Role of NADPH Oxidase in Arsenic-Induced Reactive Oxygen Species Formation and Cytotoxicity in Myeloid Leukemia Cells

Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells Wen-Chien Chou*†‡, Chunfa Jie§, Andrew A. Kenedy¶, Richard J. Jonesʈ, Michael A. Trush¶, and Chi V. Dang*†¶ʈ** *Program of Human Genetics and Molecular Biology, †Department of Medicine, §McKusick–Nathans Institute of Genetic Medicine, and ʈSidney Kimmel Comprehensive Cancer Center, School of Medicine, ¶Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205 Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved January 21, 2004 (received for review October 16, 2003) Arsenic has played a key medicinal role against a variety of associated and cytosolic subunits, can be stimulated by phorbol ailments for several millennia, but during the past century its myristate acetate (PMA) through protein kinase C-mediated prominence has been displaced by modern therapeutics. Recently, phosphorylation of the p47PHOX subunit (17, 18). This complex attention has been drawn to arsenic by its dramatic clinical efficacy is responsible for the production of superoxide anion (respira- against acute promyelocytic leukemia. Although toxic reactive tory burst) of professional phagocytes encountering microbial oxygen species (ROS) induced in cancer cells exposed to arsenic pathogens, and its importance in host immunity is underscored could mediate cancer cell death, how arsenic induces ROS remains by the immunocompromised congenital disease, chronic gran- undefined. Through the use of gene expression profiling, interfer- ulomatous disease (CGD), which results from mutations in one ence RNA, and genetically engineered cells, we report here that of the subunits of NADPH oxidase (19, 20). Our biochemical and NADPH oxidase, an enzyme complex required for the normal molecular biological studies reported here have uncovered a antibacterial function of white blood cells, is the main target of major role of this enzyme complex in arsenic-induced ROS arsenic-induced ROS production. Because NADPH oxidase enzyme production and cytotoxicity. We have also exploited the syner- activity can also be stimulated by phorbol myristate acetate, a gistic induction of NADPH oxidase activity and ROS production synergism between arsenic and the clinically used phorbol myris- by arsenic and PMA to provide proof-of-concept that this tate acetate analog, bryostatin 1, through enhanced ROS produc- synergy may be clinically applicable. tion can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can Methods effectively kill leukemic cells. Our findings pinpoint the arsenic Cell Lines. NB4, U937, PLB-985, X-CGD, and HL60 cells were target of ROS production and provide a conceptual basis for an cultured in RPMI medium 1640 supplemented with 10% FBS. anticancer regimen. ML1 was maintained in RPMI medium 1640 with 7.5% FBS and 3.4 g of Hepes͞500 ml, pH 7.4. lthough arsenic has played a significant therapeutic role in Ͼ Avarious diseases for 2,000 years (1, 2), it was not used Microarray Analysis. NB4 cells were grown to a density of 105͞ml clinically for decades, until recently when clinical trials world- and were treated with 0.75 ␮M arsenic trioxide for 10 days. wide confirmed its dramatic therapeutic effects in acute promy- mRNA was isolated with the Qiagen RNeasy minikit and was elocytic leukemia (APL) (3, 4). APL is a subtype of acute subjected to Affymetrix oligonucleotide microarray analysis by myelocytic leukemia with most cases carrying the characteristic using an HG࿝U133A chip. Five replicates, including two control chromosomal translocation t(15, 17) that results in the PML- and three arsenic-treated NB4 samples, were studied. With the RAR␣ fusion protein (5). Although APL is highly responsive to ␣ expectation that only a small fraction of genes is differentially arsenic, the presence of PML-RAR fusion protein is neither expressed between samples under different treatments, the absolutely necessary nor sufficient for sensitivity to arsenic (3, 6, brightness of chips for the samples was adjusted to comparable 7). The mechanism by which arsenic is effective against APL level by normalizing the CEL file of signal values and the probe remains elusive, despite studies suggesting that arsenic can pair (perfect match and mismatch) level data of the Affymetrix promote degradation of the oncogenic PML-RAR␣ fusion expression chips, with the method of ‘‘invariant set normaliza- protein (8, 9). Paradoxically, arsenic is also an established human tion’’ (21). The normalized CEL data were then used to estimate carcinogen that can induce reactive oxygen species (ROS), the perfect match͞mismatch-model-based expression index leading to DNA damage or cell death (10–13). Some previous mechanistic studies (14, 15) were limited to (with SE) for the probe sets (22), leading to the further com- exposure of cells other than myeloid cells, or to arsenite rather putation of the fold changes and their 90% confidence intervals than arsenic trioxide for brief periods, and hence do not reflect (21). The lower bound of a 90% confidence interval, a conser- the clinical setting for cytotoxic effects of arsenic on APL cells. vative estimate of the fold change, was then used to identify To explore the molecular mechanisms of arsenic’s therapeutic differentially expressed genes. The computing was performed effects in the treatment of APL patients with daily continuous with DCHIP 1.2. infusion of arsenic trioxide, we treated a human APL cell line, NB4, for Ͼ1 week with arsenic trioxide at a dose lower than the This paper was submitted directly (Track II) to the PNAS office. plasma trough level achieved in APL patients. We reported Abbreviations: APL, acute promyelocytic leukemia; ROS, reactive oxygen species; PMA, previously that arsenic at this dose was able to down-regulate phorbol myristate acetate; DPI, diphenyleneiodonium; HRP, horseradish peroxidase; siRNA, human telomerase hTERT transcription (16). In this report, we small interference RNA; NAC, N-acetylcysteine. determined changes in gene expression profiles by using oligo- ‡Present address: Department of Internal Medicine, National Taiwan University Hospital, nucleotide microarrays, and we found that NADPH oxidase Taipei 100, Taiwan. components were dramatically up-regulated within days in my- **To whom correspondence should be addressed at: Ross Research Building, Room 1032, eloid cells treated with low-dose arsenic. NADPH oxidase, which 720 Rutland Avenue, Baltimore, MD 21205. E-mail: [email protected]. is an enzyme complex consisting of multiple membrane- © 2004 by The National Academy of Sciences of the USA 4578–4583 ͉ PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306687101 Downloaded by guest on September 26, 2021 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 Ն5) 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 cor- responding 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 MEDICAL SCIENCES up-regulated genes, based on the lower bound fold change. Real-Time PCR. Detection of hTERT was described (16). Expres- sion of other genes was determined by reverse transcription followed by SYBR green real-time PCR. All primer sequences for the genes tested are available on request. cDNA was gener- ated by first heating a 15-␮l mixture containing 15 ␮g of total RNA and 1 ␮g of random primers (Promega) to 70°C for 5 min. After immediate chilling on ice, 5 ␮lof5ϫ reaction buffer, 5 ␮l of dNTP (2.5 mM each), 40 units of RNase inhibitor, and 200 Fig. 2. Up-regulation of NADPH oxidase and eosinophil peroxidase expres- units of Moloney murine leukemia virus reverse transcriptase sion by arsenic. (A) Absent immunohistochemical staining in control cells (Left) (Promega) were added, and the mixture was incubated at 37°C and intense staining of eosinophil peroxidase in arsenic-treated NB4 cells for 1 h. Ten nanograms of cDNA was subjected to SYBR green (Right). The staining in arsenic-treated cells depended on the primary anti- PHOX quantitative real-time PCR. Every tube of 20 ␮l contained 500 body (data not shown). As, arsenic. (B) Immunoblotting of p47 and p67PHOX shows dramatic increases in the protein levels in NB4 cells after arsenic nM each of primer, 200 ␮M dNTP, and PCR buffer with 1.75 ͞ ␮ treatment. ␣-Tubulin was used as a loading control. (C) Arsenic increased mM MgCl2 0.5 l of 15,000-fold diluted SYBR (Molecular ͞ mRNA of all of the NADPH oxidase subunits in NB4 cells as measured by SYBR Probes) 0.5 units of PlatinumTaq (Invitrogen). All primers were green quantitative real-time PCR. designed to cross introns and span Ͻ400 base pairs of the mRNA. All PCRs were performed at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 123 (Sigma) for 30 min at 37°C. The ROS was determined by the sec. The signals were detected with the ABI 7700 sequence fluorescent intensity by flow cytometry with excitation at 490 nm detection system. All of the signals were normalized by the and emission at 520 nm. expression levels of large acidic ribosomal protein (RPLP0). ROS Detection by Luminol Chemiluminescence. To detect extracel- ␮ ϫ 6 Immunohistochemical Staining. The cells were prepared by cyto- lular and intracellular ROS, 10 M luminol was added to 1 10 cells in 2 ml of aerated complete PBS (PBS with 0.5 mM spin and were fixed with methanol and then acetone for 2 min ͞ ͞ MgCl2 0.7 mM CaCl2 0.1% glucose) supplemented with 10 each.

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