Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC62
Melatonin induces calcium release from agonist-sensitive cytosolic stores and reduces pancreatic tumour cell viability by altering mitochondrial physiology
A. Gonzalez1, A. Miro-Moran1, J. A. Tapia1, G. M. Salido1
1. Department of Physiology (Cell Physiology Research Group), University of Extremadura, Caceres, Spain.
Melatonin is considered a promising antitumor agent, promoting apoptosis in tumour cells while preserving viability of normal cells. Herein, we examined the effects of melatonin on the pancreatic AR42J tumour cell line in culture. AR42J cells (ECACC N° 93100618) were cultured in RPMI 1640 supplemented with 2 mM glutamine, 10% FBS and antibiotics (0.1 mg/mL streptomycin, 100 IU penicillin) at 37 °C in a humidified incubator (5% CO2). We have analysed cytosolic free Ca2+ concentration ([Ca2+]c) by single cell imaging analysis, and mitochondrial free Ca2+ concentration ([Ca2+]m), mitochondrial membrane potential (Ψm) and mitochondrial FAD oxidative state, by confocal microscopy. Loading of cells with the fluorescent ratiometric Ca2+ indicator fura-2, with rhod-2/AM, or TMRM was employed to monitor [Ca2+]c, [Ca2+]m and Ψm, respectively. For FAD determination no dye loading was necessary, and autofluorescence of cells was monitored. We have also analyzed cellular viability, by monitoring of reduction of alamarBlue® employing an ELISA spectrofluorimeter, and caspase-3 activity, which was calculated from the cleavage of the caspase-3 specific fluorogenic substrate (AC-DEVD-AMC) and measured with a fluorescence spectrophotometer. Our results show that melatonin (at the concentrations 1 µM, 10 µM, 100 µM and 1 mM) induced changes in [Ca2+]c, that consisted of single or short lasting spikes in form of oscillations, or a slow transient increase followed by a slow decrease towards a value close to the resting level. Depletion of intracellular Ca2+ stores by stimulation of cells with 1 nM CCK-8 or 1 µM thapsigargin blocked changes in [Ca2+]c evoked by melatonin in the majority of cells. Conversely, prior stimulation of cells with 1 mM melatonin, in the absence of extracellular Ca2+, inhibited Ca2+ mobilization in response to a secondary application of CCK-8 or thapsigargin. Additionally, melatonin induced an increase in [Ca2+]m, indicating accumulation of released Ca2+ into the organelle. Melatonin also induced depolarization of Ψm and led to a reduction in the level of oxidised FAD. In addition, melatonin reduced AR42J cell viability. Finally, we found a Ca2+-dependent caspase-3 activation in response to melatonin. Collectively, these data support the likelihood that melatonin reduces viability of tumour AR42J cells via its action on mitochondrial activity and caspase-3 activation, by Ca2+-dependent mechanisms.
Where applicable, experiments conform with Society ethical requirements