Inhibition of Ca Influx Is Required for Mitochondrial Reactive Oxygen Species-Induced Endoplasmic Reticulum Ca Depletion and Cell Death in Leukemia Cells

Disturbances of endoplasmic reticulum (ER) Ca homeostasis or protein processing can lead to ER stress-induced cell death. Increasing evidence suggests that oxidative stress (OS) plays an important role in a variety of cell death mechanisms. To investigate the role of OS in ER stress, we measured OS in response to three ER stress agents: econazole (Ec), which stimulates ER Ca release and blocks Ca influx; thapsigargin (Tg), a sarco(endo)plasmic reticulum Ca ATPase inhibitor that releases ER Ca and stimulates Ca influx; and tunicamycin (Tu), a glycosylation inhibitor that causes protein accumulation in the ER. Ec, but not Tg or Tu, caused a rapid increase in OS. Reactive oxygen species (ROS) generation was observed within mitochondria immediately after exposure to Ec. Furthermore, Ec hyperpolarized the mitochondrial membrane and inhibited adenine nucleotide transport in cell-free mitochondria, suggesting a mitochondrial target. Antimycin A, an inhibitor of complex III in electron transport, reversed mitochondrial hyperpolarization, OS generation, ER Ca depletion, and cell death by Ec, suggesting complex III dependence for these effects. Antioxidants butylated hydroxytoluene and NAcetyl-L-cysteine prevented ER Ca depletion and cell death by Ec. However, inhibition of Ca influx by Ec was unaffected by either antimycin A or the antioxidants, suggesting that this target is distinct from the mitochondrial target of Ec. Atractyloside, an adenine nucleotide transport inhibitor, generated ROS and stimulated ER Ca release, but it did not block Ca influx, deplete the ER or induce cell death. Taken together, these results demonstrate that combined mitochondrial ROS generation and Ca influx blockade by Ec is required for cell death. The endoplasmic reticulum (ER) is a major intracellular calcium store and the organelle responsible for the synthesis and post-translational modification of proteins destined for secretion or surface expression. These post-translational processes include protein folding, glycosylation, disulfide bond formation, and ER-Golgi protein trafficking. Disturbances of ER calcium homeostasis and protein processing cause accumulation of unfolded or misfolded proteins in the ER lumen and initiate the unfolded protein response (Kaufman, 1999; Ferri and Kroemer, 2001; Patil and Walter, 2001). Cells exhibit a variety of responses in their attempt to mitigate such ER stress. These include increased expression of ERresident Ca -dependent molecular chaperones such as GRP78 (BiP) and GRP94 (Kozutsumi et al., 1988) and suppression of protein synthesis to reduce the unfolded protein load. Preconditioning with sublethal levels of ER stress has been shown to protect cells, in part through up-regulation of chaperones (Liu et al., 1998; Hung et al., 2003). However, sustained ER stress will eventually result in prolonged protein synthesis inhibition that leads to cell death (Soboloff and Berger, 2002; Zhang and Berger, 2004). Important mediators of ER stress-associated death include the cleavage and activation of the ER-associated caspase-12 (Szegezdi et al., 2003) This work was supported by grants from the National Cancer Institute of Canada (to S.A.B.). 1 Current affiliation: Department of Hematology, Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology, Wuhan China, 430030. 2 Current affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.024323. ABBREVIATIONS: ER, endoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca ATPase; Tg, thapsigargin; Ec, econazole; Atra, atractyloside; ROS, reactive oxygen species; OS, oxidative stress; Tu, tunicamycin; FBS, fetal bovine serum; AA, antimycin A; BHT, butylated hydroxytoluene; Nac, N-Acetyl-L-cysteine; PI, propidium iodide; CM-H2DCFDA, 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate; AM, acetoxymethyl ester; MMP, mitochondrial membrane potential; JC-1, 5,5 6,6 -tetraethylbenzimidazolcarbocyanine iodide; FCCP, carbonyl cyanide p-(trifluomethoxy) phenylhydrazone; ANT, adenine nucleotide transporter; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; DCF, 2 ,7 -dichlorofluorescein. 