Water Induces Autocrine Stimulation of Tumor Cell Killing Through ATP Release and P2 Receptor Binding
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Cell Death and Differentiation (2004) 11, S172–S180 & 2004 Nature Publishing Group All rights reserved 1350-9047/04 $30.00 www.nature.com/cdd Water induces autocrine stimulation of tumor cell killing through ATP release and P2 receptor binding N Selzner1,3, M Selzner1,3, R Graf 1, U Ungethuem1, JG Fitz2 Introduction and P-A Clavien*,1 Cell volume is maintained within a narrow physiologic range despite large changes in solute transport and extracellular 1 Department of Visceral and Transplantation Surgery, Zurich University Hospital, Zurich, Switzerland osmolalities. However, isolated cells undergo lysis after 2 Department of Medicine, University of Colorado Health Science Center, prolonged exposure to hypotonic media. This vulnerability of Denver, CO, USA cells to a hypotonic milieu has been used occasionally in 3 These authors contributed equally to this work patients. For example, for years surgeons have washed the * Corresponding author: P-A Clavien, Department for Visceral Surgery and abdominal cavity with distilled water with the intent of lysing Transplantation, University of Zurich, Ra¨mistr. 100, 8091 Zu¨rich, Switzerland isolated cancer cells left after surgery.1,2 No data are available Tel: þ 411-255-3300; Fax: þ 411-255-4449; E-mail: [email protected] supporting this practice or evaluating the potential mecha- Received 19.1.04; revised 17.6.04; accepted 20.7.04; published online 1.10.04 nisms of cancer cell injury related to hypotonic exposure. Edited by M Piacentini In most epithelial cells, transient exposure to hypotonic media stimulates compensatory mechanisms such as open- ing of ion channels which mediate solute efflux and restoration Abstract of cell volume toward basal values. Hypotonic challenge and Although exposure of cells to extreme hypotonic stress appears cell swelling also affect numerous regulators of cell metabo- to be a purely experimental set up, it has found an application in lism and transport in a cell type-specific manner. Recent clinical routine. For years, surgeons have washed the abdominal evidence indicates that release of adenosine triphosphate cavity with distilled water to lyse isolated cancer cells left (ATP) to the extracellular space is necessary in certain cell types to recover from swelling.3 ATP in the extracellular after surgery. No data are available supporting this practice space, functions as an autocrine signal by binding to or evaluating the potential mechanisms of cell injury under membrane P2 receptors.4,5 The resulting efflux of solutes these circumstances. Recent evidence indicates that increases favors passive water loss, thereby restoring cell volume. in cell volume stimulate release of adenosine triphosphate Notably, there are a number of P2 receptor subtypes which and autocrine stimulation of purinergic (P2) receptors in the are activated by extracellular ATP. Thus, a broad range of plasma membrane of certain epithelial cell types. Under cellular functions are targeted by ATP/P2 receptors.6 More- physiological conditions, purigenic stimulation can contribute over, most cells exhibit more than one P2 receptor type, to cell volume recovery through activation of solute efflux. In including members of the P2X family of ligand-regulated addition, adenosine triphosphate-P2 receptor binding might cation channels and the P2Y family of G protein coupled trigger other mechanisms affecting cell viability after profound receptors. Consequently, extracellular ATP could elicit more hypotonic stress. This study demonstrates a novel pathway of than one response depending on the P2 receptor subtype expressed and could trigger mechanisms affecting cell cell death by apoptosis in human colon cancer cells following viability after hypotonic shock. a short hypotonic stress. This pathway is induced by transitory We tested in human colon cancer cells, the effects of a short cell swelling which leads to extracellular release of adenosine period of hypotonic shock on cell viability and related triphosphate (ATP) and specific binding of ATP to P2 receptors mechanisms of injury. We first studied cell volume recovery (probably P2X7). Extracellular ATP induced activation of after a short exposure to distilled water and investigated caspases 3 and 8, annexin V, release of cytochrome c,and subsequent viability. We then measured ATP release and eventually cell death. The effect of ATP can be blocked by blocked the ATP-P2 receptor signaling using three different addition of (i) apyrase to hydrolyse extracellular ATP and (ii) agents with a focus on the induction of the apoptotic pathway. suramin, a P2 receptor antagonist. Finally, (iii) gadolinium Our results demonstrate a new pathway of cell death related pretreatment, a blocker of ATP release, reduces sensitivity of the to temporary cell swelling involving the release of extracellular cells to hypotonic stress. The