Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: How vanilloids cause non-vanilloid receptor mediated cell death

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Abstract

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.

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Materials and methods

Cells and chemicals. H460 cells (a human non-small cell lung cancer line) were obtained from the American Type Culture Collection, Manassas, VA. Drugs were dissolved in 100% ethanol at a stock concentration of 10 mM. All chemicals used were of the highest grade available and were from Sigma Chemical Company, Merck Biosciences, Tocris, or InVitrogen. For all vanilloid receptor ligands used, the diluent (ethanol) was never present at >1.0%, and control culture flasks with H460 cells or

TRPV1 ligands induce morphological features of apoptosis and necrosis in H460 cells

Figs. 1A and B show single images taken at time = 0 and time = 2 h in the time-lapse microscope in the presence of 50 μM (E)-capsaicin or 50 μM resiniferatoxin, respectively. There was a general loss of cells in the fields of view (due to their detaching from the growing surface of the cell culture plate); rounding of some individual cells (indicated by an S on the images), swelling of other individual cells (indicated by a W on the images), with an increase in cytoplasmic granularity, particularly in

Discussion

In this report, we have identified a novel mechanism for vanilloid receptor-mediated cancer cell death, which targets mitochondrial function rather than the cell-surface vanilloid receptor.

Our data showing an absence of TRPV1 immunoreactivity in mitochondria are consistent with data from MitoP2, the mitochondrial proteome database [15], which show an absence of TRPV1 channel-like proteins in the mitochondrial proteomes of man, mouse, or the model laboratory organisms Saccharomyces cerevisiae,

Acknowledgments

We thank the Medical Research Council (UK) and the School of Molecular Medical Sciences M.Sc. program in oncology, for financial support. We also thank Mr. Liaque Latif for his excellent technical assistance with SDS–PAGE and immunoblotting, and Mr. Ian Ward for outstanding technical assistance with time-lapse microscopy.

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