Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate
1 Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and CSIC, C/Sanz y Forés s/n. 47003-Valladolid, Spain
Abstract
Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway involved in control of multiple cellular and physiological processes including cell proliferation. Recent evidence has shown that SOCE depends critically on mitochondrial sinking of entering Ca2+ to avoid Ca2+-dependent inactivation. Thus, a role of mitochondria in control of cell proliferation could be anticipated. We show here that activation of SOCE induces cytosolic high [Ca2+] domains that are large enough to be sensed and avidly taken up by a pool of nearby mitochondria. Prevention of mitochondrial clearance of the entering Ca2+ inhibited both SOCE and cell proliferation in several cell types including Jurkat and human colon cancer cells. In addition, we find that therapeutic concentrations of salicylate, the major metabolite of aspirin, depolarize partially mitochondria and inhibit mitochondrial Ca2+ uptake, as revealed by mitochondrial Ca2+ measurements with targeted aequorins. This salicylate-induced inhibition of mitochondrial Ca2+ sinking prevented SOCE and impaired cell growth of Jurkat and human colon cancer cells. Finally, direct blockade of SOCE by the pyrazole derivative BTP-2 was sufficient to arrest cell growth. Taken together, our results reveal that cell proliferation depends critically on mitochondrial Ca2+ uptake and suggest that inhibition of tumour cell proliferation by salicylate may be due to interference with mitochondrial Ca2+ uptake, which is essential for sustaining SOCE. This novel mechanism may contribute to explaining the reported anti-proliferative and anti-tumoral actions of aspirin and dietary salicylates.
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Introduction
Much evidence indicates that Ca2+ signals participate in the control of cell proliferation (Berridge, 1995; Kahl & Means, 2003). They activate, for example, immediate early genes responsible for inducing resting cells to enter the cell cycle, promote DNA synthesis and stimulate different events at mitosis (Berridge, 1995). Particular attention has been paid to the role of Ca2+ entry through store-operated channels (SOCs, Parekh & Putney, 2005). Store-operated Ca2+ entry (SOCE), also termed capacitative Ca2+ entry (Putney, 1986), is the most important Ca2+ influx pathway in non-excitable cells (Parekh & Penner, 1997). SOCE is activated by emptying of intracellular Ca2+ stores, either by inositol 1,4,5 trisphosphate produced upon activation of G-protein-coupled or tyrosine kinase receptors or by pharmacological emptying of the Ca2+ stores with, for instance, thapsigargin (Montero et al. 1990, 1991; Alvarez et al. 1994; Parekh & Putney, 2005). The involvement of SOCE in cell proliferation has been studied in detail in Jurkat T cells. In these cells, T cell receptor stimulation induces sustained SOCE through the Ca2+ release-activated current (ICRAC) resulting in sustained activation of the nuclear factor of activated T cells (NFAT) that promotes proliferation (Lewis, 2001). Many observations in this and other cell models relate SOCE and cell proliferation: (i) entry in cell cycle is preceded by SOC activation (Sugioka & Yamashita, 2003); (ii) growth factor stimulation of cell proliferation is accompanied by increased activity and/or expression of transient receptor potential (TRP) channels that are related to SOCE (Golovina et al. 2001); (iii) SOCE inhibition by different means (Ca2+ removal, inorganic channel blockers and ICRAC antagonists) abolishes tumour cell proliferation (Weiss et al. 2001; Zitt et al. 2004).
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Two decades after the discovery of SOCE (Putney, 1986), the link between emptying of intracellular Ca2+ stores and the increased Ca2+ influx is still unknown (Parekh & Putney, 2005). Likewise, the nature of the SOCs responsible for SOCE remains controversial. A number of members of the TRP family of cation channels, including several canonical TRPs (TRPCs) and TRPV6, have been proposed to be involved in SOCE (Parekh & Putney, 2005). An important characteristic of SOCE, ICRAC and the TRP channels related to SOCE is the strong Ca2+-dependent inactivation that limits Ca2+ entry (Singh et al. 2002; Parekh & Putney, 2005). In fact, ICRAC is hardly recorded unless the Ca2+-dependent inactivation is prevented by a strong intracellular Ca2+ buffer (Gilabert & Parekh, 2000). Thus, intracellular Ca2+ extrusion systems are important for sustaining SOCE. Mitochondria play an important role as a Ca2+ sinking system able to shape the cytosolic Ca2+ signals (Rizzuto et al. 2000; Montero et al. 2000; Villalobos et al. 2002). With regard to SOCE, a series of reports from the laboratories of Lewis (Hoth et al. 1997, 2000) and Parekh (Gilabert & Parekh, 2000; Gilabert et al. 2001, 2002) have shown that sustained SOCE requires normal mitochondrial Ca2+ uptake in Jurkat and rat basophilic leukaemia (RBL-1) cells. Protonophores such as CCCP or FCCP prevent SOCE since they collapse the mitochondrial potential (), the driving force for Ca2+ entry into mitochondria. The inhibition of SOCE has been attributed to Ca2+-dependent inactivation of ICRAC due to generation of high [Ca2+]cyt domains secondary to a lack of mitochondrial Ca2+ uptake (Hoth et al. 1997, 2000; Glitsch et al. 2002; Malli et al. 2003; Parekh, 2003 but see also Varadi et al. 2004; Frieden et al. 2004). We have reported recently that activation of voltage-gated Ca2+ channels induces in excitable cells large [Ca2+]cyt increases that are sensed by subplasmalemmal mitochondria (Montero et al. 2000; Villalobos et al. 2001). This pool amounting to about 50% of the mitochondria, takes up tremendous amounts of Ca2+ (driving [Ca2+]mit to near millimolar levels) and clears efficiently the high [Ca2+]cyt domains (Villalobos et al. 2002). Whether SOCE induces such high [Ca2+]cyt domains leading to large [Ca2+]mit increases in a mitochondrial pool has not been studied.
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If SOCE is essential for cell proliferation and depends on mitochondrial Ca2+ uptake, then it follows that drugs interfering with mitochondrial Ca2+ uptake may prevent cell proliferation via SOC inactivation. Here we tested whether this rationale can explain the effects of salicylate, the major metabolite of aspirin (acetylsalicylic acid, ASA). Salicylate acts as a proton carrier and uncouples mitochondria (Pachman et al. 1971; Gutknecht, 1990). According to the above rationale, salicylate should interfere with mitochondrial Ca2+ uptake, SOCE and cell proliferation. In fact, aspirin and salicylate have been reported to inhibit tumour cell growth and to prevent colon and other cancers (Hanif et al. 1996; Molina et al. 1999; Perugini et al. 2000; Smith et al. 2000; Baron et al. 2003; Sandler et al. 2003), although the action mechanism is unknown. Salicylate has been recently reported to also inhibit proliferation of normal vascular smooth muscle cells (Marra & Liao, 2001). Here we have investigated (i) whether SOCE is regulated by mitochondria in colon cancer cells, (ii) whether proliferation of tumour cells depends on mitochondrial Ca2+ uptake and (iii) whether salicylate inhibits tumour cell proliferation by acting on mitochondrial control of SOCE. For these studies we have used HT29 human colon cancer cells and Jurkat T cells where mitochondrial control of SOCE and the role of SOCE in proliferation were first described (Hoth et al. 1997; Lewis, 2001).
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Methods
Materials
Fura-2 AM, Fura-4 AM, JC-1 (a cationic dye that indicates mitochondrial polarization) and tetramethylrhodamine ethyl ester perchlorate (TMRE) were purchased from Molecular Probes. Ru360 (a calcium signalling tool) and BTP2 (an immunomodulator) were purchased from Calbiochem. Thapsigargin was obtained from Alomon Laboratories. Other reagents and chemicals were obtained either from Sigma or Merck. Mutated aequorin was kindly donated by M. Montero (Valladolid University, Valladolid). Jurkat cells were obtained from the American Type Culture Collection. HT29 cells were donated by J. C. Fernández-Checa (CSIC, Barcelona). SW480 cells were donated by A. Muoz (CSIC, Madrid). NIH 3T3 cells expressing luciferase under control of the topoisomerase-II promoter were a kind gift of M. Sehested (Rigshospitalet, Copenhagen).
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Store-operated Ca2+ entry
Cells were plated at about 0.5 x 106 cells ml–1 on 12 mm glass coverslips treated with poly L-lysine (HT29) or fibronectin (Jurkat) and loaded with 4 μM fura-2 /AM or fura-4 AM for 60 min at room temperature. Cells were then incubated with 1 μM thapsigargin for 10 min in Ca2+-free standard medium containing (mM): NaCl, 145; KCl, 5; MgCl2, 1; EGTA, 0.5; glucose, 10; Hepes/NaOH, 10 (pH 7.42) and placed on the stage of an inverted microscope (Nikon Diaphot) maintained at 37°C. They were then perfused with prewarmed Ca2+-free medium, and epi-illuminated alternately at 340 and 380 nm. Light emitted above 520 nm was recorded with a Magical Image Processor (Applied Imaging). Pixel-by-pixel ratios of consecutive frames were captured and [Ca2+]cyt was estimated from these ratios as previously reported (Villalobos et al. 2002). Re-addition of standard, Ca2+-containing (CaCl2, 1 mM; no EGTA) medium evoked large [Ca2+]cyt increases revealing store-operated Ca2+ entry (Montero et al. 1990; Villalobos & García-Sancho, 1995; Hoth et al. 2000; Glitsch et al. 2002). In some experiments, SOCE induced by physiological stimulation with carbachol (100 μM) in HT29 cells and TCR stimulation in Jurkat cells was tested. For TCR stimulation, Jurkat cells were suspended in external medium and loaded with Fura-4 AM (4 μM). Then, cells were plated on fibronectin-coated glass coverslips previously incubated with IgG2a for 4 h at room temperature. Cells were then placed in the fluorescence microscope and stimulated with 0.5 μg ml–1 anti-CD3 (the T cell receptor) and 5 μg ml–1 anti-CD28 (a co-receptor) to elicit the cross-linking activation of TCR.
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Mitochondrial potential
HT29 cells were loaded with JC-1 (1 μg ml–1) for 10 min and subjected to confocal microscopy with a Bio-Rad laser scanning system (Radiance 2100) coupled to a Nikon eclipse TE2100U inverted microscope. For TMRE measurements, HT29 cells were loaded with TMRE (100 nM) for 30 min at room temperature, placed on the perfusion chamber of a Zeiss Axiovert 100 TV inverted microscope and superfused continuously with prewarmed (37°C) standard medium. Fluorescence images were taken at 5 s intervals with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor. Traces from individual cells were expressed as the percentage value of fluorescence before addition of salicylate and averaged. In some experiments, cells were loaded with TMRE (100 nM) and fluorescence from mitochondria and nearby cytosol was analysed with the confocal system. Localization of TMRE fluorescence in mitochondria was tested by co-loading cells with Mitotracker Red. To quantify mitochondrial depolarization, the effect of salicylate and FCCP on the ratio of TMRE fluorescence in mitochondria relative to that surrounding cytosol was calculated as reported by Collins et al. (2002).
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Mitochondrial calcium uptake
HT29 cells were transfected (lipofectamin) with a plasmid containing either the wild type or the mutated (Asp119Ala), low-Ca2+ affinity aequorin targeted to mitochondria (Montero et al. 2000). After 24 h, cells were incubated in standard medium (see above) containing 1 μM of either wild type or coelenterazine (n, acquorin cofactors) for 2 h at room temperature. The coverslips were then placed in a luminometer (Cairn Research), perfused continuously with warm (37°C) standard medium and subjected to photon counting at 1 s intervals. For some experiments, cells were permeabilized with 20 μM digitonin in ‘intracellular’ medium (130 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM K3PO4, 0.2 mM EGTA, 1 mM ATP, 20 μM ADP, 2 mM succynate, 20 mM Hepes/KOH, pH 6.8). The cells were then incubated with the same medium containing 200 nM Ca2+ (buffered with EGTA) with or without salicylate for 5 min. Finally, perfusion was switched to ‘intracellular’ medium containing 6 μM Ca2+ (with or without salicylate) for 1 min. Photonic emissions were converted to mitochondrial-free Ca2+ concentration ([Ca2+]mit) values as reported previously (Montero et al. 2000; Villalobos et al. 2001; Alvarez & Montero, 2001).
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Cell growth, apoptosis and cell viability
Cells (HT29, SW480, Jurkat and NIH 3T3) were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI 1640 media (Gibco) containing 10% fetal bovine serum and antibiotics. Cells were plated in wells at about 5 x 104 cells ml–1 and incubated with test solutions for 96 h. Triplicate wells were counted daily or after 96 h. Cell death was estimated in the same samples by trypan blue exclusion. Data correspond to at least three independent experiments and are expressed as cell number-fold increase, percentage growth rate relative to control or cells per hour. Differences were considered significant at P < 0.05 (Student's t test). For determination of apoptotic cells at single cell level, cells were plated at about 5 x 104 cells ml–1 and incubated with test solutions for 96 h. Apoptotic cells were revealed by the terminal deoxynucleaotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method by means of fluorescence microscopy and the in situ cell death detection kit (Roche Diagnostics, Penzberg, Germany) following the protocol provided by the manufacturer.
