Carcinoma-specific expression of P2Y11 receptor and its contribution in ATP-induced purinergic signalling and cell migration in human hepatocellular carcinoma cells

Abstract

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.