Transient Receptor Potential Ankyrin 1 (TRPA1) Modulation by 4-Hydroxynonenal (4-HNE) in Pancreatic Adenocarcinoma Cell Lines: Putative Roles for Therapies.

Abstract

Transient receptor potential channels (TRP) are overexpressed in some pancreatic adenocarcinoma (PDAC) patients and cell lines, settling them as putative therapeutic targets in this disease. Reactive oxygen species (ROS), with levels increased in PDAC, modulate some members of the TRP family renamed "redox channels". Here, we investigate the direct effects of 4-hydroxinonenal (4-HNE) on TRPA1, natively expressed in PDAC cell lines and in association with cell migration and cell cycle progression. We performed microfluorimetry experiments, while the activation of resident membrane channels was investigated using confocal microscopy. We applied a prospective molecular docking of 4-HNE using Autodock and AutoDock Tools4. Also, we simulated the diffusion of 4-HNE through the membrane from the extracellular space with the Permeability of Molecules across Membranes (PerMM) web server. The analysis of cell migration was performed using the wound healing assay, and cell cycle progression was acquired using a Beckman Coulter CytoFlex flow cytometer. Our results show, for the first time in PDAC, that 4-HNE diffuses through the cell membrane and rapidly activates Ca2+ uptake in PDAC cells. This process depends on TRPA1 activation, as 4-HNE forms a covalent binding with a pocket-like region within the intracellular N-terminal of the channel, shaped by the cysteine residues 621, 641, and 665. The activation of TRPA1 by 4-HNE inhibits cell migration and induces cell cycle arrest in the G2/M phase. Our study brings new insights into the effects of 4-HNE, highlighting the activation of the TRPA1 channel, a druggable, putative target for PDAC-expressing tumors.