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ATP measurements
HT29 cells were plated in 6-well plates and cultured in medium containing either vehicle or different salicylate concentrations (10–2000 μM). After 24 h, cells were washed twice with phosphate-buffered saline (PBS) at 37°C and 1 ml of boiling 20 mM Tris, pH 7.75, 4 mM EDTA solution was added. After 2 min, samples were centrifuged for 4 min at 10 000 g. ATP was measured later from the supernatant by the luciferin–luciferase assay (Sánchez, 1985). Readings were taken using a scintillation counter (Wallac model 1409/11) over 20 s intervals with the windows wide open. ATP levels were determined with the aid of a standard curve prepared using pure ATP over a 10–5–10–9M concentration range.
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Bioluminescence imaging of transcription dynamics and cell division
NIH 3T3 cells stably transfected with the luciferase reporter gene under control of the topoisomerase-II gene promoter (Falck et al. 1999) were synchronized in medium containing 0.5% serum for 3 days. Then, 10% serum and 1 mM luciferin were added and the Petri dish containing the cells was placed in an incubator (Zeiss CTI-controller 3700) controlling temperature, CO2 and humidity, attached over the stage of an inverted microscope (Zeiss Axiovert 100 TV). Photonic emissions and bright field images were captured concurrently with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor at 15 min intervals for 72 h. Transcription activity is expressed as total photonic emissions in each image minus background photonic emissions, as previously reported (Villalobos et al. 1999).
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Results
SOCE was shown by the increase in cytosolic Ca2+ concentration ([Ca2+]cyt) that follows addition of extracellular Ca2+ to fura-2-loaded Jurkat cells pre-treated with thapsigargin in Ca2+-free medium. We found that FCCP reversibly reduced the initial rate and the size of SOCE in Jurkat cells (Fig. 1A). SOCE was also shown in HT29 human colon carcinoma cells by the same [Ca2+]cyt overshoot protocol (Fig. 1B). No Ca2+ overshoot was observed in cells not pre-treated with thapsigargin (data not shown). A second pulse of extracellular Ca2+ evoked a similar [Ca2+]cyt increase and FCCP reversibly prevented SOCE (Fig. 1B). We noticed that the fura-2 signals were often saturated during SOCE in many individual cells. This would result in the underestimation of both [Ca2+]cyt peaks and inhibition by FCCP. To avoid this problem we repeated the experiments using the lower affinity Ca2+ probe fura-4 F instead of fura-2 (Villalobos et al. 2002). [Ca2+]cyt increases measured by fura-4 F ranged between 1.3 and 5.8 μM (3.2 ± 1.8 μM, mean ±S.E.M., n= 9) in HT29 cells and were also inhibited by FCCP (Fig. 1C). Similar results were obtained in Jurkat cells (data not shown). The SOCE-induced increase in [Ca2+]cyt as well as the FCCP-induced inhibition were not affected by the presence of F0F1–ATP synthase blocker oligomycin (Fig. 1D) indicating that the effects of FCCP are not due to ATP depletion. Oligomycin had no effect on subsequent control pulses either (not shown). In order to interpret the lack of effect of oligomycin, it must be remembered that mitochondrial respiration is very much inhibited in tumour cells simply by incubation in glucose-containing media (Crabtree, 1929). The inhibition is due to blocking of the F0F1–ATPase, but the mitochondrial potential is not lost or is even increased (Wojtczak et al. 1999). Treatment with the respiratory chain inhibitor antimycin A plus oligomycin, a cocktail that collapses and prevents SOCE and ICRAC in Jurkat and RBL-1 cells (Glitsch et al. 2002), also inhibited SOCE in HT29 cells (Fig. 1E). Valinomycin, a K+ ionophore that collapses the mitochondrial but hyperpolarizes the plasma membrane, also inhibited SOCE in HT29 cells (Fig. 1F) suggesting that the effects of FCCP are due to collapse of the mitochondrial rather than to plasma membrane depolarization. In medium containing high K+ (50 mM), a condition that should hold the membrane potential in a quite depolarized state, FCCP also inhibited SOCE (data not shown). Carbachol activates M3 muscarinic receptors in HT29 cells provoking a biphasic [Ca2+]cyt increase, a peak due to the transient Ca2+ release from intracellular stores followed by a plateau due to sustained Ca2+ entry via SOC (Kerst et al. 1995). FCCP did not affect the Ca2+ release induced by carbachol indicating that FCCP does not affect the ATP-dependent refilling of intracellular Ca2+ stores. Similar results have been reported previously in Jurkat cells (Makowska et al. 2000). However, FCCP inhibited the carbachol-induced Ca2+ entry (Fig. 1G and H). FCCP also inhibited SOCE in SW480 cells, a human colon cancer cell lacking cyclooxygenase-2 (COX-2) gene expression (Smith et al. 2000) (data not shown). Thus, human colon carcinoma cells display a robust SOCE that is prevented by mitochondrial depolarization and this effect is independent of ATP, plasma membrane depolarization and COX-2 gene expression.
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SOCE was started by addition of Ca2+ to thapsigargin-treated (1 μM, 10 min), fura-2-loaded cells incubated in Ca2+-free medium. A and B, effects of FCCP (10 μM) in Jurkat (A) and HT29 cells (B). C and D, effects of FCCP (10 μM) and oligomycin (0.12 μM) plus FCCP (10 μM) on Ca2+ entry in fura-4 F-loaded HT29 cells. E and F, effects of oligomycin (0.12 μM) plus antimycin A (0.5 μg ml–1) and valinomycin (10 μM) on SOCE in HT29 cells. G and H, carbachol (100 μM) elicited a biphasic [Ca2+]cyt increase in HT29 cells loaded with fura-4 F. In G, cells were perfused in Ca2+-containing medium with or without (control; ) FCCP (10 μM). FCCP inhibited the sustained but not the transient [Ca2+]cyt increase. In H, cells were perfused with Ca2+-free medium, except for the period indicated (Ca) in which perfusion was shifted to Ca2+-containing medium. All traces of [Ca2+]cyt are averaged values (mean ±S.E.M.) of 28–56 cells and representative of 3–9 similar experiments.
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We next studied whether SOCE increases [Ca2+]mit in colon cancer cells. We transfected HT29 cells with mitochondria-targeted aequorin and measured mitochondrial Ca2+ uptake by photon counting (Montero et al. 2000) during SOCE induced by the same Ca2+ re-addition protocol used above. The addition of extracellular Ca2+ to thapsigargin-treated cells produced massive photoluminescence emission with consumption of about 50% of aequorin (45 ± 5%, n= 3). The estimated [Ca2+]mit reaches about 2 μM. A second and subsequent stimuli produced a very small photonic emission (3 ± 1% of the total emissions) corresponding to a negligible [Ca2+]mit increase (Fig. 2A). However, the second stimulus produced a [Ca2+]cyt increase of the same extent as the first one (Fig. 1B). These results are similar to those obtained by sequential stimulation with high K+ pulses in chromaffin (Montero et al. 2000) and GH3 (Villalobos et al. 2001) cells. These results were interpreted in terms of two pools of mitochondria. The first pool, amounting to about 50% of mitochondria and located strategically near the sites of Ca2+ entry, is able to sense the high [Ca2+]cyt domains required for Ca2+ uptake through the mitochondrial Ca2+ uniporter (Montero et al. 2000). Massive Ca2+ uptake by this pool results in huge [Ca2+]mit increases near the millimolar level that dampens progression of the Ca2+ wave towards the cell core. This huge [Ca2+]mit increase by the first mitochondrial pool burns out all the aequorin present in this pool during the first stimulus. As a result, the remaining mitochondria, probably located far away from the channels, sense much smaller [Ca2+]cyt domains and take up negligible Ca2+ amounts so that the second pool does not produce photoluminescence emission. This interpretation was tested in HT29 cells by transfecting them with a mutated, mitochondria-targeted aequorin with much lower affinity for Ca2+ (Montero et al. 2000). The measurements revealed that SOCE actually induced a huge [Ca2+]mit increase of several hundred micromolar (425 ± 85 μM, n= 5, Fig. 2B). These values approach the ones measured in mitochondria from excitable cells upon activation of voltage-gated Ca2+ channels (Montero et al. 2000; Villalobos et al. 2001). Taken together, these results support the view that SOCE induces high [Ca2+]cyt domains that are sensed and cleared by a mitochondrial pool probably located nearby. Collapse of abolish mitochondrial Ca2+ uptake and impairs this clearance leading to local increase of [Ca2+]cyt and SOCE inhibition.
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A, HT29 cells were transfected with mitochondria targeted aequorin and subjected to photon counting measurements for estimation of [Ca2+]mit (see Methods). Cells were first treated with 1 μM thapsigargin for 10 min in Ca2+-free medium and Ca2+ entry was started by addition of external Ca2+ (1 mM; Ca). The top panel shows aequorin consumption and the bottom one the [Ca2+]mit estimate (see Methods for details). Data are representative of 3 similar experiments. B, HT29 cells were transfected with the low Ca2+ affinity mutated mitochondria-targeted aequorin, reconstituted with coelenterazine n and subjected to photon counting measurements. Other details as in A. For calibration of [Ca2+]mit, the size of the mitochondrial pool was assumed to contain 45% of the aequorin. Data are representative of 5 similar experiments.
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A chemist's look at salicylic acid shows it is a weak acid bound to an aromatic, hydrophobic ring, a lipophilic acid structure resembling that of mitochondrial uncouplers. We have studied the effects of salicylate on and mitochondrial Ca2+ uptake. We have used two different mitochondrial potential-sensitive probes, JC-1 and TMRE, to study the effects of salicylate on in intact HT29 and Jurkat cells. Confocal microscopy of JC1-loaded HT29 cells revealed that salicylate de-energised mitochondria, as shown by the shift of the emitted fluorescence from red to green in a manner comparable to FCCP (Fig. 3A and B). A more quantitative estimate was attempted using TMRE as probe. Results are shown in Fig. 3C and D. Salicylate induces a concentration-dependent loss of at therapeutic concentrations, as indicated by the decrease of TMRE fluorescence (see also Supplemental movie 1). Figure 3E shows confocal images of TMRE-loaded HT29 cells. To quantify further mitochondrial depolarization we calculated the ratio of TMRE fluorescence from mitochondria relative to the surrounding cytosol as reported by Collins et al. (2002). This ratio was largely decreased by salicylate (500 μM) and FCCP (10 μM) in both HT29 (Fig. 3F) and Jurkat (Fig. 3G) cells.
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A, confocal images of HT29 cells loaded with JC-1 (1 μg ml–1, 10 min) in vehicle (Control) or salicylate (+Sal, 500 μM, 10 min). Emitted red and green fluorescence corresponds to energised and depolarized mitochondria, respectively. Bar represents 10 μm. B, values of ratios (mean ±S.E.M.; n= 4) of red/green fluorescence in control, salicylate- and FCCP- (10 μM, 10 min) treated cells. P < 0.05 versus control (Student's t test). C, decrease of TMRE fluorescence induced by FCCP (10 μM) and different salicylate concentrations (in μM) in HT29 cells. The arrow indicates addition of either vehicle, FCCP or salicylate. Fluorescence values for each individual cell were normalized relative to the value before treatment. Traces correspond to the averaged (mean ±S.E.M.), normalized recordings of 48–76 cells. Pictures show TMRE fluorescent images of the same cells before (Control) and 5 min after addition of 500 μM salicylate (+Sal) or 10 μM FCCP (see also Supplemental movie 1). Bar represents 10 μm. D, dose–response relation of TMRE fluorescence quenching by treatment with salicylate; values are mean ±S.E.M. of three independent experiments. All points were significantly different from control (P < 0.05). E, confocal images of bright field and TMRE-loaded HT29 cells in vehicle (Control), 10 min after 500 μM salicylate (+Sal) and 10 min after 10 μM FCCP (+FCCP). Bar represents 10 μm. The ratio of TMRE fluorescence in mitochondria relative to the surrounding cytosol was calculated as reported by Collins et al. (2002). Salicylate and FCCP largely decreased this ratio in both HT29 (F) and Jurkat (G) cells. Data correspond to 12 HT29 and 15 Jurkat cells studied in 3 independent experiments for each cell type.
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Next we asked whether the salicylate-induced mitochondrial depolarization would affect mitochondrial Ca2+ uptake. The relation between mitochondrial Ca2+ uptake and , the driving force for Ca2+ entry into mitochondria, is exponential. Thus, the effects of salicylate on mitochondrial Ca2+ uptake should be even larger than on . The Nernst relation predicts that every 30 mV depolarization (which is less than a 20% decrease of the resting –180 mV potential) diminishes 10-fold the equilibrium [Ca2+]mit (Bernardi, 1999). In order to test directly the effects of salicylate on Ca2+ transport we measured the mitochondrial Ca2+ uptake in HT29 cells using the mutated, low Ca2+ affinity aequorin targeted to mitochondria (Montero et al. 2000). Exposure of permeabilized HT29 cells to Ca2+ concentrations below 2 μM failed to induce any mitochondrial Ca2+ uptake (data not shown) indicating the requirement for high [Ca2+]cyt concentrations, such as those reached in high [Ca2+] microdomains, to activate the mitochondrial Ca2+ uniporter (Montero et al. 2000). Exposure of permeabilized HT29 cells to 6 μM Ca2+ induced a huge [Ca2+]mit near 800 μM within less than 30 s (Fig. 4A, control). This mitochondrial [Ca2+] uptake was inhibited by ruthenium compounds (Ruthenium red or Ru360, data not shown) which have been reported to block the mitochondrial Ca2+ uniporter (Kirichok et al. 2004) and by collapsing with FCCP (Fig. 4A, FCCP). Salicylate inhibited mitochondrial Ca2+ uptake in a dose-dependent manner (Fig. 4A and B) with an IC50 below 10 μM, a salicylate concentration that is surpassed by the therapeutic concentrations and comparable to the plasma values achieved with dietary salicylates (Paterson & Lawrence, 2001).
