Original Research Article

Reactive Oxygen Species-dependent Nitric Oxide Production in Reciprocal Interactions of Glioma and Microglial Cells† SHING-CHUAN SHEN1#, MING-SHUN Wu2#, HUI-YI LIN3, LIANG-YO YANG4, YI-HSUAN CHEN1, YEN-CHOU CHEN1,5* 1

Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan. Department of Gastroenterology, Taipei Medical University Wan Fang Hospital, Taipei 116, Taiwan. Department of Cosmetic Application and Management, St. Mary’s Medicine Nursing and Management College, Yilan 265, Taiwan 4 Department of Physiology and Graduate Institute of Neuroscience, Taipei Medical University, Taipei 110, Taiwan. 5 Cancer Research Center and Orthopedics Research Center, Taipei Medical University Hospital, Taipei110, Taiwan. 2

*Corresponding author: Dr. Yen-Chou Chen Tel: 886-2-27361661 ext. 3421 Fax: 886-2-23778620 E-mail: [email protected] (Y.C.Chen) # Dr. Shen and Dr. Wu equally contribute to the present work.

Running title: Reciprocal activation of microglia and glioma cells

Abbreviations

†

This article has been accepted for publication and undergone full peer review but has not been through

the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.24659] Additional Supporting Information may be found in the online version of this article. Received 30 October 2013; Revised 28 March 2014; Accepted 25 April 2014 Journal of Cellular Physiology © 2014 Wiley Periodicals, Inc. DOI: 10.1002/jcp.24659

CSF-1, colony-stimulating factor-1; CM, conditioned medium; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2; IκB, inhibitor of NF-κB; ROS, reactive oxygen species; JNK, c-Jun N-terminal protein kinase; ERK, extracellular signalregulated kinase; MAPK, mitogen-activated protein kinase; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase; NAC, N-acetylcysteine; NAME, N-nitro L-arginine methyl ester; NF-κB, nuclear factor-kappa B; NO, nitric oxide; SNP, sodium nitroprusside; TAMs, tumor-associated macrophages; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; Vit C, vitamin C; MTT, 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

Abstract Conditional mediums (CMs) from glioma cells U87, GBM-8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV-2 but without altering the cell viability or cell-cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87-CM and C6-CM were detected by a DCHF-DA assay, and vitamin (Vit) C and N-acetyl cysteine (NAC)-reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signalregulated kinase (ERK) inhibitor, U0126, and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed U87-CM- and C6-CM-induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87-CM and C6-CM. Increased migration of U87 and C6 glioma cells by a co-culture with BV-2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV-2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 messenger (m)RNA in U87 and C6 cells were detected by an RT-PCR, and TNF-α and MCP-1 induced iNOS protein expression in time- and concentration-dependent manners. Neutralization of TNF-α or MCP-1 in U87-CM and C6-CM using a TNF-α or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF-α or MCP-1 was identified in BV-2 cells. The reciprocal activation of glioma cells and microglia via ROS-dependent iNOS/NO elevation at least partially mediated by TNF-α and MCP-1 is elucidated. Keywords: Glioma, Microglia, Migration, iNOS, Reactive Oxygen Species

INTRODUCTION Microglial cells mediate immune responses during central nervous system (CNS) repair, injury, and infection, and are the most susceptible sensors of brain pathologies that produce morphological and functional alterations (Graeber and Streit, 2010; Yang et al., 2011). Activated microglial cells can migrate to sites of inflammation and neurogenesis, and contribute to those processes. Gliomas are the most common and aggressive primary brain cancer, and the tumor microenvironment plays a critical role in glioma invasion and progression (Jackson et al., 2001; Ohgaki and Kleihues, 2005). Microglia are some of the most abundant immune cells in gliomas, and histopathological studies showed infiltration of gliomas with numerous myeloid cells including microglia and macrophages (Hussain et al., 2006; Yang et al., 2010). There is emerging evidence that microglia play a role in aiding glioma progression, and gliomas contain substantial numbers of microglia at the invasive border (Bettinger et al., 2002; Markovic et al., 2005). A direct positive correlation between the number of microglia and the grade of gliomas was found (Roggendorf et al., 1996). Data of an in vivo study showed that depletion of microglia of mice showed a significant slower tumor growth of gliomas (Jacobs et al., 2012; Markovic et al., 2005). Contrary to the enhancement of microglia activity on gliomas, the mechanism of gliomas in activating microglia is still unclear. Nitric oxide (NO), which is liberated from NO synthase (NOS), is a key regulatory molecule that is highly induced during inflammation (Chien et al., 2012; Tsai et al., 2013). Three isoforms of NOS including endogenous (e)NOS, inducible (i)NOS, and neuronal (n)NOS were identified, and a positive correlation of iNOS activation with cancer progression was indicated. In addition, expression of iNOS in tumor cells can trigger genetic instability (Hoki et al., 2007; Yang et al., 2009) induce the expressions of matrix metalloproteinase (MMP)-1 and -2, and vascular endothelial growth factor

