1598 Note Biol. Pharm. Bull. 35(9) 1598–1602 (2012) Vol. 35, No. 9

9,10-Phenanthrenequinone Induces Monocytic Differentiation of U937 Cells through Regulating Expression of Aldo-Keto Reductase 1C3 Toshiyuki Matsunaga,*,a Mika Hosogai,a Marina Arakaki,a Satoshi Endo,a Ossama El-Kabbani,b and Akira Haraa a Laboratory of Biochemistry, Gifu Pharmaceutical University; Gifu 501–1196, Japan: and b Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences; Victoria 3052, Australia. Received March 9, 2012; accepted June 5, 2012

Persistent inhalation of diesel exhaust particles results in damaged lung cells through formation of re- active oxygen species (ROS), but the details of the toxicity mechanism against monocytes are poorly under- stood. In this study, we used human promyelomonocytic U937 cells as surrogates of monocytes and investi- gated the toxicity mechanism initiated by exposure to 9,10-phenanthrenequinone (9,10-PQ), a major quinone component in diesel exhaust particles. A 24-h incubation with 9,10-PQ provoked apoptotic cell death, which was due to signaling through the enhanced ROS generation and concomitant caspase activation. Flow cy- tometric analyses of U937 cells after long-term exposure to 9,10-PQ revealed induction of differentiation that was evidenced by increasing expression of CD11b/CD18, a cell-surface marker for monocytic differentiation into macrophages. The 9,10-PQ-induced differentiation was significantly abolished by ROS inhibitors, sug- gesting that ROS generation contributes to cell differentiation. The 9,10-PQ treatment increased the expres- sion of aldo-keto reductase (AKR) 1C3, which reached a peak at 1 to 2 d post-treatment and then declined. The bell-shaped curve of the AKR1C3 expression by 9,10-PQ resembled that caused by phorbol 12-myristate 13-acetate, a differentiation inducer. Additionally, the concomitant treatment with tolfenamic acid, a selec- tive AKR1C3 inhibitor, sensitized the differentiation induced by 9,10-PQ. These results suggest that ROS formation during 9,10-PQ treatment acutely leads to apoptosis of U937 cells and the initiation of monocytic differentiation, which proceeds after the provisional overexpression of AKR1C3. Key words 9,10-phenanthrenequinone; differentiation; aldo-keto reductase 1C3; U937; oxidative stress

Diesel exhaust particles (DEP) are believed to be harmful administration15) However, detrimental effect of 9,10-PQ on air pollutants that cause significant impediments to respiratory monocytes remains unknown. function leading to asthma and carcinogenesis.1,2) The mech- Enzymes belonging to the aldo-keto reductase (AKR) anisms underlying their disorders is very complicated due to superfamily are generally cytosolic coenzyme-dependent the involvement of a variety of lung cells such as epithelial oxidoreductases that metabolize a variety of carbonyl com- cells, macrophages and lymphocytes.2) In the acute phase, pounds including endogenous carbohydrates, steroids and macrophages are directly activated in response to stimulation prostaglandins (PGs).16) Among the 15 human enzymes with DEP resulting in the regulation of the immune signaling belonging to this superfamily, three structurally similar en- networks through secretion of pro-inflammatory mediators, zymes, AKR1C1, AKR1C2 and AKR1C3, are expressed in cytokines3) and chemokines.4) In addition, the inhalation normal ubiquitous tissues at lower levels, but highly induced of DEP was shown to increase the number of lung macro- with progression of several cancers including lung, prostate phages,5) which may be involved in clearing the particles by and breast cancers.16) Thus, the enzymes are therefore consid- phagocytotic ingestion.6) Thus, macrophages are believed to ered to act as diagnostic markers relevant to the carcinogenesis play pivotal roles in the cytoprotective responses against DEP. and progression of these cancer cells. In particular, AKR1C3 On the other hand, several researchers found that the admin- that is known as type 5 17β-hydroxysteroid dehydrogenase17) 18) istration of DEP lowers both antimicrobial and mitochon- and PGF2α synthase, functions in the formation of andro- drial functions in alveolar macrophages through generation gens and PGF2α isomers (PGF2α and 9α,11β-PGF2), which are of reactive oxygen species (ROS) in addition to activating involved in promotion of the cellular growth potential through apoptotic signaling.7,8) DEP are heterogeneous species that signaling via receptors for steroid and prostanoid.19,20) In fact, consist of a variety of organic and inorganic chemicals, such a recent cell-based study has showed that ectopic overexpres- as polycyclic aromatic hydrocarbons, nitroaromatic hydrocar- sion of AKR1C3 induces proliferation of breast cancer MCF-7 bons, heterocycles, aldehydes and quinones.9) Among these cells through the production of estrogens and a decrease in chemicals, 9,10-phenanthrenequinone (9,10-PQ), one of the the levels of anti-proliferative PGD2 and peroxisome prolifera- 12,14 19) most cytotoxic quinones found in DEP, induces severe injury tor-activated receptor (PPAR) γ ligand 15-deoxy-Δ -PGJ2. toward various cells at very low concentrations,10,11) mainly In addition, down-regulation of AKR1C3 promotes differenti- due to excessive ROS generation during the redox cycling ation of HL-60 and K562 leukemia cells.21,22) Thus, AKR1C3 process.10–14) It was previously reported that intratracheal in- is suggested to play a key role in controlling proliferation and oculation of 9,10-PQ into mice resulted in significant elevation differentiation of the hormone-sensitive cancers and leukemia in the number of macrophages present in the bronchoalve- cells. olar lavage fluid, which mimics that observed by the DEP In this study, we have investigated the toxic effect of 9,10- PQ on human promyelomonocytic U937 cells as surrogates of The authors declare no conflict of interest. monocytes, and the cytotoxic mechanism related to apoptotic