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Effects of salicylate (concentrations in μM) and FCCP (10 μM) on the Ca2+ uptake by mitochondria. A, HT29 cells expressing mutated mitochondrial aequorin were permeabilized and [Ca2+]mit was calculated from photonic emissions (details as in Fig. 2). Ca2+ uptake was started by perfusion with 6 μM Ca2+ (filled bar). B, average effects of different salicylate concentrations on mitochondria Ca2+ uptake in 3 experiments similar to that shown in A. Data are mean ±S.E.M. All points were significantly different from control.
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As SOCE depends on mitochondrial Ca2+ uptake, we tested next the effects of the same salicylate concentrations used in the above experiments on SOCE. Results are shown in Fig. 5. Salicylate inhibited SOCE in a concentration-dependent manner in both HT29 and Jurkat cells (Fig. 5A and B). The IC50 values were about 100 μM, well within the therapeutical range (Fig. 5C and D). The inhibition of SOCE by salicylate was not immediate but required a pre-incubation time of up to 10–12 min, consistent with the TMRE fluorescence decay (data not shown). In addition, whereas the effects of FCCP were quickly reversible (Fig. 1A and B), reversion of the inhibitory effects of salicylate required extensive washout of the drug (data not shown). The inhibiton of SOCE induced by salicylate was not affected by pre-incubation with oligomycin (not shown). Salicylate also inhibited SOCE induced by physiological simulation with carbachol in HT29 cells (Fig. 5E) and by TCR activation in Jurkat cells (Fig. 5F). Finally, salicylate also inhibited SOCE in SW480 cells lacking COX-2 gene expression (data not shown).
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Effects of different salicylate concentrations (in μM) on SOCE in thapsigargin-treated, fura-4 F-loaded, HT29 (A) and Jurkat (B) cells. Each couple of traces correspond to [Ca2+]cyt recordings either before and after incubation (10 min) with different salicylate concentrations in the same cells. Each trace is the mean ±S.E.M. of 29–59 cells. C and D, dose–response curves of effects of salicylate on SOCE inhibition induced by salicylate in HT29 (C) and Jurkat (D) cells. Each value is the mean ±S.E.M. of 3 experiments. E, effects of salicylate (2 mM) on Ca2+ entry induced by carbachol (100 μM) in HT29 cells (traces are the mean ±S.E.M. of 23 and 32 cells and representative of 3 similar experiments). F, effects of salicylate (2 mM) on Ca2+ entry induced by TCR stimulation in Jurkat cells (0.5 μg ml–1 anti-CD3 and 5 μg ml–1 anti-CD28 cross-linked with IgG2a). Traces are mean ±S.E.M. of 12 and 22 cells and representative of 3 similar experiments.
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Since SOCE has been reported to be essential for tumour cell proliferation (Lewis, 2001) we tested next the effects of salicylate on cell growth. Results are shown in Fig. 6. Salicylate inhibited growth of both HT29 and Jurkat cells (Fig. 6A and B) at therapeutic concentrations. Salicylate also inhibited growth of SW480 cells lacking COX-2 gene expression by 41 ± 8% at 500 μM (mean ±S.E.M., n= 4). Most interestingly, the IC50 values for salicylate-induced inhibition of growth were similar to those found for SOCE inhibition (Fig. 6C and D). The correlation between SOCE prevention and growth inhibition was very good in both Jurkat and HT29 cells (r= 0.94–0.96, P < 0.005–0.05; Fig. 6E and F). It could be argued that the inhibition of growth by salicylate could be due to induction of cell death. However, at the same low concentrations, salicylate did not promote significant cell death (Fig. 6G and H). At 2 mM, cell death was only slightly increased whereas at 5 mM, a concentration 10 times above the therapeutic range and considered toxic, salicylate induced extensive cell death (Fig. 6G and H). It could also be argued that the inhibitory effects of salicylate may be due to a decrease in cell ATP. We tested the effects of different concentrations of salicylate on the cell ATP contents and found that at concentrations of 10–2000 μM it did not affect significantly the cell ATP levels in HT29 cells (Fig. 7A). Finally, we carried out a tunel assay to ascertain whether salicylate may prevent cell growth by promoting apoptosis. We found that salicylate, at the concentrations used (10 μM–2 mM) to prevent SOCE or cell growth, does not induce apoptosis (Fig. 7B). At larger concentrations (5 mM), salicylate promoted apoptosis (Fig. 7B). These results indicate that low, therapeutic concentrations of salicylate prevent tumour cell growth and this effect is not due to changes in ATP levels or cell death.
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Effects of different concentrations of salicylate (in μM) on HT29 (A) and Jurkat (B) cell growth, expressed as cell number fold increase. All the groups differed statistically from each other except for the effects of 500 μM salicylate (not shown) that were not significantly different from the effects of 100 μM salicylate. Dose–response curve of the effects of salicylate on HT29 (C) and Jurkat (D) cell growth. E and F, correlation between growth inhibition (%) and SOCE inhibition (%) in HT29 (E) and Jurkat (F) cells. Lines are best linear fit (r= 0.94–0.96; P < 0.005–0.05). G and H, effects of salicylate on percentage of dead cells (cells stained with trypan blue) after 96 h incubation with the different salicylate concentrations (in μM) in HT29 (G) and Jurkat (H) cells. Cell death induced by 10, 100 or 500 μM salicylate were not significantly different from control (P > 0.05).
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A, effects of salicylate on cell ATP concentrations in HT29 cells measured by the luciferin/luciferase assay. Salicylate (10– 2000 μM) did not affect ATP concentrations (P > 0.05). B, the effect of salicylate on the per cent of apoptotic cells was tested by the tunel assay. Pictures show HT29 cell nuclei (blue) and apoptotic cells (red) in vehicle (control) and cells treated with different concentrations of salicylate. The bar represents 10 μm. B, bars show the per cent of apoptotic cells in each condition (mean ±S.E.M., n= 3). Salicylate did not induce apoptosis at concentrations of 10– 2000 μM (P > 0.05).
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To investigate further inhibition of proliferation, we monitored concurrently transcription dynamics of the topoisomerase-II gene, a cell cycle-driven gene and marker of proliferation (Falck et al. 1999), together with cell division in individual, living cells. This was achieved by a novel methodology based on bioluminescence imaging of transcription dynamics in single, living cells (Villalobos et al. 1999). We adapted the method for concurrent monitoring of cell division and gene transcription in NIH3T3 cells stably expressing the luciferase reporter gene under control of the topoisomerase-II gene promoter (Falck et al. 1999). Figure 8 shows the pseudocolour-coded bioluminescence images superimposed on the bright field image at the beginning of the experiment and after 24, 48 and 72 h for cells incubated in either control or salicylate-containing medium. Promoter activity was not detectable in resting cells but was turned on during progression of the cell cycle to peak before cell division and turned off right after mitosis to start a new cycle (Fig. 8; see also Supplemental movie 2). Salicylate blocked transcription cycling of topoisomerase-II gene (Fig. 8) and inhibited proliferation of NIH3T3 cells by 78 ± 8% (mean ±S.E.M., n= 3).
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Transcription activity of topoisomerase-II and cell division were monitored for 72 h at 15 min intervals by concurrent bioluminescence imaging and bright field microscopy in NIH 3T3 cells expressing luciferase under control of the topoisomerase-II gene promoter. The pictures show accumulated photonic emissions (15 min integration period) superimposed on bright field images in control (top) and salicylate-treated cells (bottom) at 0, 24, 48 and 72 h. Bar represents 10 μm. Traces show photonic emissions at 15 min intervals for 72 h in vehicle (control) and salicylate (500 μM) containing medium. Data are representative of 15 (control) and 4 (salicylate) experiments. See also Supplemental movie 2.
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Our results suggest that tumour cell proliferation requires mitochondrial Ca2+ uptake. If this hypothesis is correct, then it follows that any other compound that prevents mitochondrial Ca2+ uptake, either by mitochondrial depolarization or by other means, should inhibit SOCE and tumour cell proliferation. Consistent with this view, Kerzic and colleagues have shown recently that diazoxide, a mitochondrial KATP channel opener that depolarizes mitochondria by promoting K+ entry into the matrix, inhibits SOCE and prevents Jurkat T cell proliferation in a mitochondria-dependent manner (Holmuhamedov et al. 2002). We found that 100 μM diazoxide inhibited proliferation to the same extent as 100 μM salicylate in both Jurkat and HT29 cells (data not shown). Ruthenium compounds prevent mitochondrial Ca2+ uptake by direct action on the mitochondrial Ca2+ uniporter without interfering with . Figure 9 shows that Ru360, a membrane-permeable analogue of ruthenium red, also inhibited SOCE (Fig. 9A) and transcription cycling of the topoisomerase-II gene (Fig. 9B). Ruthenium compounds also inhibited proliferation in both Jurkat and HT29 cells (Fig. 9C and D). Thus, inhibition of mitochondrial Ca2+ uptake by different means (uncouplers, diazoxide, salicylate, ruthenium compounds) prevents SOCE and tumour cell proliferation.
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A, effects of 6 μM Ru360 on SOCE in HT29 cells loaded with fura-4 F. Each trace is the mean ±S.E.M. of 54 cells (data representative of 3 experiments). B, transcription cycling of topoisomerase-II in 3T3 cells treated with vehicle (control) or 6 μM Ru360. Data representative of 15 (control) and 3 (Ru360) independent experiments. C and D, effects of ruthenium red (RR, 100 μM, 96 h) on Jurkat (C) and HT29 (D) cell growth (n= 3). P < 0.05 versus control (Student's t test). Similar results were obtained with Ru360 (data not shown).
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We asked finally whether or not the mitochondria-dependent inhibition of SOCE is sufficient to explain the inhibition of tumour cell proliferation. To answer this question, we tested the effects of blocking directly ICRAC and SOCE on cell proliferation, cell-cycle driven gene transcription and Ca2+ entry. We used BTP-2 ([N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide]) which has been reported recently to inhibit ICRAC and cell proliferation in Jurkat cells at the same concentrations (Zitt et al. 2004). Figure 10 shows that BTP-2 blocked both SOCE (Fig. 10A and B) and proliferation in HT29 cells (Fig. 10C). The correlation between both effects was remarkably good (r= 0.999, P < 0.0001; Fig. 9D). In addition, BTP-2 also blocked transcription cycling of topoisomerase-II (data not shown). These results reinforce the idea that SOCE inhibition is sufficient to prevent proliferation. Furthermore, we found that salicylate (500 μM) did not have an additive effect on inhibition of proliferation induced by BTP-2 (Fig. 10E). Finally, the inhibitory effects of salicylate on proliferation were essentially reverted simply by increasing extracellular Ca2+ concentration (Fig. 10E), a condition that increases SOCE (not shown).
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A, effects of different BTP-2 concentrations (in μM) on SOCE in HT29 cells loaded with fura-4 F. Each trace is the average (mean ±S.E.M.) of 29–83 cells. B, dose dependency of the effects of BTP-2 on SOCE (%). Each value, expressed as percentage of control, is the mean ±S.E.M. of 3 experiments. Each bar was significantly different from the others (P < 0.05). C, dose dependence of the effects of BTP-2 on HT29 cell growth. Each bar represents the mean ±S.E.M. of 3 experiments. Each bar was significantly different from the others (P < 0.05). D, correlation between SOCE inhibition (%) and growth inhibition (%) induced by BTP-2 in HT29 cells. The line is the best linear fit of experimental data shown in B and C (r= 0.999; P < 0.0001). E, effects of BTP-2 (BTP2, 10 μM), salicylate (500 μM, Sal) and both (BTP2 + Sal) on cell growth in HT29 cells. Salicylate did not increase inhibition of cell growth induced by BTP-2. Effects of increasing extracellular Ca2+ concentration from 1.8 mM (control) to 3.8 mM (+Ca2+). High Ca2+ essentially restored the inhibitory effects of salicylate. Each bar represents the mean ±S.E.M. of 3 experiments. Each bar was significantly different (P < 0.05) from the others except for BTP-2 alone versus BTP-2 plus salicylate.