(VEGF)-C and -D, and is associated with tumor growth, invasion, and lymphangiogenesis (Nakamura et al., 2006; Viswanathan et al., 2008). Microglial cells can generate large amounts of NO after exposure to inflammatory compounds such as lipopolysaccharide, peptidoglycan, and β-amyloid (Roy et al., 2008). Kim et al. reported that C6 glioma cell matrix enhanced interferon (INF)-γ- but inhibited LPS-induced iNOS/NO production, in BV-2 microglial cells (Kim et al., 2008). Yao et al. indicated that inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)1α released by activated macrophages stimulate lung cancer angiogenesis (Yao et al., 2005). Our previous study (Lin et al., 2010b) showed that increased NO production participates in the reciprocal activation of macrophages and breast carcinoma cells mediated by colony stimulating factor (CSF)-1 production. The biological significance and roles of NO produced by glioma-activated microglial cells in promoting the migration of glioma cells are still unclear. Reactive oxygen species (ROS) are central players in the physiological control of cellular functions as modulators of proinflammatory processes in microglia-associated neurodegenerative diseases (Dokic et al., 2012). At sites of inflammation, activated microglial cells are able to cause the overproduction of ROS and various proinflammatory cytokines such as NO and TNF-α, which contribute to inflammationmediated

neurological

disorders

(Yeh

et

al.,

2012).

In

the

presence

of

lipopolysaccharide (LPS) stimulation, increased iNOS/NO production is mediated by the elevated intracellular ROS production in macrophages. Additionally, three welldefined subgroups of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the p38 MAPKs are involved in cell growth, cell death, and the migration of tumor cells. Activation of MAPKs not only by receptor ligand interactions but also by various stimulus on the cells has been implicated in tumorigenesis, and targeting malignant

glioma MAPKs signaling may improve clinical outcomes (Wong et al., 2007). ROS were reported to activate ERKs, JNKs, and p38 MAPKs, and oxidative modifications of MAPK signaling proteins and/or inactivation of MAPK phosphatases may provide plausible mechanisms for ROS activation of MAPKs in cells (Chiu et al., 2010). However, the contributions of ROS and MAPK to interactions between microglial and glioma cells are still undefined. Coculture of BV-2 microglial cells with glioma cells including GBM-8401, U87-MG, and C6 cells was used to investigate the interaction between microglia and glioma cells in the present study. Increased iNOS/NO production was observed in BV-2 cells when cocultured with glioma cells or treated with conditioned medium (CM) from the indicated glioma cells. The contributions of ROS and MAPKs to iNOS/NO elevation by glioma-activated BV-2 microglial cells and their roles in glioma cell migration were investigated.

Materials and Methods Cell culture GBM8401 (BCRC60163), U87MG (BCRC60360), and C6 (BCRC60046) glioblastoma cells were purchased from BCRC (Hsinchu, Taiwan), and BV-2 microglial cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, and 0.1 mM non-essential amino acids (NEAAs) at 37 °C in a humidified incubator containing 5% CO2. Primary cultured astrocytes were derived from newborn

Sprague-Dawley rats, and were cultured in DMEM-F12 medium containing 10% FBS. All culture reagents were purchased from Life Technologies (Gaithersburg, MD, USA). Chemicals Antibodies against iNOS (sc-650), cyclooxygenase (COX)-2 (sc-1745), total ERK (tERK; sc-154), total JNK (tJNK; sc-571), Jun (sc-1694), α-tubulin (sc-5286), and total inhibitor of nuclear factor (NF)-κB (IκB) (tIκB; sc-371) proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for detecting phosphoJNK (pJNK), phospho-ERK (pERK), phospho-IκBα (pIκB), monocyte chemoattractant protein (MCP)-1, phospho-Jun (pJun), and TNF-α proteins were from Cell Signaling Technology (Beverly, MA, USA). U0126 (U0), SP600125 (SP), and SB203580 (SB) were obtained from Calbiochem (La Jolla, CA, USA). N-Acetyl cysteine (NAC), vitamin (Vit) C, N-nitro L-arginine methyl ester (L-NAME), protease inhibitor cocktail, phosphatase inhibitor cocktail, and sodium nitroprusside (SNP) were purchased from Sigma (St. Louis, MO, USA). Recombinant CSF-1, MCP-1, TGF-β, and TNF-α were purchased from R&D (Minneapolis, MN, USA).

