Characterization of Functional Transient Receptor Potential Melastatin 8 Channels in Human Pancreatic Ductal Adenocarcinoma Cells

ORIGINAL ARTICLE

Characterization of Functional Transient Receptor Potential Melastatin 8 Channels in Human Pancreatic Ductal Adenocarcinoma Cells Dana Cucu, PhD,* Gabriela Chiritoiu, MSc,Þ Stefana Petrescu, PhD,Þ Alexandru Babes, PhD,þ Luciana Stanica, MSc,þ Dan G. Duda, MD, PhD,§ Akira Horii, MD, PhD,|| Simona Olimpia Dima, MD, PhD,* and Irinel Popescu, MD, PhD, FACS*

Objective: Recently, the transient receptor potential melastatin 8 (TRPM8) channel has emerged as a putative biomarker for pancreatic ductal adenocarcinoma (PDA). This study aimed to evaluate the expression of TRPM8 and its modulation by specific agonists and antagonists in PDA cells. Methods: We examined the protein expression of TRPM8 in 3 different PDA cell lines and compared it with a nontumoral epithelial cell line of human pancreatic origin using Western blotting and immunocytochemical analysis. To assess the function of TRPM8 channels, we measured the TRPM8 currents in whole-cell mode of the patch clamp technique. To explore the putative involvement of TRPM8 in cell migration, we investigated the motility of PDA cells using the scratch-wound assay. Results: Pancreatic ductal adenocarcinoma cells express functional plasma membrane TRPM8 channels, which are responsive after exposure to agonists (menthol and icilin) and antagonists N-(3-aminopropyl)-2{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt. The silencing of TRPM8 expression by small interfering RNA augments the migration of PDA cells. Conversely, the activated form of TRPM8 inhibits PDA cell motility. Conclusions: An unglycosylated TRPM8 protein is expressed and is functional in the membrane of PDA cells. Transient receptor potential melastatin 8 inhibits the migration of PDA cells, suggesting a putative role as a biomarker or target for this channel for PDA therapy. Key Words: pancreatic adenocarcinoma, transient receptor potential melastatin 8, menthol, migration (Pancreas 2014;00: 00Y00)

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ancreatic cancer is an aggressive disease and the fourth leading cause of cancer-related deaths worldwide. Most pancreatic cancers are pancreatic ductal adenocarcinomas (PDAs). The 5-year survival rate after surgical resection for patients with localized PDA is 20%. The poor survival rate is mainly attributed to the late detection of PDA because 85% of the patients are being diagnosed with advanced, unresectable disease.1 From the *Center of Digestive Disease and Liver Transplantation, Fundeni Clinical Institute; †Department of Molecular and Cell Biology, Romanian Academy Institute of Biochemistry; ‡Department of Anatomy, Physiology, and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania; §Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA; and ||Division of Molecular Pathology, Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan. Received for publication November 30, 2012; accepted January 14, 2014. Reprints: Dana Cucu, PhD, Center of Digestive Disease and Liver Transplantation, Fundeni Clinical Institute, Funndeni 258, Bucharest, 022328, Romania (e