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Discussion
We show here that colon carcinoma cells exhibit a robust SOCE that is regulated by mitochondrial Ca2+ uptake in a manner similar to that described in other non-excitable cells including Jurkat cells. We found that collapse of mitochondrial with either FCCP, valinomycin or antimycin A plus oligomycin prevents SOCE. Similar results have been reported in other cells including Jurkat (Hoth et al. 1997, 2000), RBL (Gilabert & Parekh, 2000; Gilabert et al. 2001; Glitsch et al. 2002), HEK293 (Thyagarajan et al. 2002) and EA.hy926 endothelial cells (Malli et al. 2003). In all these cases, the effects of FCCP have been attributed to inhibition of mitochondrial Ca2+ uptake leading to Ca2+-dependent inactivation of SOCs and not to either ATP depletion or plasma membrane depolarization. Consistently, we found that the effects of FCCP on SOCE were not affected by oligomycin, used here to prevent ATP hydrolysis in the uncoupled mitochondria by reverse running of the F0F1–ATP synthase. On the other hand, valinomycin, a K+ ionophore that collapses mitochondrial potential but hyperpolarizes plasma membrane, also prevented SOCE. These results suggest that the effects of FCCP are due to the collapse of mitochondrial rather than to depolarization of the plasma membrane. Hoth et al. (1997) showed that readmisssion of Ca2+ to thapsigargin-treated cells resulted in a robust initial Ca2+ influx that was unafffected by FCCP. However, FCCP did affect the subsequent Ca2+ plateau. In our hands FCCP, antimycin A or valinomcyin all reduced both the initial rate and the size of the Ca2+ influx. Although at variance with Hoth et al. our results agree with most further works that showed clearly that CCCP or FCCP indeed reduced the initial rate and size of Ca2+ influx in Jurkat (Zablocki et al. 2003, 2005; Thyagarajan et al. 2002) and RBL-1 cells (Glitsch et al. 2002).
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According to our results, SOCE generates high [Ca2+]cyt domains that are sensed and cleared by nearby mitochondria, thus allowing sustained Ca2+ entry in colon cancer cells. Massive Ca2+ uptake by a fraction of mitochondria takes place through the Ca2+ uniporter, which is activated by high [Ca2+]cyt domains generated by nearby SOCs. This situation is similar to the one reported previously in adrenal chromaffin (Montero et al. 2000; Villalobos et al. 2002) and anterior pituitary cells (Villalobos et al. 2001). In fact, we showed previously that most of the Ca2+ entering excitable cells upon activation of voltage-gated Ca2+ channels was cleared by nearby mitochondria rather than by other extrusion systems (Villalobos et al. 2002). Our present results suggest that Ca2+ entry in non-excitable cells through SOCs is similarly cleared by nearby mitochondria. As a consequence, inhibition of mitochondrial Ca2+ uptake by different means (FCCP, ruthenium compounds or antimycin A plus oligomycin) impairs Ca2+ clearance and leads to SOCE inhibition, probably by inactivation of SOC by high [Ca2+] domains.
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If sustained SOCE requires mitochondrial Ca2+ uptake, then it follows that any compound that prevents mitochondrial Ca2+ uptake should inhibit SOCE. ASA and specially its major metabolite salicylate are mitochondrial uncouplers (Pachman et al. 1971). Salicylate enters mitochondria as salicylic acid (driven by the concentration gradient) and exits as salicylate anion (driven by voltage and concentration gradients) thus producing net proton entry (Gutknecht, 1990). Salicylate is much more potent than ASA because it contains an internal hydrogen bond that delocalizes the negative charge, thus increasing the lipid solubility and permeability of the anion. Model calculations predicted that, at therapeutic concentrations, salicylate may cause a net H+ influx into mitochondria enough to explain the reported ‘loose coupling’ effect (Gutknecht, 1990). Consistently, we find that salicylate induces a partial mitochondrial depolarization in intact cells. As stated above, the Nernst equilibrium predicts that even a small drop in should greatly affect equilibrium [Ca2+]mit and the driving force for mitochondrial Ca2+ uptake. This prediction was confirmed here as salicylate concentrations as low as 10 μM induced a modest mitochondrial depolarization (< 15%) that contrasted with a much larger inhibition of mitochondrial Ca2+ uptake (> 70%; Fig. 3). This disparity between the effects on and on mitochondrial Ca2+ uptake had not been experimentally shown before because lack of reliable measurements of Ca2+ uptake in mitochondria of living cells. Most of the previous measurements underestimated the actual [Ca2+]mit increases reached upon cell stimulation. The recent introduction of mitochondria-targeted, low affinity mutated aequorin (Montero et al. 2000) has enabled [Ca2+]mit measurements in the high micromolar to millimolar range. Using this novel methodology, we have shown here that therapeutic concentrations of salicylate produce a large inhibition of mitochondrial Ca2+ uptake. Since prevention of mitochondrial Ca2+ uptake leads to SOCE inhibition (Hoth et al. 1997, 2000; Glitsch et al. 2002), it follows that therapeutic concentrations of salicylate should also prevent SOC operation. Again, this expectation was confirmed in both Jurkat and colon carcinoma cells. Moreover, we also show that salicylate prevented SOCE elicited by physiological stimulation in both HT29 and Jurkat cells. In addition, the effects of salicylate on SOCE, like those of FCCP, were not affected by oligomycin, suggesting that the salicylate-induced inhibition of SOCE is not due to ATP depletion. In support of this view, we show here that salicylate concentrations that inhibited SOCE fully had no effect on ATP levels in HT29 cells. It must be remembered that tumour cells use glycolysis more than mitochondrial respiration, which is blocked by glucose (Crabtree, 1929), for ATP synthesis. Taken together, our results indicate that salicylate inhibits SOCE by preventing mitochondrial Ca2+ uptake, and that this leads to Ca2+-dependent inactivation of SOC.
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If SOCE is essential for proliferation in Jurkat and other tumour cells then salicylate should inhibit tumour cell proliferation at the same low concentrations that prevent SOCE. Our results fulfil this prediction as salicylate inhibits both SOCE and cell growth with the same concentration dependence. The lack of effects of salicylate on cell viability and apoptosis together with the effects of salicylate on cell division and transcription cycling of topoisomerase-II, a cell-cycle-related gene and marker of cell proliferation, supports the view that salicylate inhibits cell proliferation at low, therapeutic concentrations, and this effect is not due to changes in ATP concentration. Furthermore, our data support the view that SOCE inhibition is sufficient to prevent tumour cell proliferation. The pyrazole derivative BTP-2, a direct ICRAC blocker (Zitt et al. 2004), inhibits SOCE and proliferation in Jurkat cells with the same concentration dependence. We show here similar results for colon cancer cells. It should be mentioned that BTP-2 has been reported not to affect mitochondria or other ion channels (Zitt et al. 2004). Moreover, salicylate effects on cell proliferation are not additive with the effects induced by BTP-2. In addition, the effects of salicylate on cell proliferation are restored by increasing the extracellular Ca2+ concentration suggesting they are not due to a possible metabolic effect but to a defect in SOCE. Finally, diazoxide and ruthenium compounds, which inhibit SOCE by interfering in other ways with mitochondrial Ca2+ uptake, also prevent tumour cell proliferation. Taken together, our data support the view that salicylate, at therapeutic concentrations, inhibits SOCE in a mitochondria-dependent manner and that this effect is sufficient to prevent cell proliferation.
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What is the mechanism by which SOCE inhibition impairs cell proliferation Stimulation of Jurkat cells induces sustained SOCE and activation of the Ca2+-dependent phosphatase calcineurin that dephosphorylates NFAT, promoting expression of interleukin-2 and proliferation (Lewis, 2001). Interestingly, inhibition of SOCE by FCCP abolishes NFAT activation (Hoth et al. 2000). Moreover, direct blockade of SOCE by BTP-2 also prevents NFAT activation and proliferation (Zitt et al. 2004). Thus, the effects of salicylate on SOCE may interfere with the NFAT signalling pathway to proliferation. Consistent with this view, salicylate has been recently reported to inhibit NFAT activation and NFAT-dependent transcription in Jurkat cells (Aceves et al. 2004). The role of SOCE in control of proliferation in colon cancer cells is scarcely known. The inducible COX-2 isozyme is up-regulated in multiple tumours including colon cancer (Molina et al. 1999) and it has been proposed that it promotes tumour cell growth. Interestingly, transcription of COX-2 is regulated by NFAT both in Jurkat (Iiguez et al. 2000) and colon cancer cells (Duque et al. 2005). Salicylate inhibits COX-2 gene transcription at therapeutic concentrations similar to those used here (Xu et al. 1999). Thus, control of proliferation by SOCE in colon cancer cells could be transduced by NFAT-mediated regulation of COX-2 gene expression. Notwithstanding, the above possibility cannot account for the anti-proliferative effects of salicylate in colon cancer cells lacking COX-2 gene expression (Hanif et al. 1996; Smith et al. 2000) and we find that salicylate prevents SOCE and tumour cell growth also in the colon cancer cells lacking COX-2 gene expression. Therefore, SOCE must regulate proliferation by a different mechanism, perhaps involving other NFAT-induced genes. Interestingly, NFAT activation induced by sustained SOCE is considered a general proliferative signal (Lipskaia & Lompre, 2004). On the other hand, since SOCE is an early event in signal transduction, it is likely that multiple downstream transducing proteins could be recruited in the proliferative signalling pathway. One important example is NFB, another transcription factor involved in control of cell proliferation, which is inhibited by salicylate and modulated by multiple signalling pathways including Ca2+ signals. However, salicylate inhibits NFB activity and NFB-mediated gene expression at concentrations (IC50 5–9 mM) far larger than those reported here to inhibit SOCE and cell proliferation. Although caution is necessary in the interpretation of the results, the multiple targets proposed for aspirin (Hardwick et al. 2004) and salicylate can be consistent with our findings.
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Our results may have important implications as salicylate, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit tumour cell growth (Hanif et al. 1996; Molina et al. 1999; Smith et al. 2000) and to protect against colon (Chan, 2003) and other cancers (Bosetti et al. 2001). Clinical trials show that ASA reduces the risk of colorectal adenomas (Baron et al. 2003; Sandler et al. 2003) but the action mechanism has remained elusive. ASA irreversibly inhibits COX activity by acetylation of constitutive COX-1 at Ser530 and inducible COX-2 at Ser516. In vivo, ASA is quickly (t1/2= 15 min) deacetylated to salicylic acid, which remains in the plasma for much longer. Salicylic acid does not directly inhibit COX activity because it lacks the acetyl group, although, as stated above, it antagonizes COX-2 gene expression at the transcription level (Xu et al. 1999). Thus, both inhibition of COX-2 activity and COX-2 gene expression have been proposed to mediate the anti-tumoral effects of ASA. Recent results, however, do not support this view. It has been shown, for example, that ASA and COX-2 inhibitors block proliferation through a prostaglandin-independent pathway in both COX-2-expressing (HT29 and HCA-7) and non-expressing (SW480 and HTC116) colon cancer cells (Hanif et al. 1996; Smith et al. 2000). In the same line, it has been reported that the anti-proliferative effects of NSAIDs are independent of the level of COX-2 expression (Molina et al. 1999) or prostaglandin E2 production (Perugini et al. 2000), but related to cell cycle quiescence. Microarray analysis has shown that ASA represses many cell-cycle-related genes and modulates multiple signalling pathways (Hardwick et al. 2004), suggesting that an early mitotic signal may be the key target for the anti-proliferative effect. This view is consistent with the results we report here where the aspirin metabolite salicylate inhibits tumour cell proliferation by promoting inactivation of SOCE, an essential early requirement for proliferation. Thus, our results could contribute to explaining the mechanism for the anti-tumoral actions of aspirin and dietary salicylates (Paterson & Lawrence, 2001). Interestingly, ruthenium compounds have also been reported to have anti-tumoral and immunosuppresive properties both in vivo and in vitro, but the action of this mechanism has also remained elusive (Sava & Bergamo, 2000). Our results support the view that the effects of ruthenium compounds on mitochondrial Ca2+ uptake might contribute towards explaining also their anti-tumoral and immunosuppresive properties.
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Salicylate has been also reported to inhibit proliferation of normal vascular smooth muscle cells in a prostaglandin-independent manner (Marra & Liao, 2001). In fact, salicylate has been proposed as a candidate for treatment of proliferative disorders of the vessel wall that lead to intimal hyperplasia and hypertension. The specific mechanism for this action is not clear and multiple downstream targets have been proposed (Marra & Liao, 2001). Interestingly, vascular smooth muscle cells show up-regulated TRP and enhanced SOCE during proliferation (Golovina et al. 2001) suggesting that SOCE is very relevant for proliferation of these cells. It is tempting to speculate that salicylate might inhibit normal vascular smooth muscle cell proliferation by the same mechanism reported here. Nevertheless, the effects of salicylate are achieved at larger concentrations suggesting the possibility that salicylate may inhibit transformed cells more efficiently than normal cells. Further research is required to ascertain both the mechanism and possible differential sensitivity to salicylate in normal cells relative to their transformed counterparts.
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In summary, our results suggest a novel and important role of mitochondria in control of cell proliferation. Specifically, we show here that tumour cell proliferation critically depends on mitochondrial Ca2+ uptake. The interference of salicylate with this mechanism may ultimately provide the basis for the reported anti-proliferative and anti-tumoral effects of aspirin and dietary salicylates. In addition, as Ca2+ is a pleiotropic signal, our results point to a novel mechanism that may help to disentangle yet other elusive effects of aspirin such as its anti-inflammatory and immunosuppresive actions.
