Cadmium Induces Ca2+-Dependent Necrotic Cell Death through

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Chem. Res. Toxicol. 2007, 20, 406-415

Cadmium Induces Ca2+-Dependent Necrotic Cell Death through Calpain-Triggered Mitochondrial Depolarization and Reactive Oxygen Species-Mediated Inhibition of Nuclear Factor-KB Activity Pei-Ming Yang, Hung-Chi Chen, Jia-Shiuan Tsai, and Lih-Yuan Lin* Department of Life Science, National Tsing Hua UniVersity, Hsinchu 300, Taiwan ReceiVed June 27, 2006

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 µM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-κB activity and led cells to necrosis. An increase of NF-κB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cdinduced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-κB activity by increasing the ROS level. Introduction Apoptosis and necrosis are two distinct pathways for cell death. Apoptosis is a form of programmed cell death that is crucial for development and tissue homeostasis. Morphologically, apoptosis is characterized by cytoplasmic shrinkage, chromatin condensation, phosphatidylserine externalization, DNA fragmentation, and formation of apoptotic bodies without loss of plasma membrane integrity (1). In contrast, necrosis was previously viewed as an accidental and passive process of cell death caused by physical or chemical damage. Necrosis is characterized by pyknotic nucleus, cytoplasmic vacuolation, breakdown of plasma membranes, and induction of inflammatory responses resulting from the released cellular content (2). Accumulating evidence from recent studies in Caenorhabditis elegans (3, 4), Drosophilae (5), and rodents (6) supports the notion that necrosis, like apoptosis, is also a regulated and programmed process. For instance, a necroptosis (programmed necrosis) inhibitor, necrostatin-1, has been implicated in an intrinsic necrotic cell death mechanism in vitro and in vivo (7). Cadmium (Cd)1 is an environmental toxicant that has been shown to induce apoptosis in vivo and in vitro, but the cellular signaling pathways for the process are not clear (8). Factors that are related to cell survival and apoptosis, such as extracellular signal-regulated kinase, p38 mitogen-activated protein * To whom correspondence should be addressed. Tel: +886-3-5742693. Fax: +886-3-5715934. E-mail: [email protected]. 1 Abbreviations: Ca2+, calcium; Cd, cadmium; CHO, Chinese hamster ovary; ROS, reactive oxygen species; MPT, mitochondrial permeability transition; MMP, mitochondrial membrane potential; CsA, cyclosporin A; CAST, calpastatin; NF-κB, nuclear factor κB; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide.

kinase, and c-Jun NH2-terminal kinase, are stimulated by Cd (9). Cd enhances H2O2 production and increases the oxidative stress of cells (10), but the contribution of reactive oxygen species (ROS) to Cd-induced apoptosis is controversial (11, 12). Cd also induces necrotic cell death in vivo (13). However, the molecular mechanisms of the process are mostly unknown. Calcium (Ca2+) overload plays a critical role in Cd-induced apoptosis in mouse thymocytes (14). Calpains are Ca2+dependent proteases that have been implicated in processes such as remodeling of cytoskeletal/membrane attachments, transduction of signaling pathways, and apoptosis (15). The calpain activity is tightly regulated by its expression level, Ca2+ availability, autoproteolysis, phosphorylation, intracellular distribution, and the endogenous inhibitor calpastatin (CAST) (15). Loss of Ca2+ homeostasis deregulates calpain activity and causes tissue damage in patients with myocardial infarct, stroke, or brain trauma (16). Mitochondria play an important role in cell death by changing their outer and inner membrane permeability. The mitochondrial outer membrane permeability is regulated by the Bcl-2 family proteins, which control the release of apoptogenic factors into cytosol. Proteins participate in the mitochondrial permeability transition (MPT) process and regulate the permeability of the mitochondrial inner membrane. These proteins are activated by Ca2+ and oxidative stress (17). MPT is involved in either apoptosis or the necrosis pathway depending on the bioenergetic condition (18). The major MPT proteins consist of the mitochondrial cis-trans peptidyl-propyl isomerase (cyclophilin D, CypD) in the matrix, the adenine nucleotide translocator in the inner membrane, and porin (VDAC) in the outer membrane (19). Cells derived from mice lacking CypD perish in response to

