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ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells Jantaratnotai, Nattinee; Choi, Hyun B; McLarnon, James G Dec 15, 2009

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ralssBioMed CentBMC CancerOpen AcceResearch articleATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cellsNattinee Jantaratnotai1,2, Hyun B Choi1 and James G McLarnon*1Address: 1Department of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada and 2Department of Pharmacology, Faculty of Science, Mahidol University, Rama VI Road, Phayathai, Bangkok 10400, ThailandEmail: Nattinee Jantaratnotai - scnjt@mahidol.ac.th; Hyun B Choi - chb1202@gmail.com; James G McLarnon* - mclarnon@interchange.ubc.ca* Corresponding author    AbstractBackground: Glioma present as one of the most challenging cancers to treat, however,understanding of tumor cell biology is not well understood. Extracellular adenosine triphosphate(ATP) could serve as a critical signaling molecule regulating tumor development. This study hasexamined pharmacological modulation of calcium (Ca2+) entry through store-operated channels(SOC) on cellular expression and production of immune-cell mobilizing chemokines in ATP-stimulated C6 glioma cells.Methods: Calcium spectrofluorometry was carried out to measure mobilization of intracellularCa2+ [Ca2+]i following ATP stimulation of rat C6 glioma cells. Pretreatment with two inhibitors ofSOC, SKF96365 or gadolinium, was used to examine for effects on [Ca2+]i. RT-PCR was performedto determine effects of purinergic stimulation on C6 cell expression of metabotropic P2Y receptors(P2YR) and the chemokines, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). ELISA was carried out to measure production of MCP-1 and IL-8 with ATP stimulation of gliomacells.Results: Application of ATP (at 100 μM) to C6 glioma induced an increase in [Ca2+]i with theresponse exhibiting two components of decay. In the presence of the SOC inhibitors, SKF96365or gadolinium, or with Ca2+-free solution, ATP responses lacked a slow phase suggesting thesecondary component was due to SOC-mediated influx of Ca2+. RT-PCR confirmed expression ofpurinergic P2Y-subtype receptors in C6 cells which would serve as a precursor to activation ofSOC. In addition, ATP-stimulated C6 cells showed enhanced expression of the chemokines, MCP-1 and IL-8, with SKF96365 or gadolinium effective in reducing chemokine expression. Gadoliniumtreatment of ATP-stimulated C6 cells was also found to inhibit the production of MCP-1 and IL-8.Conclusion: These results suggest ATP-induced Ca2+ entry, mediated by activation of SOC in C6glioma, as a mechanism leading to increased cellular expression and release of chemokines. Elevatedlevels of MCP-1 and IL-8 are predicted to enhance the mobility of tumor cells and promoterecruitment of microglia into developing tumors thereby supporting tumor growth.Published: 15 December 2009BMC Cancer 2009, 9:442 doi:10.1186/1471-2407-9-442Received: 7 July 2009Accepted: 15 December 2009This article is available from: http://www.biomedcentral.com/1471-2407/9/442© 2009 Jantaratnotai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442BackgroundGliomas are a common form of human brain tumor butremain essentially incurable due to their innate character-istic of extreme invasiveness [1]. The development andprogression of gliomas include reciprocal interactionsbetween glioma cells with resident immune respondingmicroglia and tumor-associated macrophages [2,3]. Inparticular, evidence suggests that tumor cells may producemobilizing factors for microglia/macrophages and thatchemokine responses of microglia could aid in establish-ing immunosuppressive environments facilitating tumorgrowth [4,5]. Among the most prominent glioma chem-okine factors are monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) which could inducerecruitment of microglia/macrophage to help supporttumor progression [6,7]. Moreover, MCP-1 has beenshown to