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Footnotes
L. Núez and R.A. Valero contributed equally to this work.
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Abstract
Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway involved in control of multiple cellular and physiological processes including cell proliferation. Recent evidence has shown that SOCE depends critically on mitochondrial sinking of entering Ca2+ to avoid Ca2+-dependent inactivation. Thus, a role of mitochondria in control of cell proliferation could be anticipated. We show here that activation of SOCE induces cytosolic high [Ca2+] domains that are large enough to be sensed and avidly taken up by a pool of nearby mitochondria. Prevention of mitochondrial clearance of the entering Ca2+ inhibited both SOCE and cell proliferation in several cell types including Jurkat and human colon cancer cells. In addition, we find that therapeutic concentrations of salicylate, the major metabolite of aspirin, depolarize partially mitochondria and inhibit mitochondrial Ca2+ uptake, as revealed by mitochondrial Ca2+ measurements with targeted aequorins. This salicylate-induced inhibition of mitochondrial Ca2+ sinking prevented SOCE and impaired cell growth of Jurkat and human colon cancer cells. Finally, direct blockade of SOCE by the pyrazole derivative BTP-2 was sufficient to arrest cell growth. Taken together, our results reveal that cell proliferation depends critically on mitochondrial Ca2+ uptake and suggest that inhibition of tumour cell proliferation by salicylate may be due to interference with mitochondrial Ca2+ uptake, which is essential for sustaining SOCE. This novel mechanism may contribute to explaining the reported anti-proliferative and anti-tumoral actions of aspirin and dietary salicylates.
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Introduction
Much evidence indicates that Ca2+ signals participate in the control of cell proliferation (Berridge, 1995; Kahl & Means, 2003). They activate, for example, immediate early genes responsible for inducing resting cells to enter the cell cycle, promote DNA synthesis and stimulate different events at mitosis (Berridge, 1995). Particular attention has been paid to the role of Ca2+ entry through store-operated channels (SOCs, Parekh & Putney, 2005). Store-operated Ca2+ entry (SOCE), also termed capacitative Ca2+ entry (Putney, 1986), is the most important Ca2+ influx pathway in non-excitable cells (Parekh & Penner, 1997). SOCE is activated by emptying of intracellular Ca2+ stores, either by inositol 1,4,5 trisphosphate produced upon activation of G-protein-coupled or tyrosine kinase receptors or by pharmacological emptying of the Ca2+ stores with, for instance, thapsigargin (Montero et al. 1990, 1991; Alvarez et al. 1994; Parekh & Putney, 2005). The involvement of SOCE in cell proliferation has been studied in detail in Jurkat T cells. In these cells, T cell receptor stimulation induces sustained SOCE through the Ca2+ release-activated current (ICRAC) resulting in sustained activation of the nuclear factor of activated T cells (NFAT) that promotes proliferation (Lewis, 2001). Many observations in this and other cell models relate SOCE and cell proliferation: (i) entry in cell cycle is preceded by SOC activation (Sugioka & Yamashita, 2003); (ii) growth factor stimulation of cell proliferation is accompanied by increased activity and/or expression of transient receptor potential (TRP) channels that are related to SOCE (Golovina et al. 2001); (iii) SOCE inhibition by different means (Ca2+ removal, inorganic channel blockers and ICRAC antagonists) abolishes tumour cell proliferation (Weiss et al. 2001; Zitt et al. 2004).
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Two decades after the discovery of SOCE (Putney, 1986), the link between emptying of intracellular Ca2+ stores and the increased Ca2+ influx is still unknown (Parekh & Putney, 2005). Likewise, the nature of the SOCs responsible for SOCE remains controversial. A number of members of the TRP family of cation channels, including several canonical TRPs (TRPCs) and TRPV6, have been proposed to be involved in SOCE (Parekh & Putney, 2005). An important characteristic of SOCE, ICRAC and the TRP channels related to SOCE is the strong Ca2+-dependent inactivation that limits Ca2+ entry (Singh et al. 2002; Parekh & Putney, 2005). In fact, ICRAC is hardly recorded unless the Ca2+-dependent inactivation is prevented by a strong intracellular Ca2+ buffer (Gilabert & Parekh, 2000). Thus, intracellular Ca2+ extrusion systems are important for sustaining SOCE. Mitochondria play an important role as a Ca2+ sinking system able to shape the cytosolic Ca2+ signals (Rizzuto et al. 2000; Montero et al. 2000; Villalobos et al. 2002). With regard to SOCE, a series of reports from the laboratories of Lewis (Hoth et al. 1997, 2000) and Parekh (Gilabert & Parekh, 2000; Gilabert et al. 2001, 2002) have shown that sustained SOCE requires normal mitochondrial Ca2+ uptake in Jurkat and rat basophilic leukaemia (RBL-1) cells. Protonophores such as CCCP or FCCP prevent SOCE since they collapse the mitochondrial potential (), the driving force for Ca2+ entry into mitochondria. The inhibition of SOCE has been attributed to Ca2+-dependent inactivation of ICRAC due to generation of high [Ca2+]cyt domains secondary to a lack of mitochondrial Ca2+ uptake (Hoth et al. 1997, 2000; Glitsch et al. 2002; Malli et al. 2003; Parekh, 2003 but see also Varadi et al. 2004; Frieden et al. 2004). We have reported recently that activation of voltage-gated Ca2+ channels induces in excitable cells large [Ca2+]cyt increases that are sensed by subplasmalemmal mitochondria (Montero et al. 2000; Villalobos et al. 2001). This pool amounting to about 50% of the mitochondria, takes up tremendous amounts of Ca2+ (driving [Ca2+]mit to near millimolar levels) and clears efficiently the high [Ca2+]cyt domains (Villalobos et al. 2002). Whether SOCE induces such high [Ca2+]cyt domains leading to large [Ca2+]mit increases in a mitochondrial pool has not been studied.
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If SOCE is essential for cell proliferation and depends on mitochondrial Ca2+ uptake, then it follows that drugs interfering with mitochondrial Ca2+ uptake may prevent cell proliferation via SOC inactivation. Here we tested whether this rationale can explain the effects of salicylate, the major metabolite of aspirin (acetylsalicylic acid, ASA). Salicylate acts as a proton carrier and uncouples mitochondria (Pachman et al. 1971; Gutknecht, 1990). According to the above rationale, salicylate should interfere with mitochondrial Ca2+ uptake, SOCE and cell proliferation. In fact, aspirin and salicylate have been reported to inhibit tumour cell growth and to prevent colon and other cancers (Hanif et al. 1996; Molina et al. 1999; Perugini et al. 2000; Smith et al. 2000; Baron et al. 2003; Sandler et al. 2003), although the action mechanism is unknown. Salicylate has been recently reported to also inhibit proliferation of normal vascular smooth muscle cells (Marra & Liao, 2001). Here we have investigated (i) whether SOCE is regulated by mitochondria in colon cancer cells, (ii) whether proliferation of tumour cells depends on mitochondrial Ca2+ uptake and (iii) whether salicylate inhibits tumour cell proliferation by acting on mitochondrial control of SOCE. For these studies we have used HT29 human colon cancer cells and Jurkat T cells where mitochondrial control of SOCE and the role of SOCE in proliferation were first described (Hoth et al. 1997; Lewis, 2001).
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Methods
Materials
Fura-2 AM, Fura-4 AM, JC-1 (a cationic dye that indicates mitochondrial polarization) and tetramethylrhodamine ethyl ester perchlorate (TMRE) were purchased from Molecular Probes. Ru360 (a calcium signalling tool) and BTP2 (an immunomodulator) were purchased from Calbiochem. Thapsigargin was obtained from Alomon Laboratories. Other reagents and chemicals were obtained either from Sigma or Merck. Mutated aequorin was kindly donated by M. Montero (Valladolid University, Valladolid). Jurkat cells were obtained from the American Type Culture Collection. HT29 cells were donated by J. C. Fernández-Checa (CSIC, Barcelona). SW480 cells were donated by A. Muoz (CSIC, Madrid). NIH 3T3 cells expressing luciferase under control of the topoisomerase-II promoter were a kind gift of M. Sehested (Rigshospitalet, Copenhagen).
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Store-operated Ca2+ entry
Cells were plated at about 0.5 x 106 cells ml–1 on 12 mm glass coverslips treated with poly L-lysine (HT29) or fibronectin (Jurkat) and loaded with 4 μM fura-2 /AM or fura-4 AM for 60 min at room temperature. Cells were then incubated with 1 μM thapsigargin for 10 min in Ca2+-free standard medium containing (mM): NaCl, 145; KCl, 5; MgCl2, 1; EGTA, 0.5; glucose, 10; Hepes/NaOH, 10 (pH 7.42) and placed on the stage of an inverted microscope (Nikon Diaphot) maintained at 37°C. They were then perfused with prewarmed Ca2+-free medium, and epi-illuminated alternately at 340 and 380 nm. Light emitted above 520 nm was recorded with a Magical Image Processor (Applied Imaging). Pixel-by-pixel ratios of consecutive frames were captured and [Ca2+]cyt was estimated from these ratios as previously reported (Villalobos et al. 2002). Re-addition of standard, Ca2+-containing (CaCl2, 1 mM; no EGTA) medium evoked large [Ca2+]cyt increases revealing store-operated Ca2+ entry (Montero et al. 1990; Villalobos & García-Sancho, 1995; Hoth et al. 2000; Glitsch et al. 2002). In some experiments, SOCE induced by physiological stimulation with carbachol (100 μM) in HT29 cells and TCR stimulation in Jurkat cells was tested. For TCR stimulation, Jurkat cells were suspended in external medium and loaded with Fura-4 AM (4 μM). Then, cells were plated on fibronectin-coated glass coverslips previously incubated with IgG2a for 4 h at room temperature. Cells were then placed in the fluorescence microscope and stimulated with 0.5 μg ml–1 anti-CD3 (the T cell receptor) and 5 μg ml–1 anti-CD28 (a co-receptor) to elicit the cross-linking activation of TCR.
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Mitochondrial potential
HT29 cells were loaded with JC-1 (1 μg ml–1) for 10 min and subjected to confocal microscopy with a Bio-Rad laser scanning system (Radiance 2100) coupled to a Nikon eclipse TE2100U inverted microscope. For TMRE measurements, HT29 cells were loaded with TMRE (100 nM) for 30 min at room temperature, placed on the perfusion chamber of a Zeiss Axiovert 100 TV inverted microscope and superfused continuously with prewarmed (37°C) standard medium. Fluorescence images were taken at 5 s intervals with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor. Traces from individual cells were expressed as the percentage value of fluorescence before addition of salicylate and averaged. In some experiments, cells were loaded with TMRE (100 nM) and fluorescence from mitochondria and nearby cytosol was analysed with the confocal system. Localization of TMRE fluorescence in mitochondria was tested by co-loading cells with Mitotracker Red. To quantify mitochondrial depolarization, the effect of salicylate and FCCP on the ratio of TMRE fluorescence in mitochondria relative to that surrounding cytosol was calculated as reported by Collins et al. (2002).
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Mitochondrial calcium uptake
HT29 cells were transfected (lipofectamin) with a plasmid containing either the wild type or the mutated (Asp119Ala), low-Ca2+ affinity aequorin targeted to mitochondria (Montero et al. 2000). After 24 h, cells were incubated in standard medium (see above) containing 1 μM of either wild type or coelenterazine (n, acquorin cofactors) for 2 h at room temperature. The coverslips were then placed in a luminometer (Cairn Research), perfused continuously with warm (37°C) standard medium and subjected to photon counting at 1 s intervals. For some experiments, cells were permeabilized with 20 μM digitonin in ‘intracellular’ medium (130 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM K3PO4, 0.2 mM EGTA, 1 mM ATP, 20 μM ADP, 2 mM succynate, 20 mM Hepes/KOH, pH 6.8). The cells were then incubated with the same medium containing 200 nM Ca2+ (buffered with EGTA) with or without salicylate for 5 min. Finally, perfusion was switched to ‘intracellular’ medium containing 6 μM Ca2+ (with or without salicylate) for 1 min. Photonic emissions were converted to mitochondrial-free Ca2+ concentration ([Ca2+]mit) values as reported previously (Montero et al. 2000; Villalobos et al. 2001; Alvarez & Montero, 2001).
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Cell growth, apoptosis and cell viability
Cells (HT29, SW480, Jurkat and NIH 3T3) were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI 1640 media (Gibco) containing 10% fetal bovine serum and antibiotics. Cells were plated in wells at about 5 x 104 cells ml–1 and incubated with test solutions for 96 h. Triplicate wells were counted daily or after 96 h. Cell death was estimated in the same samples by trypan blue exclusion. Data correspond to at least three independent experiments and are expressed as cell number-fold increase, percentage growth rate relative to control or cells per hour. Differences were considered significant at P < 0.05 (Student's t test). For determination of apoptotic cells at single cell level, cells were plated at about 5 x 104 cells ml–1 and incubated with test solutions for 96 h. Apoptotic cells were revealed by the terminal deoxynucleaotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method by means of fluorescence microscopy and the in situ cell death detection kit (Roche Diagnostics, Penzberg, Germany) following the protocol provided by the manufacturer.