* To whom correspondence should be addressed. e-mail: [email protected] © 2012 The Pharmaceutical Society of Japan September 2012 1599 signaling including ROS production and caspase activation. with the specific primers (Toyobo, Osaka, Japan). We have also found that long-term treatment with 9,10-PQ Western Blot Analysis The cells were suspended in ice- induces the monocytic differentiation into macrophages and cold DPBS containing 0.1% Triton X-100 and 0.3 mM phenyl- reduces AKR1C3 expression. Additionally, we examined the methanesulfonyl fluoride, and their lysates were prepared by relationship between cell differentiation and both ROS pro- the homogenization and centrifugation as described above. duction and AKR1C3 expression. After their protein concentrations were determined in the bicinchoninic acid assay, the proteins (40 µg) in the lysates MATERIALS AND METHODS were applied to Western blotting using the anti-AKR1C3 anti- body.27) The β-actin level is also detected as a loading control Materials U937 cells were purchased from Amer- using the specific antibody (Cell Signaling Technology, Dan- ican Type Culture Collection (Manassas, VA, U.S.A.). vers, MA, U.S.A.). 6-Carboxy-2′,7′-dichloro-dihydrofluorescein diacetate-di(ace- Statistical Analysis Statistical evaluation of the data was toxymethylester) (DCFH-DA) was obtained from Molecular performed by using the unpaired Student’s t-test and analysis probes (Eugene, OR, U.S.A.). 3-Chloro-5-phenylsalicylic acid of variance (ANOVA) followed by Fisher’s test. A p value (CPSA) was synthesized according to the method described <0.05 was considered statistically significant. previously.23) Polyethylene glycol-conjugated catalase (PEG- CAT) was generously gifted from Dr. Yoshito Kumagai (Uni- RESULTS AND DISCUSSION versity of Tsukuba, Japan). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.), 9,10-PQ Induces U937 Cell Apoptosis through a Invitrogen (Carlsbad, CA, U.S.A.), Amersham Biosciences ROS-Dependent Mechanism Incubation with 9,10-PQ for (Piscataway, NJ, U.S.A.), Nacalai Tesque (Kyoto, Japan) and 24 h provoked U937 cell damage, which was remarkable at Wako Pure Chemical Industries, Ltd. (Osaka, Japan). concentrations more than 1 µM (Fig. 1A). In addition, apoptotic Cell Culture and Measurement of ROS and Caspase Ac- morphological alterations such as chromatin condensation and tivity U937 cells were grown in Dulbecco’s modified Eagle’s formation of apoptotic bodies were observed in almost all medium (DMEM) supplemented with 10% fetal bovine serum of the cells treated with 2 µM 9,10-PQ (supplemental Fig. 1), (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) at demonstrating that the 9,10-PQ toxicity toward the monocytes