directly induce angiogenesis [8] while bothchemokines also act as autocrine factors to drive the inva-sive phenotypes of the gliomas [6,9].A spectrum of stimulatory signals is likely present indeveloping gliomas which activate microglial chemotacticresponses and promote an overall immunosuppressivemicroenvironment. In particular, purinergic signalingpathways in glioma may be highly relevant in enhance-ment of chemotactic factors to recruit microglia to helpsustain tumor growth [10,11]. Glioma cells both releaseand respond to ATP [12,13] with catabolism of ATPextremely low in glioma cells, compared to astrocytes, dueto a marked reduction in expression and activity ofenzymes that degrade ATP [14]. Importantly, depletion ofATP has been reported to reduce the size and invasivecharacteristics of tumors in an animal glioma model [15].Evidence suggests that mobilization of intracellular cal-cium ([Ca2+]i), mediated by activation of the purinergicsubtype receptor, P2X7R by the ligand 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), serves as a link between ATP stim-ulation of glioma and cellular expression of chemokineand cytokine factors [16]. However, roles of other purin-ergic family members were also suggested since antago-nism of P2X7R had no effect to inhibit factor expressionand only partially suppressed calcium responses [16]. Inparticular, it was speculated that subtype P2YR could alsobe activated by BzATP thereby increasing [Ca2+]i by arapid release from endoplasmic stores followed by a sub-sequent sustained influx of the ion through store-oper-ated channels (SOC). These findings suggested therelevance of study using the endogenous compound ATPas an activator of C6 cells to determine effects of pharma-cological modulation of SOC-mediated Ca2+ entry on cel-lular production of chemokines.MethodsMaterialsAll chemicals were purchased from Sigma (St.Louise, MO)unless otherwise stated. Two inhibitors of SOC wereemployed in this work; gadolinium [17,18] andSKF96365 [19].Cell cultureC6 glioma cells were obtained from the American TypeCulture Collection (ATCC, Manassas, VA). Cells from pas-sage number 39-59 were used in this work. Cells were cul-tured in Kaighn's modification of Ham's F12 medium(F12K) with 2 mM l-glutamine modified by ATCC to con-tain 1.5 g/l sodium bicarbonate. The medium was thensupplemented with 15% horse serum, 2.5% fetal bovineserum, 0.5 μg/ml fungizone (Invitrogen: GIBCO, GrandIsland, NY) and 0.02 mg/ml gentamicin (Invitrogen:GIBCO). Cells were maintained in 100 mm culture dishes(SARSTEDT, Newton, NC) at 37°C in a humidified 5%CO2 air atmosphere.Calcium spectrofluorometryThe detailed procedure for calcium imaging was carriedout as published [20]. Briefly, cultured C6 glioma cellswere incubated with fura-2 acetoxymethyl ester (fura-2AM at 1 μM; Molecular Probes, Eugene, OR) andpluronic acid (at 1 μM) in normal physiological salinesolution (PSS) for 20 min at room temperature (20-22°C). Cells were then washed with PSS solution contain-ing (in mM): 126 NaCl, 5 KCl, 1 CaCl2, 1.2 MgCl2, 10HEPES and 10 glucose (pH 7.4). In some experiments,Ca2+-free PSS was used; this solution had the same com-position as PSS with the exception that EGTA was added(at 1 mM) with no CaCl2. Coverslips were placed in a per-fusion chamber mounted on an inverted microscope(Zeiss, Jena, Germany) and fluorescence was measuredthrough a 40× quartz objective lens. Alternating wave-lengths (340/380 nm) of ultraviolet light were applied at6-s intervals for excitation and fluorescence signals weremeasured at 510 nm of emission light. Signals wereacquired from a digital camera (DVC-1310, DVC Co. Aus-tin, TX) and were processed using an imaging system(Empix, Mississauga, ON, Canada) to determine ratios ofthe 340 and 380 nm intensities which were used as quan-titative measures of fluorescence levels in this work.Semiquantitative RT-PCRC6 glioma cells (8 × 105 cells/well) were plated in serum-free media and the experiment was carried out the follow-ing