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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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Expression 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) showing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group.
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Figure 3: Expression 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) showing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group.

Mentions: Chemokines, such as MCP-1 and IL-8, released from ATP-stimulated glioma could act to mobilize immune cells nearby tumor microenvironments. Representative RT-PCR for these two chemokines is presented in Fig. 3A for the different treatments (4 h exposure) applied to C6 glioma cells. Application of ATP (100 μM) markedly enhanced expression of MCP-1 and IL-8 relative to control. Treatments of SKF96365 (25 μM) or gadolinium (1 μM) with ATP attenuated chemokine expression compared with ATP alone. Application of SKF96365 or gadolinium alone showed mRNA for MCP-1 and IL-8 similar to control. Quantification for the relative expression of the two chemokines with the different treatments (N = 4/treatment) of C6 glioma is presented in Fig. 3B. ATP stimulation increased expression of MCP-1 by 4.4-fold and IL-8 by 3.2-fold relative to control. Expression of MCP-1 was significantly reduced with SKF96365 (by 78%) and gadolinium (by 64%) treatment of ATP-stimulated C6 relative to ATP application alone. The corresponding decreases for IL-8 were 46% with SKF96365 and 40% with gadolinium with both values representing significant effects of the SOC antagonists.


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

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

Expression 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) showing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Expression 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) showing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group.
Mentions: Chemokines, such as MCP-1 and IL-8, released from ATP-stimulated glioma could act to mobilize immune cells nearby tumor microenvironments. Representative RT-PCR for these two chemokines is presented in Fig. 3A for the different treatments (4 h exposure) applied to C6 glioma cells. Application of ATP (100 μM) markedly enhanced expression of MCP-1 and IL-8 relative to control. Treatments of SKF96365 (25 μM) or gadolinium (1 μM) with ATP attenuated chemokine expression compared with ATP alone. Application of SKF96365 or gadolinium alone showed mRNA for MCP-1 and IL-8 similar to control. Quantification for the relative expression of the two chemokines with the different treatments (N = 4/treatment) of C6 glioma is presented in Fig. 3B. ATP stimulation increased expression of MCP-1 by 4.4-fold and IL-8 by 3.2-fold relative to control. Expression of MCP-1 was significantly reduced with SKF96365 (by 78%) and gadolinium (by 64%) treatment of ATP-stimulated C6 relative to ATP application alone. The corresponding decreases for IL-8 were 46% with SKF96365 and 40% with gadolinium with both values representing significant effects of the SOC antagonists.

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