37°C in a humidified incubator containing 5% CO2. Viable is due to apoptotic cell death. To determine whether ROS cell number was evaluated by the trypan blue dye-exclusion resulting from the redox cycling of 9,10-PQ participate in an method.24) Intracellular ROS level was estimated by oxida- apoptotic mechanism(s) that involve the quinone, we mea- 25) tion of DCFH-DA as previously reported. The activities sured H2O2 concentrations in the quinone-treated cells using of caspase-3 and caspase-9 in extracts from U937 cell were a fluorogenic probe DCFH-DA (Fig. 1B). The treatment with measured using acetyl Asp-Glu-Val-Asp p-nitroanilide and 1 µM 9,10-PQ considerably increased the DCF-positive cells, acetyl Leu-Glu-His-Asp p-nitroanilide, respectively, as the in which marginal activation of caspases (caspase-3 and -9), substrates.25) key effectors for the ROS-dependent apoptotic induction,10,25) Induction and Evaluation of Differentiation U937 cells was also validated (Fig. 1C). Moreover, pretreating with ROS were suspended in the growth medium, seeded into a 60- inhibitors, N-acetyl-L-cysteine (NAC) and PEG-CAT, resulted mm dish at a density of 1×105 cells/mL and cultured for 5 d in significant protection against the cellular damage elicited in the medium with or without the agents. After fixing with by the 9,10-PQ treatment (Fig. 1D). These results suggest that Dulbecco’s phosphate-buffered saline (DPBS) containing 1% ROS production and its downstream caspase activation par- paraformaldehyde, the cells were incubated for 30 min with ticipate in the 9,10-PQ-initiated apoptotic signaling of mono- phycoerythrin-labeled anti-CD11b/CD18 antibody. Following cytes, similar to the 9,10-PQ-induced apoptosis in other cells the sufficient wash with DPBS, the population of the CD11b/ including T-lymphocytes10) and aortic endothelial cells.11) CD18-positive cells was detected by a Becton Dickinson Facilitation of Monocytic Differentiation of U937 Cells FACScan flow cytometer. To examine the ability of cells to re- by 9,10-PQ Treatment Long-term exposure (5 d) to 9,10- duce nitroblue tetrazolium (NBT), the cells were suspended in PQ made U937 cells more sensitive to quinone toxicity the growth medium containing 0.1% NBT and then incubated compared to a 24-h exposure, resulting in a 1.6-fold decrease at 37°C for 30 min with shaking, and the percentage of cells in LD50 value for the quinone (data not shown). In addition, with blue-black formazan deposits was microscopically deter- considering that homotypic aggregation of U937 cells was mined on at least 200 cells. microscopically observed during the long-term treatment with Reverse Transcription-Polymerase Chain Reaction (RT- 9,10-PQ, it is assumed that the quinone is capable of trig- PCR) Analysis Total RNAs were isolated using TRIzol gering the monocytic differentiation in addition to the acute reagent and single-stranded cDNAs were prepared from the apoptotic signaling pathway mentioned above. To test this total RNA samples as described previously.26) The cDNAs assumption, we monitored the expression of a differentiation for AKR1C1, AKR1C2 and AKR1C3 were amplified from marker protein CD11b/CD18 during the exposure to 9,10-PQ the single-stranded cDNA sample (5 µg) by PCR using the by flow cytometry (the flow cytometric patterns are shown in gene-specific primers.26) The amplification of the NQO1 supplemental Fig. 2). While the expression level of the marker cDNA was similarly performed using the sense and antisense was slightly elevated even in the cells treated with dimeth- primers, 5′-atggtcggcagaagagcactg-3′ and 5′-tcattttctagcttt- ylsulfoxide (vehicle) and phenanthrene (9,10-PQ derivative gatctggttg-3′. The PCR products were analyzed on 1% agarose lacking the ortho-quinone structure), the 9,10-PQ treatment gel electrophoresis and ethidium bromide staining. The cDNA for 5 d dose-dependently increased the population of CD11b/ for human β-actin was also amplified as an internal control CD18-positive cells (Fig. 2). The differentiation was confirmed 1600 Vol. 35, No. 9