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ATP measurements
HT29 cells were plated in 6-well plates and cultured in medium containing either vehicle or different salicylate concentrations (10–2000 μM). After 24 h, cells were washed twice with phosphate-buffered saline (PBS) at 37°C and 1 ml of boiling 20 mM Tris, pH 7.75, 4 mM EDTA solution was added. After 2 min, samples were centrifuged for 4 min at 10 000 g. ATP was measured later from the supernatant by the luciferin–luciferase assay (Sánchez, 1985). Readings were taken using a scintillation counter (Wallac model 1409/11) over 20 s intervals with the windows wide open. ATP levels were determined with the aid of a standard curve prepared using pure ATP over a 10–5–10–9M concentration range.
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Bioluminescence imaging of transcription dynamics and cell division
NIH 3T3 cells stably transfected with the luciferase reporter gene under control of the topoisomerase-II gene promoter (Falck et al. 1999) were synchronized in medium containing 0.5% serum for 3 days. Then, 10% serum and 1 mM luciferin were added and the Petri dish containing the cells was placed in an incubator (Zeiss CTI-controller 3700) controlling temperature, CO2 and humidity, attached over the stage of an inverted microscope (Zeiss Axiovert 100 TV). Photonic emissions and bright field images were captured concurrently with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor at 15 min intervals for 72 h. Transcription activity is expressed as total photonic emissions in each image minus background photonic emissions, as previously reported (Villalobos et al. 1999).
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Results
SOCE was shown by the increase in cytosolic Ca2+ concentration ([Ca2+]cyt) that follows addition of extracellular Ca2+ to fura-2-loaded Jurkat cells pre-treated with thapsigargin in Ca2+-free medium. We found that FCCP reversibly reduced the initial rate and the size of SOCE in Jurkat cells (Fig. 1A). SOCE was also shown in HT29 human colon carcinoma cells by the same [Ca2+]cyt overshoot protocol (Fig. 1B). No Ca2+ overshoot was observed in cells not pre-treated with thapsigargin (data not shown). A second pulse of extracellular Ca2+ evoked a similar [Ca2+]cyt increase and FCCP reversibly prevented SOCE (Fig. 1B). We noticed that the fura-2 signals were often saturated during SOCE in many individual cells. This would result in the underestimation of both [Ca2+]cyt peaks and inhibition by FCCP. To avoid this problem we repeated the experiments using the lower affinity Ca2+ probe fura-4 F instead of fura-2 (Villalobos et al. 2002). [Ca2+]cyt increases measured by fura-4 F ranged between 1.3 and 5.8 μM (3.2 ± 1.8 μM, mean ±S.E.M., n= 9) in HT29 cells and were also inhibited by FCCP (Fig. 1C). Similar results were obtained in Jurkat cells (data not shown). The SOCE-induced increase in [Ca2+]cyt as well as the FCCP-induced inhibition were not affected by the presence of F0F1–ATP synthase blocker oligomycin (Fig. 1D) indicating that the effects of FCCP are not due to ATP depletion. Oligomycin had no effect on subsequent control pulses either (not shown). In order to interpret the lack of effect of oligomycin, it must be remembered that mitochondrial respiration is very much inhibited in tumour cells simply by incubation in glucose-containing media (Crabtree, 1929). The inhibition is due to blocking of the F0F1–ATPase, but the mitochondrial potential is not lost or is even increased (Wojtczak et al. 1999). Treatment with the respiratory chain inhibitor antimycin A plus oligomycin, a cocktail that collapses and prevents SOCE and ICRAC in Jurkat and RBL-1 cells (Glitsch et al. 2002), also inhibited SOCE in HT29 cells (Fig. 1E). Valinomycin, a K+ ionophore that collapses the mitochondrial but hyperpolarizes the plasma membrane, also inhibited SOCE in HT29 cells (Fig. 1F) suggesting that the effects of FCCP are due to collapse of the mitochondrial rather than to plasma membrane depolarization. In medium containing high K+ (50 mM), a condition that should hold the membrane potential in a quite depolarized state, FCCP also inhibited SOCE (data not shown). Carbachol activates M3 muscarinic receptors in HT29 cells provoking a biphasic [Ca2+]cyt increase, a peak due to the transient Ca2+ release from intracellular stores followed by a plateau due to sustained Ca2+ entry via SOC (Kerst et al. 1995). FCCP did not affect the Ca2+ release induced by carbachol indicating that FCCP does not affect the ATP-dependent refilling of intracellular Ca2+ stores. Similar results have been reported previously in Jurkat cells (Makowska et al. 2000). However, FCCP inhibited the carbachol-induced Ca2+ entry (Fig. 1G and H). FCCP also inhibited SOCE in SW480 cells, a human colon cancer cell lacking cyclooxygenase-2 (COX-2) gene expression (Smith et al. 2000) (data not shown). Thus, human colon carcinoma cells display a robust SOCE that is prevented by mitochondrial depolarization and this effect is independent of ATP, plasma membrane depolarization and COX-2 gene expression.
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SOCE was started by addition of Ca2+ to thapsigargin-treated (1 μM, 10 min), fura-2-loaded cells incubated in Ca2+-free medium. A and B, effects of FCCP (10 μM) in Jurkat (A) and HT29 cells (B). C and D, effects of FCCP (10 μM) and oligomycin (0.12 μM) plus FCCP (10 μM) on Ca2+ entry in fura-4 F-loaded HT29 cells. E and F, effects of oligomycin (0.12 μM) plus antimycin A (0.5 μg ml–1) and valinomycin (10 μM) on SOCE in HT29 cells. G and H, carbachol (100 μM) elicited a biphasic [Ca2+]cyt increase in HT29 cells loaded with fura-4 F. In G, cells were perfused in Ca2+-containing medium with or without (control; ) FCCP (10 μM). FCCP inhibited the sustained but not the transient [Ca2+]cyt increase. In H, cells were perfused with Ca2+-free medium, except for the period indicated (Ca) in which perfusion was shifted to Ca2+-containing medium. All traces of [Ca2+]cyt are averaged values (mean ±S.E.M.) of 28–56 cells and representative of 3–9 similar experiments.
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We next studied whether SOCE increases [Ca2+]mit in colon cancer cells. We transfected HT29 cells with mitochondria-targeted aequorin and measured mitochondrial Ca2+ uptake by photon counting (Montero et al. 2000) during SOCE induced by the same Ca2+ re-addition protocol used above. The addition of extracellular Ca2+ to thapsigargin-treated cells produced massive photoluminescence emission with consumption of about 50% of aequorin (45 ± 5%, n= 3). The estimated [Ca2+]mit reaches about 2 μM. A second and subsequent stimuli produced a very small photonic emission (3 ± 1% of the total emissions) corresponding to a negligible [Ca2+]mit increase (Fig. 2A). However, the second stimulus produced a [Ca2+]cyt increase of the same extent as the first one (Fig. 1B). These results are similar to those obtained by sequential stimulation with high K+ pulses in chromaffin (Montero et al. 2000) and GH3 (Villalobos et al. 2001) cells. These results were interpreted in terms of two pools of mitochondria. The first pool, amounting to about 50% of mitochondria and located strategically near the sites of Ca2+ entry, is able to sense the high [Ca2+]cyt domains required for Ca2+ uptake through the mitochondrial Ca2+ uniporter (Montero et al. 2000). Massive Ca2+ uptake by this pool results in huge [Ca2+]mit increases near the millimolar level that dampens progression of the Ca2+ wave towards the cell core. This huge [Ca2+]mit increase by the first mitochondrial pool burns out all the aequorin present in this pool during the first stimulus. As a result, the remaining mitochondria, probably located far away from the channels, sense much smaller [Ca2+]cyt domains and take up negligible Ca2+ amounts so that the second pool does not produce photoluminescence emission. This interpretation was tested in HT29 cells by transfecting them with a mutated, mitochondria-targeted aequorin with much lower affinity for Ca2+ (Montero et al. 2000). The measurements revealed that SOCE actually induced a huge [Ca2+]mit increase of several hundred micromolar (425 ± 85 μM, n= 5, Fig. 2B). These values approach the ones measured in mitochondria from excitable cells upon activation of voltage-gated Ca2+ channels (Montero et al. 2000; Villalobos et al. 2001). Taken together, these results support the view that SOCE induces high [Ca2+]cyt domains that are sensed and cleared by a mitochondrial pool probably located nearby. Collapse of abolish mitochondrial Ca2+ uptake and impairs this clearance leading to local increase of [Ca2+]cyt and SOCE inhibition.
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A, HT29 cells were transfected with mitochondria targeted aequorin and subjected to photon counting measurements for estimation of [Ca2+]mit (see Methods). Cells were first treated with 1 μM thapsigargin for 10 min in Ca2+-free medium and Ca2+ entry was started by addition of external Ca2+ (1 mM; Ca). The top panel shows aequorin consumption and the bottom one the [Ca2+]mit estimate (see Methods for details). Data are representative of 3 similar experiments. B, HT29 cells were transfected with the low Ca2+ affinity mutated mitochondria-targeted aequorin, reconstituted with coelenterazine n and subjected to photon counting measurements. Other details as in A. For calibration of [Ca2+]mit, the size of the mitochondrial pool was assumed to contain 45% of the aequorin. Data are representative of 5 similar experiments.
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A chemist's look at salicylic acid shows it is a weak acid bound to an aromatic, hydrophobic ring, a lipophilic acid structure resembling that of mitochondrial uncouplers. We have studied the effects of salicylate on and mitochondrial Ca2+ uptake. We have used two different mitochondrial potential-sensitive probes, JC-1 and TMRE, to study the effects of salicylate on in intact HT29 and Jurkat cells. Confocal microscopy of JC1-loaded HT29 cells revealed that salicylate de-energised mitochondria, as shown by the shift of the emitted fluorescence from red to green in a manner comparable to FCCP (Fig. 3A and B). A more quantitative estimate was attempted using TMRE as probe. Results are shown in Fig. 3C and D. Salicylate induces a concentration-dependent loss of at therapeutic concentrations, as indicated by the decrease of TMRE fluorescence (see also Supplemental movie 1). Figure 3E shows confocal images of TMRE-loaded HT29 cells. To quantify further mitochondrial depolarization we calculated the ratio of TMRE fluorescence from mitochondria relative to the surrounding cytosol as reported by Collins et al. (2002). This ratio was largely decreased by salicylate (500 μM) and FCCP (10 μM) in both HT29 (Fig. 3F) and Jurkat (Fig. 3G) cells.
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A, confocal images of HT29 cells loaded with JC-1 (1 μg ml–1, 10 min) in vehicle (Control) or salicylate (+Sal, 500 μM, 10 min). Emitted red and green fluorescence corresponds to energised and depolarized mitochondria, respectively. Bar represents 10 μm. B, values of ratios (mean ±S.E.M.; n= 4) of red/green fluorescence in control, salicylate- and FCCP- (10 μM, 10 min) treated cells. P < 0.05 versus control (Student's t test). C, decrease of TMRE fluorescence induced by FCCP (10 μM) and different salicylate concentrations (in μM) in HT29 cells. The arrow indicates addition of either vehicle, FCCP or salicylate. Fluorescence values for each individual cell were normalized relative to the value before treatment. Traces correspond to the averaged (mean ±S.E.M.), normalized recordings of 48–76 cells. Pictures show TMRE fluorescent images of the same cells before (Control) and 5 min after addition of 500 μM salicylate (+Sal) or 10 μM FCCP (see also Supplemental movie 1). Bar represents 10 μm. D, dose–response relation of TMRE fluorescence quenching by treatment with salicylate; values are mean ±S.E.M. of three independent experiments. All points were significantly different from control (P < 0.05). E, confocal images of bright field and TMRE-loaded HT29 cells in vehicle (Control), 10 min after 500 μM salicylate (+Sal) and 10 min after 10 μM FCCP (+FCCP). Bar represents 10 μm. The ratio of TMRE fluorescence in mitochondria relative to the surrounding cytosol was calculated as reported by Collins et al. (2002). Salicylate and FCCP largely decreased this ratio in both HT29 (F) and Jurkat (G) cells. Data correspond to 12 HT29 and 15 Jurkat cells studied in 3 independent experiments for each cell type.
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Next we asked whether the salicylate-induced mitochondrial depolarization would affect mitochondrial Ca2+ uptake. The relation between mitochondrial Ca2+ uptake and , the driving force for Ca2+ entry into mitochondria, is exponential. Thus, the effects of salicylate on mitochondrial Ca2+ uptake should be even larger than on . The Nernst relation predicts that every 30 mV depolarization (which is less than a 20% decrease of the resting –180 mV potential) diminishes 10-fold the equilibrium [Ca2+]mit (Bernardi, 1999). In order to test directly the effects of salicylate on Ca2+ transport we measured the mitochondrial Ca2+ uptake in HT29 cells using the mutated, low Ca2+ affinity aequorin targeted to mitochondria (Montero et al. 2000). Exposure of permeabilized HT29 cells to Ca2+ concentrations below 2 μM failed to induce any mitochondrial Ca2+ uptake (data not shown) indicating the requirement for high [Ca2+]cyt concentrations, such as those reached in high [Ca2+] microdomains, to activate the mitochondrial Ca2+ uniporter (Montero et al. 2000). Exposure of permeabilized HT29 cells to 6 μM Ca2+ induced a huge [Ca2+]mit near 800 μM within less than 30 s (Fig. 4A, control). This mitochondrial [Ca2+] uptake was inhibited by ruthenium compounds (Ruthenium red or Ru360, data not shown) which have been reported to block the mitochondrial Ca2+ uniporter (Kirichok et al. 2004) and by collapsing with FCCP (Fig. 4A, FCCP). Salicylate inhibited mitochondrial Ca2+ uptake in a dose-dependent manner (Fig. 4A and B) with an IC50 below 10 μM, a salicylate concentration that is surpassed by the therapeutic concentrations and comparable to the plasma values achieved with dietary salicylates (Paterson & Lawrence, 2001).