day with addition of ATP (100 μM) in the absence orpresence of SKF96365 (25 μM) or gadolinium (1 μM) for4 h. Total RNA was isolated using TRIzol (Invitrogen:GIBCO) and then processed for the first strand comple-Page 2 of 10(page number not for citation purposes)mentary DNA (cDNA) synthesis using Moloney murineleukemia virus (M-MLV) reverse transcriptase (Invitrogen:BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442GIBCO). The cDNA products were then amplified by PCRusing a GeneAmp thermal cycler (Applied Biosystems,Foster City, CA). Specific sense and antisense primers withthe expected product sizes are shown in Table 1. PCR con-ditions were as follows: initial denaturation at 95°C for 6min followed by a 25- to 35- cycle amplification programconsisting of denaturation at 95°C for 45 s, annealing at55-60°C for 1 min and extention at 72°C for 1 min. Afinal extention was carried out at 72°C for 10 min. ForP2YR expression study, the PCR cycle was set at 35 cyclesfor all subtype receptors. Amplification of the constitu-tively expressed enzyme D-glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as a reaction standard.The amplified PCR products were identified using 1.5%agarose gels containing ethidium bromide (final concen-tration 0.5 μg/ml) and visualized under ultraviolet light.The intensities of each band were measured by densitom-etry using NIH Image J 1.37b software (National Instituteof Health, Bethesda, MD) and expressed as relative mRNAlevels (mRNA levels normalized to GAPDH).ELISAC6 cells were plated using serum-free media in a 24-wellplate at 3 × 105 cells/well and were allowed to acclimatefor 24 h before starting the experiment. ATP was appliedto the cells with or without gadolinium for 48 h and cul-ture media were collected and analyzed with rat MCP-1and rat IL-8 (GRO/KC) ELISA kit (Peprotech, Rockey Hill,NJ) according to the manufacturer's protocol. The detec-tion limits were 47 and 16 pg/ml for MCP-1 and IL-8,respectively.Statistical analysisData are presented as means ± standard error of mean(SEM). Statistical significance (p < 0.05) was evaluatedusing one-way analysis of variance followed by Student-Newman-Keuls multiple comparison test (GraphPadPrism 3.0; San Diego, CA).ResultsATP-induced changes in Ca2+ mobilizationApplication of ATP (100 μM) to C6 glioma cells resultedin an early phase of increased [Ca2+]i which rapidlydeclined and was followed by a sustained component ofresponse (Fig. 1A). In the presence of Ca2+-free PSS (0PSS), ATP administration led to a single phase of [Ca2+]imobilization representing only the rapid portion of theresponse (Fig. 1B). These results suggested ATP binding toa P2YR activating an inositol 1,4,5-trisphosphate (IP3)-mediated intracellular release of Ca2+ and a subsequententry of divalent ion through SOC [21,22]. To test thispossibility two inhibitors of SOC, gadolinium andSKF96365, were applied prior to ATP stimulation. Theconcentrations of gadolinium (at 1 μM) [18] andSKF96365 (at 25 μM) [23,24] were chosen according toones used in previous studies which were found effectivefor SOC inhibition. Representative ATP responses are pre-sented with pre-treatment (3 min) with gadolinium (Fig.1C) or SKF96365 (Fig. 1D). Exposure of C6 to either SOCinhibitor yielded a pattern of ATP response close to thatobserved in Ca2+-free PSS (Fig. 1B) with attenuation of theprolonged component of [Ca2+]i. Peak amplitudes of[Ca2+]i responses did not appreciably differ between con-trol or the different treatments. Overall results are summa-rized in Fig. 1E (N = 4/treatment) and show durations(measured at half-maximal of peak value) of ATPresponses were similar between Ca2+-free PSS and treat-ments with the two SOC inhibitors. Respective inhibi-tions of response durations relative to control ATPresponse were 44% (0 PSS), 49% (gadolinium) and 55%(SKF96365).Expression of purinergic P2YR in C6 gliomaRT-PCR experiments were undertaken to determine theexpression of P2YR in unstimulated and ATP-stimulatedC6 