Fig. 1. Incubation with 9,10-PQ Triggers U937 Cell Apoptosis (A) Toxicity of 9,10-PQ. The cells were treated for 24 h with the indicated concentrations of 9,10-PQ. The viability values are expressed as percentages of the value in the control cells treated with the vehicle dimethylsulfoxide (DMSO) at the final concentration of 0.1%. (B) ROS generation. The cells were treated for 24 h with 0, 0.5 or 1 µM 9,10-PQ. The intracellular ROS levels were estimated by the oxidation of DCFH-DA, and are expressed as percentages of the DCF fluorescence-positive cells to total cells. (C) Caspase activation. The cells treated for 16 h with 0, 0.2, 0.5 or 1 µM 9,10-PQ. The activities of caspase-9 (open bar) and caspase-3 (closed bar) in the cell lysates are expressed as percentages of the activities in the control cells treated with DMSO alone (shown as 0 µM). (D) Effect of ROS inhibitors on the 9,10-PQ-induced cytotoxic- ity. The cells were pretreated for 2 h with 0.2 mM NAC or 200 U/mL PEG-CAT, and then treated for 24 h with 1 µM 9,10-PQ. * Significant difference from the group treated with 9,10-PQ alone (‘No inhibitor’), p<0.05. Values are means±S.D. of 4 samples in each group. by measuring the NBT-reductase activity, one of the earliest markers for differentiation of monocytes into macrophages.28) The NBT-reductase activity in the CD11b/CD18-positive cells was approximately 6-fold higher than that in the CD11b/ CD18-negative cells (data not shown). The 5-days treatment with 1 µM 9,10-PQ conferred a severe loss of the cell viability compared to the short-term (24 h) treatment (21% versus 46% of 1 d), suggesting an involvement of differentiation in the 9,10-PQ-mediated toxic mechanism. To our knowledge, this is the first report that 9,10-PQ induces the differentiation of monocytes into macrophages. The 9,10-PQ-induced differ- entiation was significantly abrogated by preincubation with the ROS inhibitors, NAC and PEG-CAT (Fig. 2). It should be noted that the ratio of CD11b/CD18-positive cells to total cells was increased by 38% when exposed to 80 µM H2O2 for 5 d. These results clearly demonstrate the participation of ROS formation in the mechanism underlying the 9,10-PQ-induced monocytic differentiation, and are in agreement with the report of Barbieri et al. that shows that the ROS driven by reduced nicotinamide adenine dinucleotide phosphate oxidase 29) play a critical role in this differentiation. Fig. 2. 9,10-PQ Treatment Promotes Differentiation of U937 Cells Alteration in AKR1C3 Expression Promotes 9,10-PQ- The cells were pretreated for 2 h with 0.2 mM NAC or 200 U/mL PEG-CAT, and Induced Monocytic Differentiation Exposure of lung then treated for 5 d with the indicated concentrations of phenanthrene (P) or 9,10- PQ. The CD11b/CD18 expression on the cells was analyzed by flow cytometry to epithelial cells to 9,10-PQ elevates the expression level of enumerate a ratio of the differentiated cells to total cells. * Significant difference 27) AKR1C3. AKR1C3 is overexpressed in prostate and breast from the group treated with 1 µM 9,10-PQ alone without the inhibitor, p<0.05. Each cancers, and is suggested to be involved in proliferation of bar is the mean±S.D. of 4 samples. cancer cells.30) Therefore, we examined whether 9,10-PQ affects the levels of AKR1C3 in U937 cells, in which the similar alteration in the expression of AKR1C1, AKR1C3 mRNAs for AKR1C3 and its isoforms AKR1C1 and AKR1C2 and NQO1 was observed when the cells were incubated with were detected by RT-PCR analysis. Among the three AKR1C phorbol 12-myristate 13-acetate (PMA), an inducer of dif- isoforms, the expression of the mRNAs for AKR1C1 and ferentiation via the enhanced ROS generation32) (Fig. 3A(b)). AKR1C3 was increased up to 2 d after the 9,10-PQ treatment, These results indicate that AKR1C1 and AKR1C3 are induced but declined at 5 d (Fig. 3A(a)). This pattern was similar to probably via the ROS-dependent pathway including Nrf2 ac- that of the nuclear factor-erythroid 2 p45-related factor 2 tivation. The ROS-mediated up-regulation of AKR1C3 is also (Nrf2)-driven gene, NQO1.31) Consistent with the time–course supported by the Western blot results that pretreating the cells of the mRNA expression, AKR1C3 protein expression induced with NAC markedly suppressed the AKR1C3 induction by by 9,10-PQ showed a bell-shaped curve (Fig. 3B). Moreover, 9,10-PQ (data not shown). In order to elucidate the relationship September 2012 1601

Fig. 3. AKR1C3 Inhibitor Sensitizes U937 Cells to 9,10-PQ-Mediated Differentiation (A) Time–course gene expression profiles of three AKR1C isoforms and NQO1. The cells were treated with 1 µM 9,10-PQ (a) or 5 nM PMA (b) for the indicated days, and the expressed mRNAs for the four enzymes were analyzed by RT-PCR using the gene specific primers and the representative pattern in 3 samples is indicated. (B) Western blotting of AKR1C3. The cells were treated with 1 µM 9,10-PQ for the indicated days, and their lysates (100 µg) were analyzed. The representative result is shown in the inset. The band density (the mean of two independent experiments) is expressed as the mean±S.D. of 4 samples. (C) Effects of AKR1C1 and AKR1C3 inhibitors on U937 cell differentiation induced by 9,10-PQ. The cells were treated for 5 d with 1 µM 9,10-PQ in the presence of 20 µM CPSA or tolfenamic acid (TOL). Each bar is the mean±S.D. of 6 samples.

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