0026-895X/06/7004-1424–1434$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 24323/3142093 Mol Pharmacol 70:1424–1434, 2006 Printed in U.S.A. 1424 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from and increased expression of CHOP/GADD153 (Wang et al., 1996), a transcription factor that sensitizes cells to apoptosis. Disruption of Ca homeostasis in the ER, such as after treatment with the SERCA inhibitor thapsigargin (Tg) (Thastrup et al., 1990), can stimulate sustained Ca influx from the extracellular milieu. In some cell types, this leads to both cytosolic and mitochondrial Ca overload (Babich et al., 1994; Soboloff and Berger, 2002), triggering apoptosis (Orrenius et al., 2003). Econazole (Ec), an imidazole antifungal, also stimulates depletion of the Tg-sensitive ER calcium store. However unlike Tg, Ec additionally blocks Ca influx, resulting in sustained ER Ca depletion (Franzius et al., 1994; Jan et al., 1999; Soboloff and Berger, 2002). In previous studies, we have shown that the Ca depletion caused by Ec induces activation enhanced cell death in leukemic cells, breast cancer cells, and murine bone marrow-derived mast cells through sustained inhibition of protein synthesis (Gommerman and Berger, 1998; Soboloff and Berger, 2002; Soboloff et al., 2002; Zhang et al., 2002; Zhang and Berger, 2004). However, the mechanism of how this compound affects the intracellular ER store and the subsequent fate of the cell remains unknown. Increasing evidence suggests that reactive oxygen species (ROS) and the oxidation-reduction (redox) state play important roles in a variety of cell death mechanisms induced by widely used antitumor drugs or by environmental toxic substances (Orrenius, 1985; Orrenius and Nicotera, 1987; Slater et al., 1995) (Feinendegen, 2002; Ueda et al., 2002). Because oxidative damage to the ER has been implicated in some forms of cell death (Hayashi et al., 2003; Lai et al., 2003; Watanabe et al., 2003), we investigated the possible role of oxidative stress (OS) in the induction of ER stress by different agents. Here, we show that ER Ca depletion and cell death induced by Ec but not Tg or tunicamycin (Tu) is dependent on ROS production at the mitochondria, thus identifying a mediating role for ROS in communicating mitochondrial disruption to the ER. Materials and Methods Cell Culture, Cell Death, and Caspase Assays. Human promyelocytic leukemia HL-60 cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 2.5 mM L-glutamine. HL-60 cells were treated with 15 M Ec in the presence or absence of 125 nM AA, 5 M BHT, or 1 mM Nac at 37°C for 2 h, in medium containing 1% FBS. Drugs were washed out, and the cells were recovered in 10% FBS drug-free RPMI 1640 medium for 6 h. Samples were costained with Annexin V-Cy5 (BioVision, Mountain View, CA) and propidium iodide (PI), and cell death was determined by flow cytometry. Early (Annexin V-positive, PI-negative) and late (Annexin V-positive, PI-positive) events were scored as dead cells. Caspase-3/7 activation was measured using the Vybrant 5-carboxyfluorescein caspase assay kit from Invitrogen (Carlsbad, CA) as described by the manufacturer. Detection of Oxidative Stress and Mitochondrial ROS. To examine the generation of OS, HL-60 cells were incubated with the indicated concentrations of Ec, Tu, or Tg in RPMI 1640 containing 2% FBS at 37°C for 2 h and then loaded with the OS indicator 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate (8 M CM-H2DCFDA; Invitrogen, Carlsbad, CA) at 37°C for 30 min. Fluorescence was measured by flow cytometry using excitation at 488 nm and emission at 530 nm at indicated intervals after treatment. Mitochondrial-specific ROS generation was measured using the MitoSox Red fluorescent dye (Invitrogen) as