cells. The study examined only those P2YR known tobe G protein coupled to IP3-dependent intracellular stores[21,22] and included P2Y1, P2Y2, P2Y4 and P2Y6 recep-Table 1: Primer sequences and product sizesP2Y1R sense 5'-TGGCGTGGTGTACCCTCTCAAGTC-3' 558 bpP2Y1R antisense 5'-CGGGACAGTCTCCTTCTGAATGTA-3'P2Y2R sense 5'-CTGCCAGGCACCCGTGCTCTACTT-3' 340 bpP2Y2R antisense 5'-CTGAGGTCAAGTGATCGGAAGGAG-3'P2Y4R sense 5'-GGCATTGTCAGACACCTTG-3' 530 bpP2Y4R antisense 5'-AAGACAGTCAGCACCACAG-3'P2Y6R sense 5'-CGCTTCCTCTTCTATGCCA-3' 478 bpP2Y6R antisense 5'-AGGCTGTCTTGGTGATGTG-3'MCP-1 sense 5'-CCTGTTGTTCACAGTTGCTGCC-3' 396 bpMCP-1 antisense 5'-TCTACAGAAGTGCTTGAGGTGGTTG-3'IL-8 sense 5'-GAAGAT AGATTGCACCGATG-3' 365 bpIL-8 antisense 5'-CATAGCCTCTCACACATTTC-3'GAPDH sense 5'-TCCCTCAAGATTGTCAGCAA-3' 309 bpPage 3 of 10(page number not for citation purposes)GAPDH antisense 5'-AGATCCACAACGGATACATT-3'BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442Page 4 of 10(page number not for citation purposes)ATP-induced changes in [Ca2+]i in C6 gliomaFigure 1ATP-induced changes in [Ca2+]i in C6 glioma. (A) Representative response for ATP (100 μM) showing rapid and slow components of decay. (B) Typical ATP response in Ca2+-free PSS. (C) ATP response following gadolinium (Gd3+) pretreatment (1 μM, for 3 min). (D) ATP response in the presence of SKF96365 (25 μM, pretreatment for 3 min). (E) Quantification of dura-tions of ATP responses for the different treatments (measured at one-half of peak response; N = 4/treatment). Values are mean ± SEM. *p < 0.001 compared with control. 100 200 300t ime (s)F340/F38000. 100 200 300t ime (s)F340/F38000. 100 200 300t ime (s)F340/F38000. 100 200 300t ime (s)F340/F380020406080100120Duration (s)con           0 PSS           Gd3+ SKFACBED** *ATP 100 PMATP 100 PMSKF96365 25 PMATP 100 PMGadolinium 1PMATP 100 PMCa2+-free PSSBMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442tors. Typical expression of these subtype receptors are pre-sented in Fig. 2 for control and ATP stimulation of C6glioma for 4 h. Expression of mRNA for any of the subtypeP2YR were unchanged between control and cells exposedto ATP treatment. The levels of band intensities occurredin the following order: P2Y2R > P2Y1R > P2Y6R > P2Y4R.Consistent results were obtained in three additionalexperiments with the intensity of P2Y2R exceeding levelstion (48 h) had no significant effect to alter expression ofany of the subtype P2YR (data not shown).Expression of chemokines MCP-1 and IL-8 in ATP-stimulated C6 cellsChemokines, such as MCP-1 and IL-8, released from ATP-stimulated glioma could act to mobilize immune cellsnearby tumor microenvironments. Representative RT-PCR for these two chemokines is presented in Fig. 3A forthe different treatments (4 h exposure) applied to C6 gli-oma cells. Application of ATP (100 μM) markedlyenhanced expression of MCP-1 and IL-8 relative to con-trol. Treatments of SKF96365 (25 μM) or gadolinium (1μM) with ATP attenuated chemokine expression com-pared with ATP alone. Application of SKF96365 or gado-linium alone showed mRNA for MCP-1 and IL-8 similarto control. Quantification for the relative expression ofthe two chemokines with the different treatments (N = 4/treatment) of C6 glioma is presented in Fig. 3B. ATP stim-ulation increased expression of MCP-1 by 4.4-fold and IL-8 by 3.2-fold relative to control. Expression of MCP-1 wassignificantly reduced with SKF96365 (by 78%) and gado-linium (by 64%) treatment of ATP-stimulated C6 relativeto ATP application alone. The corresponding decreases forIL-8 were 46% with SKF96365 and 40% with gadoliniumwith both values representing significant effects of theSOC antagonists.Production of MCP-1 and IL-8 in ATP-stimulated C6 cellsWe next examined if the enhancing effect of ATP and theinhibitory effect of SOC inhibitors on chemokine expres-sion were translated at the protein levels. PreliminaryELISA studies showed that production of both MCP-1 andIL-8 was detectable as early as 4 h but in the low