Carcinoma-specific expression of P2Y11 receptor and its contribution in ATP-induced purinergic signalling and cell migration in human hepatocellular carcinoma cells
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Madiha Khalid1,2,*, Lucie Brisson3,*, Menahil Tariq1, Yunjie Hao1, Roseline Guibon4, Gaëlle Fromont4, Sharifah Alawieyah Syed Mortadza1, Fatema Mousawi1, Sobia Manzoor2, Sébastien Roger3,5 and Lin-Hua Jiang1,3,6,7
1School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK
2Atta-ur-Rahman School of Applied Biosciences, National University of Science and Technology, Islamabad, Pakistan
3Inserm UMR1069, Nutrition, Croissance et Cancer, Université François-Rabelais de Tours, Tours, France
4Centre Hospitalo-Universitaire de Tours, Tours, France
5Institut Universitaire de France, Paris Cedex 05, France
6Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, P. R. China
7Sino-UK Joint Laboratory of Brain Function and Injury, Xinxiang Medical University, Xinxiang, P. R. China
*These authors have contributed equally to this work
Lin-Hua Jiang, email: [email protected]
Sébastien Roger, email: [email protected]
Sobia Manzoor, email: [email protected]
Keywords: HCC cells, extracellular ATP, P2Y11 receptor, cytosolic Ca2+, cell migration
Received: March 19, 2016 Accepted: February 15, 2017 Published: March 14, 2017
Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.
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