UTP Inhibites Gastric Cancer Via Ca²⁺ Signaling And PTEN Pathway

Part 1 Comparision of P2Y6 receptor expression in human primary gastric cancer tissues with adjacent non-tumorous stomach tissues,and in gastric cancer cells with normal GES-1 cells.Objectives: To investigate the expression of P2Y6 receptors in gastric cancer and adjacent non-tumorous stomach tissues,gastric cancer cells and normal GES-1 cells.Methods: Immunohistochemistry was applied for examining the protein expression of P2Y6 receptors in primary gastric cancer and adjacent non-tumorous stomach tissues.In addition,correlations of P2Y6 receptor proteins in gastric cancer tissues and the clinicopathological feature and prognosis were also analyzed.qRT-PCR and Western blot were carried out to detect the expression of P2Y6 receptor mRNA and proteins in gastric cancer and adjacent non-tumorous stomach tissues,gastric cancer cells and normal GES-1 cells.Results: The P2Y6 receptor expression at both mRNA and protein levels were reduced in primary gastric cancer tissues and gastric cancer cells compared to adjacent non-tumorous stomach tissues and normal GES-1 cells.The P2Y6 protein levels in gastric cancer tissues were significantly correlated with tumor size?tumor differentiation?tumor lymph nodes metastasis and survival time.The patients with high protein expression of P2Y6 receptors in gastric cancer tissues had small tumor size?<4.5cm?,good differentiation,less lymph nodes metastasis and long survival time.Conclusion: The P2Y6 receptor expression is reduced in primary gastric cancer tissues and gastric cancer cells,suggesting that P2Y6 receptors may serve as tumor suppressor genes in gastric cancer.Part 2 The influence of UTP and UDP on gastric cancer cell behaviors.Objectives: To study the influence of UTP and UDP,two P2Y6 receptor agonists,on gastric cancer cell proliferation,migration and invasion in vitro as well as tumor growth in vivo.Methods: CCK8 was carried out to detect the proliferation of gastric cancer cells.Flow cytometry was used to analyze cell cycle.Wound scratch assay and transwell migration assay were applied for examining cell migration and invasion,respectively.Subcutaneous implanted tumors to nude mice were carried to examine tumor growth in vivo.Results: The P2Y6 receptor agonists UTP and UDP,and two intracellular Ca²⁺mobilizers Spiperone and CPA,inhibited the proliferation of MKN-45 and SGC-7901 gastric cancer cells.UTP,UDP,Spiperone and CPA arrested cell cycle in G1 phase of SGC-7901 gastric cancer cells.UTP suppressed the migration and invasion of MKN-45 and SGC-7901 gastric cancer cells,but not normal GES-1 cells.UTP also attenuated the subcutaneous implanted tumor sizes in nude mice.Conclusion: UTP not noly suppressed the growth of gastric cancer in vitro and in vivo,but also inhibited migration and invasion of MKN-45 and SGC-7901 gastric cancer cells.Part 3 Mechanisms of UTP-induced inhibition of gastric cancerThe Objective 1: To elucidate the mechanisms of UTP suppression of cell proliferation of gastric cancer.Methods: single cell calcium imaging was used to detect the changes of [Ca²⁺]cyt in gastric cancer cells after treatment with UTP,UDP,MRS2578,U73122,2-APB,CPA or Spiperone in 0Ca²⁺ or 2m M Ca²⁺.Western blot was carried out to examine effects of ?-catenin Ser675 by UTP,UDP,Spiperone,MRS2578 and 2-APB.Results: Both UTP and UDP increased cytosolic free Ca²⁺ concentration([Ca²⁺]cyt)in 0 Ca²⁺ or 2mM Ca²⁺ solutions,which was reversed by MRS-2578,U73122 and 2-APB.UTP,CPA and Spiperone induced [Ca²⁺]cyt in 0Ca²⁺ solutions;however,in 0Ca²⁺ solutions UTP could not induce change in [Ca²⁺]cyt after treatment with CPA or Spiperone.UTP,UDP and Spiperone inhibited ?-catenin Ser675,which was reversed by MRS2578 or 2-APB.Conclusion: UTP increases [Ca²⁺]cyt in gastric cancer cells through activation of P2Y6 receptors and inhibites ?-catenin signaling.Objectives 2: To elucidate the mechanisms of UTP suppression of cell migration and invasion of gastric cancer.Methods: Western blot was used to examine expression of PTEN in GES-1?MKN-45 and SGC-7901 cells,in additional,Phospho-PTEN and Vimentin treating with UTP.Results: The expression of PTEN is significantly higher in GES-1 than in MKN-45 and SGC-7901 cells.UTP increased Phospho-PTEN but inhibited Vimentin.Conclusion: UTP inhibited cell migration and invasion of gastric cancer likely through activation of PTEN/Vimentin.