range ofdetection limits for up to 24 h after ATP stimulation. Sub-sequent experiments used a 48 h exposure of C6 glioma toATP with gadolinium studied as an SOC inhibitor. Sus-tained exposure of C6 cells to SKF96365 (25 μM) causedcytotoxicity to cells thus precluding use of this compoundfor longer-term treatment. The overall results (N = 4/treat-ment) show MCP-1 production was increased by 131%with ATP stimulation compared with control (Fig. 4A).Inclusion of gadolinium with ATP significantly attenuatedMCP-1 (by 64%) relative to ATP alone. Levels of IL-8 werealso enhanced by ATP (by 92%) compared to control (Fig.4B). Addition of gadolinium with ATP was effective inreducing levels of IL-8 by 54% compared to ATP appliedalone.DiscussionThe results from this work demonstrate that ATP stimula-tion of C6 glioma causes an increased cellular expressionand production of the chemokines, MCP-1 and IL-8.Expression of P2YR linked to calcium stores in rodent C6 gli-omaFigure 2Expression of P2YR linked to calcium stores in rodent C6 glioma. The levels of receptor subtype P2Y1, P2Y2, P2Y4 and P2Y6 were examined in control and ATP-stimulated C6 cells (100 μM; 4 h exposure). The number of PCR cycles was 35 for all receptor subtypes. GAPDH served as a reaction standard. Data are representatives from 4 inde-pendent experiments.con        ATPP2Y1P2Y2P2Y4P2Y6GAPDHPage 5 of 10(page number not for citation purposes)for the other subtype P2YR for both control and ATP-stim-ulated glioma. Exposure of cells to longer-term ATP solu-These chemokines could serve as potent factors to mobi-lize tumor cells and also recruit microglia to help promoteBMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442Page 6 of 10(page number not for citation purposes)Expression of MCP-1 and IL-8 in C6 cellsFigure 3Expression of MCP-1 and IL-8 in C6 cells. (A) Representative levels for MCP-1 (upper row) and IL-8 (middle row) for the different treatments (4 h) applied to C6 glioma. Concentrations of ATP, SKF96365 and gadolinium (Gd3+) were the same as used in Ca2+ studies (Fig. 1). GAPDH served as a reaction standard (lower row). (B) Overall results (N = 4 experiments) show-ing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group. 				 BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442Page 7 of 10(page number not for citation purposes)Production of MCP-1 and IL-8 in C6 gliomaFigure 4Production of MCP-1 and IL-8 in C6 glioma. C6 cells were treated with ATP (100 μM) in the presence, or absence, of gadolinium (Gd3+, 1 μM) for 48 h. (A) Levels of MCP-1 production (ng/ml) and (B) levels of IL-8 production (pg/ml) for the dif-ferent treatments. Data are presented as mean ± SEM from 4 independent experiments. * indicates significant difference from control (p < 0.001) and ** indicates significant difference from ATP-treated group (p < 0.05). (ng/mL)020406080100120140IL-8 (pg/mL)*** ** *controlGd3+ATPATP+Gd3+ABcontrolGd3+ATPATP+Gd3+BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442tumor development. Both expression and release ofchemokines were inhibited by antagonists for SOC-medi-ated influx of Ca2+ suggesting ATP induced the activationof a SOC-dependent pathway. Our findings lead to thenovel suggestion that ATP binding to a metabotropicP2YR, coupled to activation of SOC, as an underlyingmechanism for chemokine production in tumor cells. Asdiscussed below, these findings are distinct from previouswork reporting increased levels of these chemokinesmediated by activation of the ionotropic P2X7R with theligand, BzATP [16].Calcium-sensitive spectrofluorometry showed ATP-induced increases in [Ca2+]i consisted of two componentsof response decay with the rapid and slow phases repre-senting respective store release and plasmalemmal entryof Ca2+. In the presence of inhibitors of SOC, SKF96365or gadolinium, the secondary but not initial, componentof response was suppressed. Both SOC inhibitors werehighly effective in blocking Ca2+ entry since durations ofATP responses with either