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ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells.

Jantaratnotai N, Choi HB, McLarnon JG - BMC Cancer (2009)

Bottom Line: 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.Gadolinium treatment of ATP-stimulated C6 cells was also found to inhibit the production of MCP-1 and IL-8.These results suggest ATP-induced Ca2+ entry, mediated by activation of SOC in C6 glioma, as a mechanism leading to increased cellular expression and release of chemokines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. scnjt@mahidol.ac.th

ABSTRACT

Background: 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 has examined 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 intracellular Ca2+ [Ca2+]i following ATP stimulation of rat C6 glioma cells. Pretreatment with two inhibitors of SOC, SKF96365 or gadolinium, was used to examine for effects on [Ca2+]i. RT-PCR was performed to 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 glioma cells.

Results: Application of ATP (at 100 microM) to C6 glioma induced an increase in [Ca2+]i with the response exhibiting two components of decay. In the presence of the SOC inhibitors, SKF96365 or gadolinium, or with Ca2+-free solution, ATP responses lacked a slow phase suggesting the secondary component was due to SOC-mediated influx of Ca2+. RT-PCR confirmed expression of purinergic P2Y-subtype receptors in C6 cells which would serve as a precursor to activation of SOC. 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. Gadolinium treatment 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 C6 glioma, as a mechanism leading to increased cellular expression and release of chemokines. Elevated levels of MCP-1 and IL-8 are predicted to enhance the mobility of tumor cells and promote recruitment of microglia into developing tumors thereby supporting tumor growth.

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Production 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 different 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).
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Figure 4: Production 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 different 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).

Mentions: We next examined if the enhancing effect of ATP and the inhibitory effect of SOC inhibitors on chemokine expression were translated at the protein levels. Preliminary ELISA studies showed that production of both MCP-1 and IL-8 was detectable as early as 4 h but in the low range of detection limits for up to 24 h after ATP stimulation. Subsequent experiments used a 48 h exposure of C6 glioma to ATP with gadolinium studied as an SOC inhibitor. Sustained exposure of C6 cells to SKF96365 (25 μM) caused cytotoxicity to cells thus precluding use of this compound for longer-term treatment. The overall results (N = 4/treatment) show MCP-1 production was increased by 131% with ATP stimulation compared with control (Fig. 4A). Inclusion of gadolinium with ATP significantly attenuated MCP-1 (by 64%) relative to ATP alone. Levels of IL-8 were also enhanced by ATP (by 92%) compared to control (Fig. 4B). Addition of gadolinium with ATP was effective in reducing levels of IL-8 by 54% compared to ATP applied alone.


ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells.

Jantaratnotai N, Choi HB, McLarnon JG - BMC Cancer (2009)

Production 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 different 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807438&req=5

Figure 4: Production 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 different 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).
Mentions: We next examined if the enhancing effect of ATP and the inhibitory effect of SOC inhibitors on chemokine expression were translated at the protein levels. Preliminary ELISA studies showed that production of both MCP-1 and IL-8 was detectable as early as 4 h but in the low range of detection limits for up to 24 h after ATP stimulation. Subsequent experiments used a 48 h exposure of C6 glioma to ATP with gadolinium studied as an SOC inhibitor. Sustained exposure of C6 cells to SKF96365 (25 μM) caused cytotoxicity to cells thus precluding use of this compound for longer-term treatment. The overall results (N = 4/treatment) show MCP-1 production was increased by 131% with ATP stimulation compared with control (Fig. 4A). Inclusion of gadolinium with ATP significantly attenuated MCP-1 (by 64%) relative to ATP alone. Levels of IL-8 were also enhanced by ATP (by 92%) compared to control (Fig. 4B). Addition of gadolinium with ATP was effective in reducing levels of IL-8 by 54% compared to ATP applied alone.

Bottom Line: 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.Gadolinium treatment of ATP-stimulated C6 cells was also found to inhibit the production of MCP-1 and IL-8.These results suggest ATP-induced Ca2+ entry, mediated by activation of SOC in C6 glioma, as a mechanism leading to increased cellular expression and release of chemokines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. scnjt@mahidol.ac.th

ABSTRACT

Background: 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 has examined 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 intracellular Ca2+ [Ca2+]i following ATP stimulation of rat C6 glioma cells. Pretreatment with two inhibitors of SOC, SKF96365 or gadolinium, was used to examine for effects on [Ca2+]i. RT-PCR was performed to 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 glioma cells.

Results: Application of ATP (at 100 microM) to C6 glioma induced an increase in [Ca2+]i with the response exhibiting two components of decay. In the presence of the SOC inhibitors, SKF96365 or gadolinium, or with Ca2+-free solution, ATP responses lacked a slow phase suggesting the secondary component was due to SOC-mediated influx of Ca2+. RT-PCR confirmed expression of purinergic P2Y-subtype receptors in C6 cells which would serve as a precursor to activation of SOC. 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. Gadolinium treatment 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 C6 glioma, as a mechanism leading to increased cellular expression and release of chemokines. Elevated levels of MCP-1 and IL-8 are predicted to enhance the mobility of tumor cells and promote recruitment of microglia into developing tumors thereby supporting tumor growth.

Show MeSH
Related in: MedlinePlus