9, 10-Phenanthrenequinone Induces Monocytic Differentiation of U937

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 reactive 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 investigated 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 expression 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 antibody.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

Load more