CM preparation and stimulation To collect CM from breast cancer, 2 x 105 glioma cells including GBM8401, U87MG, and C6 were seeded overnight; the medium was collected, centrifuged at 2000 rpm to separate out the debris, and stored at 4 °C for no more than 1 week. For stimulation with glioma-CM, BV-2 cells were replaced with serum-free medium (SFM) and supplemented with CM from glioma cells to different concentrations (v/v), and the control group was supplemented with an equal volume of uncultured CM. Coculture experiments and glioma migration assay

In the transwell coculture system, glioma cells U87 and C6 (4x104) suspended in 200 l of DMEM were seeded onto transwell inserts (with a 0.8-μm pore size; Corning, Corning, NY, USA), and BV-2 cells (2×105) were seeded in the lower chambers and incubated in a humidified incubator with 5 % CO2.for different times for respective purposes. In BV-2 cells, the peroxide levels by DCHF-DA labeling at 5 h, p(t)ERK/p(t)JNK protein at 6 h, and iNOS/NO production at 16 h after coculture were detected by flow cytometric analysis, Western blotting, and Griess reaction, respectively. Glioma cells that had not invaded were removed with a cotton swab, and the migrated glioma cells U87 and C6 attached to the lower surface of membrane were fixed and stained with 10% Giemsa at room temperature for 30 min and the number of cells on the lower surface of the filters was counted under the microscope. Cells which had migrated were counted using three randomly selected fields at 200x magnification for each transwell filter. Reverse-transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated with an RNA extraction kit (Amersham Pharmacia, Buckinghamshire, UK), and the concentration of total RNA was measured spectrophotometrically. RNA (2 μg) was converted to complementary (c)DNA by an RT-PCR Bead kit (Amersham Pharmacia) according to the manufacturer’s protocol. The amplification sequence was 30 cycles of 94 °C for 30 s, 56~60 °C for 30 s, and 72 °C for 1 min. The PCR product of each sample was analyzed by electrophoresis in a 1.5% agarose gel, and visualized by ethidium bromide staining. The oligonucleotide primer sequences were as follows: MCP-1, 5'-GCTCATAGCAGCCACCTTCATTC-3' (forward)

and

5'-GGACACTTGCTGCTGGTGATTC-3'

ATGTGCTCTGAAAGCATGTCTCCAAA-3' CCCTTCACAGAGCAATGACTCCAAA-3'

(reverse);

(forward) (reverse);

and

TNF-α, and

GAPDH,

5'5'5′-

TGAAGGTCGGTGTGAACGGATTTGGC-3′

(forward)

and

5′-

CATGTAGGCCATGAGGTCCACCAC-3′ (reverse). Measurement of NO The extent of NO production was detected with the Griess reagent. Briefly, 100 μl of medium from stimulated RAW264.7 macrophages was mixed with the same volume of Griess

reagent

(1%

sulfanilamide

in

5%

phosphoric

acid

and

0.1%

naphthylethylenediamine dihydrochloride in water). The absorbance of the mixture was detected with an enzyme-linked immunosorbent assay (ELISA) at an optical density (OD) of 530 nm, with sodium nitrite as the standard. Western blotting Cells lysates were prepared by suspending cells in lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 1 mM EGTA, 0.025% sodium deoxycholate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). An equal amount of protein was added to 5x sodium dodecylsulfate (SDS) sample buffer with 2-mecaptoethanol and separated on 10% SDS-polyacrylamide gels, and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedfore, MA). Membranes were incubated at 4 C with 1% bovine serum albumin and then incubated with the indicated antibodies for a further 3 h at room temperature followed by incubation with an alkaline phosphatase-conjugated immunoglobulin G (IgG) antibody for 1 h. Protein was visualized by incubating with the colorimetric substrates, NBT and BCIP. Measurement of ROS generation by intact cells Intracellular production of ROS by BV-2 cells under different treatments was measured by oxidation of DCFH-DA to DCF. DCFH-DA is a non-polar compound that readily diffuses into cells, where it is hydrolyzed to the non-fluorescent polar derivative, DCFH, and thereby trapped within cells. If DCFH-DA is oxidized, it turns into the highly

fluorescent DCF. After different treatments, cells were incubated in the dark for 10 min at 37 °C with 50 μM DCFH-DA, then harvested, and resuspended in plain medium. Each sample consisted of 104 cells, and the fluorescence in BV-2 cells was analyzed using a FACScan (Becton Dickinson, Sunnyvale, CA, USA) flow cytometer with excitation at 488 nm and emission at 530 nm. Increases in the peroxide levels were quantitated by measuring the percentage of cells (%) at the M1 intervals. MTT assay The viability of BV-2 microglial cells was measured as previously described using a colorimetric method based on the metabolic reduction of the soluble yellow tetrazolium dye, MTT, to the insoluble purple formazan by the action of mitochondrial succinyl dehydrogenase. BV-2 cells were treated with different components for 16 h, then incubated at 37 ºC for 4 h containing 100 mM MTT under 5% CO2/95% air, followed by dissolution in 500 μl of a lysis solution (10% SDS in 0.01 N HCl). The absorbance of the solution was read at 595 nm using a multiplate reader, and the cell viability was expressed as a percentage of the control (%). Statistical analysis Values are expressed as the mean±standard error (SE) of triplicate experiments. The significance of the difference from the respective controls for each experimental condition was assayed using a one-way analysis of variance (ANOVA) for a post-hoc Bonferroni analysis when applicable, and a p value of