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Effects of salicylate (concentrations in μM) and FCCP (10 μM) on the Ca2+ uptake by mitochondria. A, HT29 cells expressing mutated mitochondrial aequorin were permeabilized and [Ca2+]mit was calculated from photonic emissions (details as in Fig. 2). Ca2+ uptake was started by perfusion with 6 μM Ca2+ (filled bar). B, average effects of different salicylate concentrations on mitochondria Ca2+ uptake in 3 experiments similar to that shown in A. Data are mean ±S.E.M. All points were significantly different from control.
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As SOCE depends on mitochondrial Ca2+ uptake, we tested next the effects of the same salicylate concentrations used in the above experiments on SOCE. Results are shown in Fig. 5. Salicylate inhibited SOCE in a concentration-dependent manner in both HT29 and Jurkat cells (Fig. 5A and B). The IC50 values were about 100 μM, well within the therapeutical range (Fig. 5C and D). The inhibition of SOCE by salicylate was not immediate but required a pre-incubation time of up to 10–12 min, consistent with the TMRE fluorescence decay (data not shown). In addition, whereas the effects of FCCP were quickly reversible (Fig. 1A and B), reversion of the inhibitory effects of salicylate required extensive washout of the drug (data not shown). The inhibiton of SOCE induced by salicylate was not affected by pre-incubation with oligomycin (not shown). Salicylate also inhibited SOCE induced by physiological simulation with carbachol in HT29 cells (Fig. 5E) and by TCR activation in Jurkat cells (Fig. 5F). Finally, salicylate also inhibited SOCE in SW480 cells lacking COX-2 gene expression (data not shown).
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Effects of different salicylate concentrations (in μM) on SOCE in thapsigargin-treated, fura-4 F-loaded, HT29 (A) and Jurkat (B) cells. Each couple of traces correspond to [Ca2+]cyt recordings either before and after incubation (10 min) with different salicylate concentrations in the same cells. Each trace is the mean ±S.E.M. of 29–59 cells. C and D, dose–response curves of effects of salicylate on SOCE inhibition induced by salicylate in HT29 (C) and Jurkat (D) cells. Each value is the mean ±S.E.M. of 3 experiments. E, effects of salicylate (2 mM) on Ca2+ entry induced by carbachol (100 μM) in HT29 cells (traces are the mean ±S.E.M. of 23 and 32 cells and representative of 3 similar experiments). F, effects of salicylate (2 mM) on Ca2+ entry induced by TCR stimulation in Jurkat cells (0.5 μg ml–1 anti-CD3 and 5 μg ml–1 anti-CD28 cross-linked with IgG2a). Traces are mean ±S.E.M. of 12 and 22 cells and representative of 3 similar experiments.
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Since SOCE has been reported to be essential for tumour cell proliferation (Lewis, 2001) we tested next the effects of salicylate on cell growth. Results are shown in Fig. 6. Salicylate inhibited growth of both HT29 and Jurkat cells (Fig. 6A and B) at therapeutic concentrations. Salicylate also inhibited growth of SW480 cells lacking COX-2 gene expression by 41 ± 8% at 500 μM (mean ±S.E.M., n= 4). Most interestingly, the IC50 values for salicylate-induced inhibition of growth were similar to those found for SOCE inhibition (Fig. 6C and D). The correlation between SOCE prevention and growth inhibition was very good in both Jurkat and HT29 cells (r= 0.94–0.96, P < 0.005–0.05; Fig. 6E and F). It could be argued that the inhibition of growth by salicylate could be due to induction of cell death. However, at the same low concentrations, salicylate did not promote significant cell death (Fig. 6G and H). At 2 mM, cell death was only slightly increased whereas at 5 mM, a concentration 10 times above the therapeutic range and considered toxic, salicylate induced extensive cell death (Fig. 6G and H). It could also be argued that the inhibitory effects of salicylate may be due to a decrease in cell ATP. We tested the effects of different concentrations of salicylate on the cell ATP contents and found that at concentrations of 10–2000 μM it did not affect significantly the cell ATP levels in HT29 cells (Fig. 7A). Finally, we carried out a tunel assay to ascertain whether salicylate may prevent cell growth by promoting apoptosis. We found that salicylate, at the concentrations used (10 μM–2 mM) to prevent SOCE or cell growth, does not induce apoptosis (Fig. 7B). At larger concentrations (5 mM), salicylate promoted apoptosis (Fig. 7B). These results indicate that low, therapeutic concentrations of salicylate prevent tumour cell growth and this effect is not due to changes in ATP levels or cell death.
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Effects of different concentrations of salicylate (in μM) on HT29 (A) and Jurkat (B) cell growth, expressed as cell number fold increase. All the groups differed statistically from each other except for the effects of 500 μM salicylate (not shown) that were not significantly different from the effects of 100 μM salicylate. Dose–response curve of the effects of salicylate on HT29 (C) and Jurkat (D) cell growth. E and F, correlation between growth inhibition (%) and SOCE inhibition (%) in HT29 (E) and Jurkat (F) cells. Lines are best linear fit (r= 0.94–0.96; P < 0.005–0.05). G and H, effects of salicylate on percentage of dead cells (cells stained with trypan blue) after 96 h incubation with the different salicylate concentrations (in μM) in HT29 (G) and Jurkat (H) cells. Cell death induced by 10, 100 or 500 μM salicylate were not significantly different from control (P > 0.05).
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A, effects of salicylate on cell ATP concentrations in HT29 cells measured by the luciferin/luciferase assay. Salicylate (10– 2000 μM) did not affect ATP concentrations (P > 0.05). B, the effect of salicylate on the per cent of apoptotic cells was tested by the tunel assay. Pictures show HT29 cell nuclei (blue) and apoptotic cells (red) in vehicle (control) and cells treated with different concentrations of salicylate. The bar represents 10 μm. B, bars show the per cent of apoptotic cells in each condition (mean ±S.E.M., n= 3). Salicylate did not induce apoptosis at concentrations of 10– 2000 μM (P > 0.05).
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To investigate further inhibition of proliferation, we monitored concurrently transcription dynamics of the topoisomerase-II gene, a cell cycle-driven gene and marker of proliferation (Falck et al. 1999), together with cell division in individual, living cells. This was achieved by a novel methodology based on bioluminescence imaging of transcription dynamics in single, living cells (Villalobos et al. 1999). We adapted the method for concurrent monitoring of cell division and gene transcription in NIH3T3 cells stably expressing the luciferase reporter gene under control of the topoisomerase-II gene promoter (Falck et al. 1999). Figure 8 shows the pseudocolour-coded bioluminescence images superimposed on the bright field image at the beginning of the experiment and after 24, 48 and 72 h for cells incubated in either control or salicylate-containing medium. Promoter activity was not detectable in resting cells but was turned on during progression of the cell cycle to peak before cell division and turned off right after mitosis to start a new cycle (Fig. 8; see also Supplemental movie 2). Salicylate blocked transcription cycling of topoisomerase-II gene (Fig. 8) and inhibited proliferation of NIH3T3 cells by 78 ± 8% (mean ±S.E.M., n= 3).
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Transcription activity of topoisomerase-II and cell division were monitored for 72 h at 15 min intervals by concurrent bioluminescence imaging and bright field microscopy in NIH 3T3 cells expressing luciferase under control of the topoisomerase-II gene promoter. The pictures show accumulated photonic emissions (15 min integration period) superimposed on bright field images in control (top) and salicylate-treated cells (bottom) at 0, 24, 48 and 72 h. Bar represents 10 μm. Traces show photonic emissions at 15 min intervals for 72 h in vehicle (control) and salicylate (500 μM) containing medium. Data are representative of 15 (control) and 4 (salicylate) experiments. See also Supplemental movie 2.
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Our results suggest that tumour cell proliferation requires mitochondrial Ca2+ uptake. If this hypothesis is correct, then it follows that any other compound that prevents mitochondrial Ca2+ uptake, either by mitochondrial depolarization or by other means, should inhibit SOCE and tumour cell proliferation. Consistent with this view, Kerzic and colleagues have shown recently that diazoxide, a mitochondrial KATP channel opener that depolarizes mitochondria by promoting K+ entry into the matrix, inhibits SOCE and prevents Jurkat T cell proliferation in a mitochondria-dependent manner (Holmuhamedov et al. 2002). We found that 100 μM diazoxide inhibited proliferation to the same extent as 100 μM salicylate in both Jurkat and HT29 cells (data not shown). Ruthenium compounds prevent mitochondrial Ca2+ uptake by direct action on the mitochondrial Ca2+ uniporter without interfering with . Figure 9 shows that Ru360, a membrane-permeable analogue of ruthenium red, also inhibited SOCE (Fig. 9A) and transcription cycling of the topoisomerase-II gene (Fig. 9B). Ruthenium compounds also inhibited proliferation in both Jurkat and HT29 cells (Fig. 9C and D). Thus, inhibition of mitochondrial Ca2+ uptake by different means (uncouplers, diazoxide, salicylate, ruthenium compounds) prevents SOCE and tumour cell proliferation.
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A, effects of 6 μM Ru360 on SOCE in HT29 cells loaded with fura-4 F. Each trace is the mean ±S.E.M. of 54 cells (data representative of 3 experiments). B, transcription cycling of topoisomerase-II in 3T3 cells treated with vehicle (control) or 6 μM Ru360. Data representative of 15 (control) and 3 (Ru360) independent experiments. C and D, effects of ruthenium red (RR, 100 μM, 96 h) on Jurkat (C) and HT29 (D) cell growth (n= 3). P < 0.05 versus control (Student's t test). Similar results were obtained with Ru360 (data not shown).
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We asked finally whether or not the mitochondria-dependent inhibition of SOCE is sufficient to explain the inhibition of tumour cell proliferation. To answer this question, we tested the effects of blocking directly ICRAC and SOCE on cell proliferation, cell-cycle driven gene transcription and Ca2+ entry. We used BTP-2 ([N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide]) which has been reported recently to inhibit ICRAC and cell proliferation in Jurkat cells at the same concentrations (Zitt et al. 2004). Figure 10 shows that BTP-2 blocked both SOCE (Fig. 10A and B) and proliferation in HT29 cells (Fig. 10C). The correlation between both effects was remarkably good (r= 0.999, P < 0.0001; Fig. 9D). In addition, BTP-2 also blocked transcription cycling of topoisomerase-II (data not shown). These results reinforce the idea that SOCE inhibition is sufficient to prevent proliferation. Furthermore, we found that salicylate (500 μM) did not have an additive effect on inhibition of proliferation induced by BTP-2 (Fig. 10E). Finally, the inhibitory effects of salicylate on proliferation were essentially reverted simply by increasing extracellular Ca2+ concentration (Fig. 10E), a condition that increases SOCE (not shown).
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A, effects of different BTP-2 concentrations (in μM) on SOCE in HT29 cells loaded with fura-4 F. Each trace is the average (mean ±S.E.M.) of 29–83 cells. B, dose dependency of the effects of BTP-2 on SOCE (%). Each value, expressed as percentage of control, is the mean ±S.E.M. of 3 experiments. Each bar was significantly different from the others (P < 0.05). C, dose dependence of the effects of BTP-2 on HT29 cell growth. Each bar represents the mean ±S.E.M. of 3 experiments. Each bar was significantly different from the others (P < 0.05). D, correlation between SOCE inhibition (%) and growth inhibition (%) induced by BTP-2 in HT29 cells. The line is the best linear fit of experimental data shown in B and C (r= 0.999; P < 0.0001). E, effects of BTP-2 (BTP2, 10 μM), salicylate (500 μM, Sal) and both (BTP2 + Sal) on cell growth in HT29 cells. Salicylate did not increase inhibition of cell growth induced by BTP-2. Effects of increasing extracellular Ca2+ concentration from 1.8 mM (control) to 3.8 mM (+Ca2+). High Ca2+ essentially restored the inhibitory effects of salicylate. Each bar represents the mean ±S.E.M. of 3 experiments. Each bar was significantly different (P < 0.05) from the others except for BTP-2 alone versus BTP-2 plus salicylate.
