Role of P2 Receptor Expression in ATP-Induced Purinergic Signaling and Cell Migration in Human Hepatocellular Carcinoma Cells

Abstract

Hepatitis C virus (HCV), a foremost public health issue, is associated with number of pathological disorders naming chronic hepatitis, cirrhosis, and fibrosis and hepatocellular carcinoma. ATP acts as a ligand for P2X receptors that are non-selective ligand-gated ion channels. P2X4 and P2X7 are the most widely expressed proteins in the liver besides other P2X receptor proteins. The basic notion behind this study was to find out the association of prevalence of purinoceptors in hepatitis C virus (HCV) and non-HCV hepatocellular carcinoma (HCC). Immunohistochemistry was used to study the expression of P2X4 and P2X7 receptors on ex-planted liver tissue samples obtained from hepatocellular carcinoma patients. There was a significant increase in P2X4 receptor expression in HCV HCC as compared to non-HCV HCV. But P2X7 receptor expression was not increased in both HCV HCC and non-HCV HCC. Docking was used to study protein-protein interactions (PPIs) of P2X4 and P2X7 receptor protein with HCV envelops protein E1. We found out few interacting residues between HCV E1 and P2X4R and P2X7R.The current study was designed to study the GPCR-associated calcium signaling with mutated downstream signaling proteins. In the next part of the study quantitative modeling of the GPCR-associated signaling was done and specific agonist and antagonist were used to activate and inhibit the calcium signaling. Moreover, we examined the P2 receptor for ATP-induced Ca2+ signaling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) exhibited an ATP induced elevation of [Ca2+]i inHuh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in elevation of [Ca2+]i concentration. Whereas, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 ABSTRACT iii receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increase in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. 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 mediated cell migration. Taken together, our results show critical role of P2Y11 in mediating ATP-inducing Ca2+ signaling and regulating cell migration in human HCC cells.