compound were similar tothose measured using Ca2+ free solution. It should benoted that no specific SOC antagonist has yet been iden-tified with both SKF96365 and gadolinium capable ofmodulating other Ca2+ entry pathways. The findings andimplication for two Ca2+ influx channels in glioma, SOC(this work) and P2X7R [16], are considered in more detailbelow.RT-PCR was used to determine expression of metabo-tropic purinergic receptors P2Y1R, P2Y2R, P2Y4R andP2Y6R which are linked to IP3-mediated mobilization of[Ca2+]i [21]. Unstimulated C6 glioma showed a promi-nent band intensity for P2Y2R with a relatively lower basalexpression for the other P2YR. The expression for any ofthe subtype P2YR was not altered with exposure of C6cells to ATP (4 h treatment). Interestingly, longer-termexposure (48 h) of C6 glioma to ATP had no appreciableeffect to alter mRNA expression for any of the P2YR (datanot shown). Although our results show prominent expres-sion of P2Y2R in glioma, future studies on protein levelsand use of specific purinergic agonists and antagonistswill have utility in linking specific P2YR with Ca2+-dependent responses. It is noteworthy that P2Y subtypereceptors exhibit differential activation with purines withP2Y1R and P2Y6R more sensitive to ADP while P2Y2R andP2Y4R are more sensitive to ATP [21].ATP stimulation of C6 glioma (4 h exposure) enhancedexpression of the potent microglial-mobilizing chemok-ines, MCP-1 and IL-8. Expression of both chemokineswere significantly reduced with SKF96365 or gadoliniumincluded with the ATP stimulation. ELISA assay demon-24 h but was enhanced with longer-term (48 h) exposure.In the latter case, levels of MCP-1 and IL-8 were signifi-cantly diminished with gadolinium included in the ATPtreatment. It is noteworthy that the stimulatory effects ofATP on MCP-1 and IL-8 production were markedly lowercompared to other agents releasing chemokines fromastrocytic cell types. These agents include the P2X7R ago-nist, BzATP [25] and the pro-inflammatory cytokinesTNF-α, and IL-1β [26,27].The increased chemokine expression and production wasnot directly attributable to changes in receptor expressionsince none of the P2YR showed changes in band intensitywith ATP treatment (Fig. 2). It is possible that the highbasal levels of subtype P2Y2R expression in control wouldpreclude RT-PCR analysis showing any increases in thissubtype receptor after exposure of C6 cells to ATP. Gado-linium treatment was demonstrated to significantlyreduce levels of MCP-1 and IL-8 in ATP-stimulated C6cells. However, the inclusion of gadolinium with ATP didnot diminish chemokine productions to control levels.Although P2X7R could contribute, this possibility isunlikely since ATP levels near 1 mM are generally requiredfor activation of this receptor [28,29]. Our results do notexclude involvement of other P2XR since it is known thatsubtype receptors from the P2X family mediate influx ofCa2+ into stimulated cells [12].Together with previous data [16], C6 glioma cells areshown to express at least two different purinergic-depend-ent Ca2+ entry pathways which modulate cellular releasesof the chemokines, MCP-1 and IL-8. Activation of SOCsubsequent to ATP binding to a P2YR or activation ofP2X7R [16] with BzATP (ATP was not studied as an ago-nist) is coupled to C6 chemokine production. The overallresults suggest that in C6 glioma, two different calciuminflux pathways converge with the same functional end-point of enhanced chemokine release. The effects of SOCinhibitors on rapid Ca2+ responses and on longer-term cellfunctional responses indicates the involvement of Ca2+-dependent transcription factors in mediating tumor cellproduction of chemokines. One possibility is that releaseof chemokines such as MCP-1 and IL-8 from C6 are mod-ulated by SOC activation with physiological ATP concen-trations whereas the purinergic P2X7R pathway onlycontributes under extremely elevated levels of ATP.Our findings suggest that pharmacological antagonism ofSOC