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.
Highlights
Hepatocellular carcinoma (HCC) is the primary liver cancer; poor prognosis and ineffective treatment of HCC with currently available anti-cancer treatments have made it to be one of the leading and most deadly causes of cancer-related mortality, with the 5-year survival rate being less than 15% [1,2,3,4]
Summary of mean P2Y11 protein expression, normalized to the HSC70 protein level and presented as % of the value in cells transfected with siCTL, from six independent experiments. *, p < 0.05. (E) Left, representative recordings of Ca2+ responses induced by 10 μM NF546 in cells transfected with indicate small interfering RNA (siRNA), with 4 wells of cells for each case
The present study provides pharmacological and genetic evidence that consistently supports functional expression of the P2Y11 receptor as a critical mechanism in mediating ATP-induced Ca2+ signalling and stimulating cell migration in human HCC cells and, importantly, provide evidence to suggest HCC-specific P2Y11 receptor expression
Summary
Hepatocellular carcinoma (HCC) is the primary liver cancer; poor prognosis and ineffective treatment of HCC with currently available anti-cancer treatments have made it to be one of the leading and most deadly causes of cancer-related mortality, with the 5-year survival rate being less than 15% [1,2,3,4]. ATP can elevate the intracellular Ca2+ concentrations ([Ca2+]i) via the P2X receptor-mediated extracellular Ca2+ influx or the P2Y receptor-PLC-IP3R signalling pathway leading to internal Ca2+ release. Further studies demonstrated that activation of the P2Y2 receptor leads to ATP-induced increase in the [Ca2+]i in human normal hepatocytes and human HCC cells [37, 38]. A separate study showed functional expression of the P2X4 receptor and possibly the P2X7 receptor in rat and mouse hepatocytes and rat HCC cells [39]. We provide pharmacological, functional and genetic evidence to support the P2Y11 receptor in ATPinduced Ca2+ signalling in human HCC cells, reveal strong HCC-specific P2Y11 receptor expression, and propose their involvement in HCC cell migration
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