described by the manufacturer. This dye accumulates in the mitochondria, is oxidized by superoxide, and emits at 580 nm. Measurement of Cytosolic Ca Concentration. Cytosolic Ca measurements were performed by flow cytometry. HL-60 cells were serum-deprived for 2 h in Tyrode’s buffer (10 mM HEPES, 100 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.05% bovine serum albumin (0.05%) containing Ec, atractyloside (Atra), antioxidants, and/or hydrogen peroxide where indicated. Cells were then incubated in loading buffer (5 M indo-1AM and 0.03% Pluronic F-127 in Tyrode’s buffer; both from Invitrogen) at 37°C for 30 min and washed and incubated (15 min at room temperature) to allow for the complete conversion of indo-1AM to Ca -sensitive indo-1 through hydrolysis. Measurements were performed using a laser tuned to 338 nm while monitoring emissions at 405 and 450 nm. FloJo software (Treestar Software, Ashland, OR) was used to analyze the flow cytometric data. The concentration of intracellular free Ca was calculated according to the following formula (Grynkiewcz et al., 1985): [Ca ]i Kd (Fmin/Fmax) (R Rmin)/(Rmax R), where R is the ratio of the fluorescence intensities measured at 405 and 450 nm during the experiments, and F is the fluorescence intensity measured at 450 nm. Rmin, Rmax, Fmin, and Fmax were determined from in situ calibration of viable cells using 4 M ionomycin in the absence (Rmin and Fmin; 10 mM EGTA) and presence of (Rmax and Fmax) of Ca 2 . Kd (250 nM) is the dissociation constant for indo-1 at 37°C. Rmin, Rmax, Fmin, and Fmax varied depending upon settings and were determined at the beginning of each experimental procedure and experimental condition. Potentiometric Measurement of Mitochondria Membrane Potential. MMP measurements were performed by flow cytometry. Cells (5 10 cells/ml) were growth factor-deprived for 2 h in Tyrode’s buffer and then incubated with 5 g/ml JC-1 (Invitrogen) for 15 min at room temperature in Tyrode’s buffer. Then, cells were washed (three times) to remove extracellular JC-1. Measurements were performed using a laser tuned to 488 nm while monitoring the emissions of JC-1 monomers at 530 nm and JC-1 aggregates at 585 nm. MMP measurements were normalized using a modification of a formula from Rottenberg and Wu (1998): MMP (R RFccp)/(R0 RFccp) 100, where R is the ratio of the fluorescence intensities measured at 530 and 585 nm during the experiments, R0 is the fluorescence ratio of untreated cells, and RFccp is the fluorescence intensity measured after the addition of 2 M carbonyl cyanide p-(trifluomethoxy) phenylhydrazone (FCCP; Sigma-Aldrich, St. Louis, MO), a procedure that collapses the mitochondrial membrane potential and was performed at the beginning of each experiment. Adenosine Nucleotide Transfer-Dependent ADP Import Assay. Adenosine nucleotide transfer (ANT) across the mitochondrial membrane in ER-stressed cells was measured as described by Vander Heiden et al. (1999). In brief, 5 10 HL-60 cells were disrupted by mechanical lysis through homogenization in a mitochondria isolation buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES, pH 7.4, and 1 mM EGTA). After centrifugation of the supernatant (750g for 10 min to remove debris followed by 10,000g for 10 min), mitochondrial pellets were resuspended in ADP import buffer (250 mM sucrose, 20 mM HEPES, pH 7.2, 10 mM KCl, 5 mM succinate, 3 mM KH2PO4, 1.5 mM MgCl2, 1 mM EGTA, and 5 M rotenone) with or without the adenine nucleotide transporter inhibitor atractyloside (50 M; Sigma-Aldrich). [C]ADP (1 Ci; PerkinElmer Life and Analytical Sciences, Boston, MA) was added to the mitochondrial suspension and incubated for 10 min on ice. After washing two times in ADP import buffer, the samples were resuspended in scintillant (PerkinElmer) and quantified using a beta counter (PerkinElmer Wallac, Gaithersburg, MD). ANT-dependent ADP transport activity was determined by calculating the difference in counts between samples that were or were not preincubated with