could serve as a rationale strategy to inhibit tumorgrowth. The SOC-dependent secretion of MCP-1 and IL-8from C6 glioma would increase tumor cell mobility andalso act as mobilization and proliferation signals to directresident microglia into expanding tumors. It has beenPage 8 of 10(page number not for citation purposes)strated that production of both chemokines was relativelylow for ATP stimulation of C6 cells for durations less thanpointed out that recruitment of microglia could aid insupporting an immunosuppressive tumor environment toBMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/442counter effects of T-cell-mediated killing of tumor cells[4]. However, recent findings also indicate that under cer-tain conditions activated microglia/macrophages arecapable of killing glioma cells [30,31]. Thus, it seemslikely that microglia/macrophages could play both pro- oranti-neoplastic roles depending on the signaling factorspresent in the tumor microenvironment.ConclusionsOur results suggest ATP-induced Ca2+ entry, mediated byactivation of SOC in C6 glioma, as a mechanism linked tothe cellular expression and release of chemokines.Increased levels of MCP-1 and IL-8 are predicted toenhance the mobility of tumor cells and promote recruit-ment of microglia into developing tumors thereby sup-porting tumor growth. Pharmacological inhibition ofSOC in astroglioma is suggested as a novel treatment strat-egy to inhibit tumor progression.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsNJ designed research, performed research, analyzed data,and wrote the manuscript. HBC analyzed data JGMdesigned research, analyzed data, and wrote the manu-script. All authors have read and approved the final man-uscript.AcknowledgementsFunding from Canadian Institute for Health Research supported this work (to JGM).References1. Louis DN: Molecular pathology of malignant gliomas.  Annu RevPathol 2006, 1:97-117.2. Hoelzinger DB, Demuth T, Berens ME: Autocrine factors thatsustain glioma invasion and paracrine biology in the brainmicroenvironment.  J Natl Cancer Inst 2007, 99(21):1583-1593.3. Badie B, Schartner J: Role of microglia in glioma biology.  MicroscRes Tech 2001, 54(2):106-113.4. Watters JJ, Schartner JM, Badie B: Microglia function in braintumors.  J Neurosci Res 2005, 81(3):447-455.5. Markovic DS, Vinnakota K, Chirasani S, Synowitz M, Raguet H, StockK, Sliwa M, Lehmann S, Kalin R, van Rooijen N, et al.: Gliomasinduce and exploit microglial MT1-MMP expression fortumor expansion.  Proc Natl Acad Sci USA 2009,106(30):12530-12535.6. Platten M, Kretz A, Naumann U, Aulwurm S, Egashira K, Isenmann S,Weller M: Monocyte chemoattractant protein-1 increasesmicroglial infiltration and aggressiveness of gliomas.  Ann Neu-rol 2003, 54(3):388-392.7. Rong Y, Durden DL, Van Meir EG, Brat DJ: 'Pseudopalisading'necrosis in glioblastoma: a familiar morphologic feature thatlinks vascular pathology, hypoxia, and angiogenesis.  J Neu-ropathol Exp Neurol 2006, 65(6):529-539.8. Stamatovic SM, Keep RF, Mostarica-Stojkovic M, Andjelkovic AV:CCL2 regulates angiogenesis via activation of Ets-1 tran-scription factor.  J Immunol 2006, 177(4):2651-2661.9. de la Iglesia N, Konopka G, Lim KL, Nutt CL, Bromberg JF, Frank DA,Mischel PS, Louis DN, Bonni A: Deregulation of a STAT3-inter-10. Fields RD, Burnstock G: Purinergic signalling in neuron-gliainteractions.  Nat Rev Neurosci 2006, 7(6):423-436.11. White N, Burnstock G: P2 receptors and cancer.  Trends in Phar-macological Sciences 2006, 27(4):211-217.12. Dubyak GR, el-Moatassim C: Signal transduction via P2-puriner-gic receptors for extracellular ATP and other nucleotides.Am J Physiol 1993, 265(3 Pt 1):C577-606.13. Harden TK, Lazarowski ER: Release of ATP and UTP fromastrocytoma cells.  Prog Brain Res 1999, 120:135-143.14. Wink MR, Lenz G, Braganhol E, Tamajusuku AS, Schwartsmann G,Sarkis JJ, Battastini AM: Altered extracellular ATP, ADP andAMP catabolism in glioma cell lines.  