adenosine triphosphate-P2 ATP and selective binding to ATP-P2 receptors, most likely to the P2X7 receptor. This autocrine or paracrine purinergic receptor cell death pathway suggests that autocrine/paracrine signaling triggers activation of caspase 8, release of cyto- signaling may contribute to regulation of viability in certain chrome c followed by caspase 3, and causes cell death by cancer cells disclosed with this pathway. apoptosis within 24 h. Cell Death and Differentiation (2004) 11, S172–S180. doi:10.1038/sj.cdd.4401505 Published online 1 October 2004 Results Keywords: ATP; colon cancer cells; P2 receptor; apoptosis Hypotonic stress decreases cell viability Abbreviations: ATP, adenosine triphosphate; LDH, lactate Abdominal lavage with distilled water is presumed to induce deshydrogenase; Gadolinium, Gd3 cell death. To assess whether this can be detected in culture Water induces tumor cell killing through ATP release N Selzner et al S173 cell models, the effects of hypotonic exposure on cell viability a were assessed in three different human colon cancer cell lines 100 (SW403, HCT 116 and Colo), chosen for their preserved phenotype and their expression of human colon cancer 80 antigens. As controls, two different nontumor cells (human fibroblasts and primary rat hepatocytes) were evaluated in 60 parallel. To establish the effect of short hypotonic stress on 40 colorectal cell viability, we exposed three tumor cells to % Viability distilled water for various periods of time (1, 3 and 5 min). Cells 20 were returned afterwards to their standard isotonic culture medium. The viability of cells was detected by Trypan blue 0 135 uptake after 24 h. Our results demonstrated a dramatic Minutes decrease in viability of cells 24 h after 1, 3 or 5 min of exposure to distilled water. As the effect on cell viability was * * intense with either period of exposure, we performed further b experiments with only 1 min of exposure to distilled water (Figure 1a). Next, we evaluated cell viability at three different 100 times points after 1 min of exposure to distilled water. Only minimal cell death was detected in all groups after 4 h 80 incubation. Afterwards, all three cell lines challenged with pure water exhibited a dramatic decrease in viability in a time- dependent manner after 12 and 24 h incubation. Finally, we 60 determined the effect of (i) gradual hypotonic dilution (0.20, 40 0.45 and 0.75%), (ii) isotonic (0.9%) saline solution and (iii) % Viability hypertonic saline solution (1.8%) on cell viability 24 h after 1 min of incubation with either of these solutions. Only minimal 20 decreases in viability were detectable in all three cell lines after exposure to 0.20, 0.45, 0.75, 0.9, or 1.8% saline 0 solutions. The results for the most sensitive cell line SW403 41224 are presented in Figure 1b. While long exposure (24 h) to Hours hypertonic media can affect cell viability8,9 our results indicate that a short hypertonic challenge is harmless. As results were c 100 similar for the other two colon cancer cell lines, these data are not shown. In contrast to colon cancer cells, only minimal 80 decreases in viability (10 and 5%) were detectable in human fibroblasts and rat hepatocytes 24 h after exposure to 1 min of distilled water (Figure 1c, n ¼ 5 for each experiments). 60 40 % Viability Hypotonic shock induces temporary cell swelling followed by release of endogenous ATP 20 To assess the effect of cell volume changes on the subsequent ATP release, we first determined the cell volume 0 after hypotonic stress. Mean cell volumes were measured rat hepatocytes human fibroblasts during hypotonic (distilled H2O, 0.20 or 0.45%) and isotonic Figure 1 Effect of exposure time and hypotonic shock on cell viability. (a) (0.9% saline solution) challenge at 0, 30 and 60 s. We further SW403 cells were exposed for 1, 3 or 5 min to distilled water (&) or isotonic evaluated cell volume recovery after 2, 5 and 20 min after solution (0.9% saline ’) followed by restitution in normal cell medium (DMEM). replacement of the media by culture medium (Figure 2). Cells Cell viability was evaluated by trypan blue staining at 24 h after hypotonic stress (n ¼ 5 in each group). (b) SW403 cells were exposed for 1 min to distilled water underwent a three-fold increase in volume within 30 s of (’) or different concentrations of saline solution (0.20 0.45 &, 0.75 , 0.9 or exposure to distilled H2O, and a five-fold increase after 1 min 1.8% ) followed by restitution in normal cell media (DMEM). Cell viability was compared to the control group (cells exposed to isotonic evaluated by trypan blue staining at 4, 12 and 24 h after osmotic stress. media). Cells exposed to 1 min of 0.20 and 0.45% hypotonic Hypotonic stress by distilled water induced 50 and 35% of SW403 cell death after media resulted in 1.4- and 2-fold increase of volume 12 and 24 h incubation (a)(n ¼ 5 in each group, Po0.001, ANOVA; * ¼ Po0.001, Bonferroni post hoc). (c) Rat hepatocytes or human fibroblasts compared to the control group.