stimulate nonphagocytic NADPH oxidase in endothelial cells (16,34), vascular smooth muscle cells (35), and in the human promyelocytic NB4 cell line (15); however, molecular mechanisms mediating its activation remain poorly understood. Chou et al (15) have shown that prolonged treatment of NB4 cells (9 days) with 0.75 M As 2 O 3 markedly up-regulate mRNA levels and membrane expression of p47 phox .…”

“…Cellular effects of iAs are frequently related to the increased production of reactive oxygen species (ROS) (4,14); recent studies have reported that NADPH oxidase could, in part, mediate ROS formation in iAs-treated cells (15,16). Interestingly, NADPH oxidase is a major superoxide-generating enzymatic system, first described in monocytes/macrophages (17).…”

Mitochondria in hematopoiesis and hematological diseases

“…For example, adenine deoxynucleoside analogs commonly used for treating some lymphoid malignancies such as cladribine and 2-chlororo-2 0 -ara-fluorodeoxyadenosine (Genini et al, 2000) and arsenic trioxide, an old cytotoxic agents that proved efficient in treating acute promyelocytic leukemia (Kroemer and de The, 1999;Larochette et al, 1999;Sordet et al, 2001). In acute promyelocytic leukemia cells, cellular toxicity of arsenic trioxide appears to mainly result from increased production of ROS, leading to the opening of the permeability transition pore complex, a decrease in mitochondrial membrane potential, cytochrome c release and caspase activation (Zhou et al, 2003;Chou et al, 2004). Ascorbic acid can enhance the efficacy of arsenic trioxide by reducing intracellular glutathione concentrations and this combination is currently tested in elderly patients with acute myeloid leukemia when these patients are not able to withstand intensive chemotherapy.…”

Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells

“…As 2 O 3 -induced apoptosis were accompanied by activation of death signals: caspase 3, 8 and 9 [10,11]. The generation of ROS appears to consistently accompany arsenic exposure and many evidences suggest that ROS may be a determinant of cellular susceptibility to arsenic [6,[12][13][14][15]. Manipulation of ROS may thus be a way to expend the anticancer therapeutic spectrum of As 2 O 3 .…”