Cancer Lett 2003,198(2):211-218.15. Morrone FB, Oliveira DL, Gamermann P, Stella J, Wofchuk S, WinkMR, Meurer L, Edelweiss MI, Lenz G, Battastini AM: In vivo glioblas-toma growth is reduced by apyrase activity in a rat gliomamodel.  BMC Cancer 2006, 6:226.16. Wei W, Ryu JK, Choi HB, McLarnon JG: Expression and functionof the P2X(7) receptor in rat C6 glioma cells.  Cancer Lett 2008,260(1-2):79-87.17. Broad LM, Cannon TR, Taylor CW: A non-capacitative pathwayactivated by arachidonic acid is the major Ca2+ entry mech-anism in rat A7r5 smooth muscle cells stimulated with lowconcentrations of vasopressin.  J Physiol 1999, 517(Pt1):121-134.18. Peppiatt CM, Holmes AM, Seo JT, Bootman MD, Collins TJ, McDon-ald F, Roderick HL: Calmidazolium and arachidonate activatea calcium entry pathway that is distinct from store-operatedcalcium influx in HeLa cells.  Biochem J 2004, 381(Pt 3):929-939.19. Choi HB, Hong SH, Ryu JK, Kim SU, McLarnon JG: Differential acti-vation of subtype purinergic receptors modulates Ca(2+)mobilization and COX-2 in human microglia.  Glia 2003,43(2):95-103.20. McLarnon JG, Choi HB, Lue LF, Walker DG, Kim SU: Perturbationsin calcium-mediated signal transduction in microglia fromAlzheimer's disease patients.  J Neurosci Res 2005,81(3):426-435.21. von Kugelgen I: Pharmacological profiles of cloned mamma-lian P2Y-receptor subtypes.  Pharmacol Ther 2006,110(3):415-432.22. Czajkowski R, Baranska J: Cross-talk between the ATP and ADPnucleotide receptor signalling pathways in glioma C6 cells.Acta Biochim Pol 2002, 49(4):877-889.23. Zhu X, Jiang M, Birnbaumer L: Receptor-activated Ca2+ influxvia human Trp3 stably expressed in human embryonic kid-ney (HEK)293 cells. Evidence for a non-capacitative Ca2+entry.  J Biol Chem 1998, 273(1):133-142.24. Giannone G, Takeda K, Kleschyov AL: Novel activation of non-selective cationic channels by dinitrosyl iron-thiosulfate inPC12 cells.  J Physiol 2000, 529(Pt 3):735-745.25. Panenka W, Jijon H, Herx LM, Armstrong JN, Feighan D, Wei T, YongVW, Ransohoff RM, MacVicar BA: P2X7-like receptor activationin astrocytes increases chemokine monocyte chemoattract-ant protein-1 expression via mitogen-activated proteinkinase.  J Neurosci 2001, 21(18):7135-7142.26. Nabors LB, Suswam E, Huang Y, Yang X, Johnson MJ, King PH:Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabili-zation and HuR.  Cancer Res 2003, 63(14):4181-4187.27. John GR, Simpson JE, Woodroofe MN, Lee SC, Brosnan CF: Extra-cellular nucleotides differentially regulate interleukin-1betasignaling in primary human astrocytes: implications forinflammatory gene expression.  J Neurosci 2001,21(12):4134-4142.28. McLarnon JG: Purinergic mediated changes in Ca2+ mobiliza-tion and functional responses in microglia: effects of low lev-els of ATP.  J Neurosci Res 2005, 81(3):349-356.29. Ogata T, Chuai M, Morino T, Yamamoto H, Nakamura Y, Schubert P:Adenosine triphosphate inhibits cytokine release fromlipopolysaccharide-activated microglia via P2y receptors.Brain Res 2003, 981(1-2):174-183.30. Mora R, Abschuetz A, Kees T, Dokic I, Joschko N, Kleber S, Geibig R,Mosconi E, Zentgraf H, Martin-Villalba A, et al.: TNF-alpha- andTRAIL-resistant glioma cells undergo autophagy-dependentPage 9 of 10(page number not for citation purposes)leukin 8 signaling pathway promotes human glioblastomacell proliferation and invasiveness.  J Neurosci 2008,28(23):5870-5878.cell death induced by activated microglia.  Glia 2009,57(5):561-581.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Cancer 2009, 9:442 http://www.biomedcentral.com/1471-2407/9/44231. Zhang L, Alizadeh D, Van Handel M, Kortylewski M, Yu H, Badie B:Stat3 inhibition activates tumor macrophages and abrogatesglioma growth in mice.  Glia 2009, 57(13):1458-1467.Pre-publication historyThe pre-publication history for this paper can be accessedhere:http://www.biomedcentral.com/1471-2407/9/442/prepubyours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 10 of 10(page number not for citation purposes)


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