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Discussion
We show here that colon carcinoma cells exhibit a robust SOCE that is regulated by mitochondrial Ca2+ uptake in a manner similar to that described in other non-excitable cells including Jurkat cells. We found that collapse of mitochondrial with either FCCP, valinomycin or antimycin A plus oligomycin prevents SOCE. Similar results have been reported in other cells including Jurkat (Hoth et al. 1997, 2000), RBL (Gilabert & Parekh, 2000; Gilabert et al. 2001; Glitsch et al. 2002), HEK293 (Thyagarajan et al. 2002) and EA.hy926 endothelial cells (Malli et al. 2003). In all these cases, the effects of FCCP have been attributed to inhibition of mitochondrial Ca2+ uptake leading to Ca2+-dependent inactivation of SOCs and not to either ATP depletion or plasma membrane depolarization. Consistently, we found that the effects of FCCP on SOCE were not affected by oligomycin, used here to prevent ATP hydrolysis in the uncoupled mitochondria by reverse running of the F0F1–ATP synthase. On the other hand, valinomycin, a K+ ionophore that collapses mitochondrial potential but hyperpolarizes plasma membrane, also prevented SOCE. These results suggest that the effects of FCCP are due to the collapse of mitochondrial rather than to depolarization of the plasma membrane. Hoth et al. (1997) showed that readmisssion of Ca2+ to thapsigargin-treated cells resulted in a robust initial Ca2+ influx that was unafffected by FCCP. However, FCCP did affect the subsequent Ca2+ plateau. In our hands FCCP, antimycin A or valinomcyin all reduced both the initial rate and the size of the Ca2+ influx. Although at variance with Hoth et al. our results agree with most further works that showed clearly that CCCP or FCCP indeed reduced the initial rate and size of Ca2+ influx in Jurkat (Zablocki et al. 2003, 2005; Thyagarajan et al. 2002) and RBL-1 cells (Glitsch et al. 2002).
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According to our results, SOCE generates high [Ca2+]cyt domains that are sensed and cleared by nearby mitochondria, thus allowing sustained Ca2+ entry in colon cancer cells. Massive Ca2+ uptake by a fraction of mitochondria takes place through the Ca2+ uniporter, which is activated by high [Ca2+]cyt domains generated by nearby SOCs. This situation is similar to the one reported previously in adrenal chromaffin (Montero et al. 2000; Villalobos et al. 2002) and anterior pituitary cells (Villalobos et al. 2001). In fact, we showed previously that most of the Ca2+ entering excitable cells upon activation of voltage-gated Ca2+ channels was cleared by nearby mitochondria rather than by other extrusion systems (Villalobos et al. 2002). Our present results suggest that Ca2+ entry in non-excitable cells through SOCs is similarly cleared by nearby mitochondria. As a consequence, inhibition of mitochondrial Ca2+ uptake by different means (FCCP, ruthenium compounds or antimycin A plus oligomycin) impairs Ca2+ clearance and leads to SOCE inhibition, probably by inactivation of SOC by high [Ca2+] domains.
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If sustained SOCE requires mitochondrial Ca2+ uptake, then it follows that any compound that prevents mitochondrial Ca2+ uptake should inhibit SOCE. ASA and specially its major metabolite salicylate are mitochondrial uncouplers (Pachman et al. 1971). Salicylate enters mitochondria as salicylic acid (driven by the concentration gradient) and exits as salicylate anion (driven by voltage and concentration gradients) thus producing net proton entry (Gutknecht, 1990). Salicylate is much more potent than ASA because it contains an internal hydrogen bond that delocalizes the negative charge, thus increasing the lipid solubility and permeability of the anion. Model calculations predicted that, at therapeutic concentrations, salicylate may cause a net H+ influx into mitochondria enough to explain the reported ‘loose coupling’ effect (Gutknecht, 1990). Consistently, we find that salicylate induces a partial mitochondrial depolarization in intact cells. As stated above, the Nernst equilibrium predicts that even a small drop in should greatly affect equilibrium [Ca2+]mit and the driving force for mitochondrial Ca2+ uptake. This prediction was confirmed here as salicylate concentrations as low as 10 μM induced a modest mitochondrial depolarization (< 15%) that contrasted with a much larger inhibition of mitochondrial Ca2+ uptake (> 70%; Fig. 3). This disparity between the effects on and on mitochondrial Ca2+ uptake had not been experimentally shown before because lack of reliable measurements of Ca2+ uptake in mitochondria of living cells. Most of the previous measurements underestimated the actual [Ca2+]mit increases reached upon cell stimulation. The recent introduction of mitochondria-targeted, low affinity mutated aequorin (Montero et al. 2000) has enabled [Ca2+]mit measurements in the high micromolar to millimolar range. Using this novel methodology, we have shown here that therapeutic concentrations of salicylate produce a large inhibition of mitochondrial Ca2+ uptake. Since prevention of mitochondrial Ca2+ uptake leads to SOCE inhibition (Hoth et al. 1997, 2000; Glitsch et al. 2002), it follows that therapeutic concentrations of salicylate should also prevent SOC operation. Again, this expectation was confirmed in both Jurkat and colon carcinoma cells. Moreover, we also show that salicylate prevented SOCE elicited by physiological stimulation in both HT29 and Jurkat cells. In addition, the effects of salicylate on SOCE, like those of FCCP, were not affected by oligomycin, suggesting that the salicylate-induced inhibition of SOCE is not due to ATP depletion. In support of this view, we show here that salicylate concentrations that inhibited SOCE fully had no effect on ATP levels in HT29 cells. It must be remembered that tumour cells use glycolysis more than mitochondrial respiration, which is blocked by glucose (Crabtree, 1929), for ATP synthesis. Taken together, our results indicate that salicylate inhibits SOCE by preventing mitochondrial Ca2+ uptake, and that this leads to Ca2+-dependent inactivation of SOC.
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If SOCE is essential for proliferation in Jurkat and other tumour cells then salicylate should inhibit tumour cell proliferation at the same low concentrations that prevent SOCE. Our results fulfil this prediction as salicylate inhibits both SOCE and cell growth with the same concentration dependence. The lack of effects of salicylate on cell viability and apoptosis together with the effects of salicylate on cell division and transcription cycling of topoisomerase-II, a cell-cycle-related gene and marker of cell proliferation, supports the view that salicylate inhibits cell proliferation at low, therapeutic concentrations, and this effect is not due to changes in ATP concentration. Furthermore, our data support the view that SOCE inhibition is sufficient to prevent tumour cell proliferation. The pyrazole derivative BTP-2, a direct ICRAC blocker (Zitt et al. 2004), inhibits SOCE and proliferation in Jurkat cells with the same concentration dependence. We show here similar results for colon cancer cells. It should be mentioned that BTP-2 has been reported not to affect mitochondria or other ion channels (Zitt et al. 2004). Moreover, salicylate effects on cell proliferation are not additive with the effects induced by BTP-2. In addition, the effects of salicylate on cell proliferation are restored by increasing the extracellular Ca2+ concentration suggesting they are not due to a possible metabolic effect but to a defect in SOCE. Finally, diazoxide and ruthenium compounds, which inhibit SOCE by interfering in other ways with mitochondrial Ca2+ uptake, also prevent tumour cell proliferation. Taken together, our data support the view that salicylate, at therapeutic concentrations, inhibits SOCE in a mitochondria-dependent manner and that this effect is sufficient to prevent cell proliferation.
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What is the mechanism by which SOCE inhibition impairs cell proliferation Stimulation of Jurkat cells induces sustained SOCE and activation of the Ca2+-dependent phosphatase calcineurin that dephosphorylates NFAT, promoting expression of interleukin-2 and proliferation (Lewis, 2001). Interestingly, inhibition of SOCE by FCCP abolishes NFAT activation (Hoth et al. 2000). Moreover, direct blockade of SOCE by BTP-2 also prevents NFAT activation and proliferation (Zitt et al. 2004). Thus, the effects of salicylate on SOCE may interfere with the NFAT signalling pathway to proliferation. Consistent with this view, salicylate has been recently reported to inhibit NFAT activation and NFAT-dependent transcription in Jurkat cells (Aceves et al. 2004). The role of SOCE in control of proliferation in colon cancer cells is scarcely known. The inducible COX-2 isozyme is up-regulated in multiple tumours including colon cancer (Molina et al. 1999) and it has been proposed that it promotes tumour cell growth. Interestingly, transcription of COX-2 is regulated by NFAT both in Jurkat (Iiguez et al. 2000) and colon cancer cells (Duque et al. 2005). Salicylate inhibits COX-2 gene transcription at therapeutic concentrations similar to those used here (Xu et al. 1999). Thus, control of proliferation by SOCE in colon cancer cells could be transduced by NFAT-mediated regulation of COX-2 gene expression. Notwithstanding, the above possibility cannot account for the anti-proliferative effects of salicylate in colon cancer cells lacking COX-2 gene expression (Hanif et al. 1996; Smith et al. 2000) and we find that salicylate prevents SOCE and tumour cell growth also in the colon cancer cells lacking COX-2 gene expression. Therefore, SOCE must regulate proliferation by a different mechanism, perhaps involving other NFAT-induced genes. Interestingly, NFAT activation induced by sustained SOCE is considered a general proliferative signal (Lipskaia & Lompre, 2004). On the other hand, since SOCE is an early event in signal transduction, it is likely that multiple downstream transducing proteins could be recruited in the proliferative signalling pathway. One important example is NFB, another transcription factor involved in control of cell proliferation, which is inhibited by salicylate and modulated by multiple signalling pathways including Ca2+ signals. However, salicylate inhibits NFB activity and NFB-mediated gene expression at concentrations (IC50 5–9 mM) far larger than those reported here to inhibit SOCE and cell proliferation. Although caution is necessary in the interpretation of the results, the multiple targets proposed for aspirin (Hardwick et al. 2004) and salicylate can be consistent with our findings.
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Our results may have important implications as salicylate, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit tumour cell growth (Hanif et al. 1996; Molina et al. 1999; Smith et al. 2000) and to protect against colon (Chan, 2003) and other cancers (Bosetti et al. 2001). Clinical trials show that ASA reduces the risk of colorectal adenomas (Baron et al. 2003; Sandler et al. 2003) but the action mechanism has remained elusive. ASA irreversibly inhibits COX activity by acetylation of constitutive COX-1 at Ser530 and inducible COX-2 at Ser516. In vivo, ASA is quickly (t1/2= 15 min) deacetylated to salicylic acid, which remains in the plasma for much longer. Salicylic acid does not directly inhibit COX activity because it lacks the acetyl group, although, as stated above, it antagonizes COX-2 gene expression at the transcription level (Xu et al. 1999). Thus, both inhibition of COX-2 activity and COX-2 gene expression have been proposed to mediate the anti-tumoral effects of ASA. Recent results, however, do not support this view. It has been shown, for example, that ASA and COX-2 inhibitors block proliferation through a prostaglandin-independent pathway in both COX-2-expressing (HT29 and HCA-7) and non-expressing (SW480 and HTC116) colon cancer cells (Hanif et al. 1996; Smith et al. 2000). In the same line, it has been reported that the anti-proliferative effects of NSAIDs are independent of the level of COX-2 expression (Molina et al. 1999) or prostaglandin E2 production (Perugini et al. 2000), but related to cell cycle quiescence. Microarray analysis has shown that ASA represses many cell-cycle-related genes and modulates multiple signalling pathways (Hardwick et al. 2004), suggesting that an early mitotic signal may be the key target for the anti-proliferative effect. This view is consistent with the results we report here where the aspirin metabolite salicylate inhibits tumour cell proliferation by promoting inactivation of SOCE, an essential early requirement for proliferation. Thus, our results could contribute to explaining the mechanism for the anti-tumoral actions of aspirin and dietary salicylates (Paterson & Lawrence, 2001). Interestingly, ruthenium compounds have also been reported to have anti-tumoral and immunosuppresive properties both in vivo and in vitro, but the action of this mechanism has also remained elusive (Sava & Bergamo, 2000). Our results support the view that the effects of ruthenium compounds on mitochondrial Ca2+ uptake might contribute towards explaining also their anti-tumoral and immunosuppresive properties.
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Salicylate has been also reported to inhibit proliferation of normal vascular smooth muscle cells in a prostaglandin-independent manner (Marra & Liao, 2001). In fact, salicylate has been proposed as a candidate for treatment of proliferative disorders of the vessel wall that lead to intimal hyperplasia and hypertension. The specific mechanism for this action is not clear and multiple downstream targets have been proposed (Marra & Liao, 2001). Interestingly, vascular smooth muscle cells show up-regulated TRP and enhanced SOCE during proliferation (Golovina et al. 2001) suggesting that SOCE is very relevant for proliferation of these cells. It is tempting to speculate that salicylate might inhibit normal vascular smooth muscle cell proliferation by the same mechanism reported here. Nevertheless, the effects of salicylate are achieved at larger concentrations suggesting the possibility that salicylate may inhibit transformed cells more efficiently than normal cells. Further research is required to ascertain both the mechanism and possible differential sensitivity to salicylate in normal cells relative to their transformed counterparts.
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In summary, our results suggest a novel and important role of mitochondria in control of cell proliferation. Specifically, we show here that tumour cell proliferation critically depends on mitochondrial Ca2+ uptake. The interference of salicylate with this mechanism may ultimately provide the basis for the reported anti-proliferative and anti-tumoral effects of aspirin and dietary salicylates. In addition, as Ca2+ is a pleiotropic signal, our results point to a novel mechanism that may help to disentangle yet other elusive effects of aspirin such as its anti-inflammatory and immunosuppresive actions.
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Footnotes
L. Núez and R.A. Valero contributed equally to this work.
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