10.1021/tx060144c CCC: $37.00 © 2007 American Chemical Society Published on Web 02/27/2007

Mechanism of Cadmium-Induced Necrosis in CHO Cells

various apoptotic stimuli but resist necrotic cell death induced by ROS and Ca2+ overload (20, 21). Nuclear factor κB (NF-κB) transcriptional factors are important in the regulation of immune and inflammatory responses. The mammalian NF-κB family has five members: NF-κB1 (p105 and p50), NF-κB2 (p100 and p52), RelA (p65), RelB, and c-Rel. They are sequestered in cytosol as inactive homoor heterodimers by binding to an inhibitory subunit, IκB. Upon stimulation, IκB is rapidly phosphorylated and degraded via the ubiquitin-proteasome pathway. The released NF-κB then enters the nucleus and regulates target gene expression (22). NF-κB promotes cell survival by inducing genes that encode antiapoptotic proteins, including FLIP, Bcl-2, Bcl-xL, A1/Bfl-1, c-IAP (cellular inhibitor of apoptosis), XIAP (X chromosomelinked inhibitor of apoptosis), TRAF1, and TRAF2. However, NF-κB also induces genes encoding antioxidant proteins that prevent programmed necrosis (23). The present study investigates the mechanism of cell death induced by Cd. We show that Cd triggers nonapoptotic cell death in CHO cells. This phenomenon allows us to investigate the mechanism of Cd-induced necrosis. Cd increases the intracellular Ca2+ level that subsequently stimulates calpain activation and ROS generation. The activated calpain reduces MPT while the elevated ROS suppresses the basal NF-κB activity, resulting in Cd-induced necrosis.

Materials and Methods Cell Culture and Chemicals. Chinese hamster ovary (CHO K1) cells were cultured as monolayers at 37 °C in McCoy’s 5A medium supplemented with 10% heat-inactivated fetal bovine serum, 0.22% sodium bicarbonate, 100 U/mL ampicillin, and 100 µg/mL streptomycin, in 5% CO2/95% air and 100% humidity. Reagents for cell culture were purchased from Gibco (Invitrogen). Cadmium chloride (CdCl2) was from Merck. BAPTA-AM [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester] was from Biomol. H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate), Fluo-3 AM {1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester}, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), Ac-DEVD-AFC, Suc-LY-AMC, calpeptin (benzyloxycarbonylleucyl-norleucinal), and PMA (phorbol-12-myristate13-acetate) were acquired from Calbiochem. BHA (butylated hydroxyanisole) and cyclosporin A (CsA) were from Sigma-Aldrich. Other chemicals were purchased from Sigma unless specified. DNA Fragmentation Assay. DNA fragmentation was analyzed by measuring the increase in sub-G1 fraction using flow cytometry and DNA ladder formation using agarose gel electrophoresis. For the analysis of sub-G1 fraction, treated cells were centrifuged at 1500 rpm for 5 min, and the cell pellet was resuspended in 70% ethanol and stored at 4 °C overnight. The cells were collected by centrifugation and resuspended in 1 mL of PBS containing RNase A (100 µg/mL). After 30 min at room temperature, the cells were spun at 1500 rpm for 5 min and the pellet was stained with 1 mL of propidium iodide (PI, 20 µg/mL in PBS) for 30 min. Flow cytometric analysis was carried out on a FACScalibur (Becton Dickinson). For DNA ladder analysis, cells were treated with 4 µM Cd or irradiated with 25 J/m2 UV. After incubation at 37 °C for 24 h, cells were washed twice with PBS and the low-molecular-weighted DNA fragments were extracted with TTE buffer (0.2% Triton X-100, 10 mM Tris, 15 mM EDTA, pH 7.6) for 15 min at room temperature. Cells were removed by centrifugation, and the supernatant containing the DNA fragments was incubated with 100 µg/mL of RNase A at 37 °C for 1 h. DNA was extracted with 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated in 0.1 volume of 3 M sodium acetate (pH 5.2) and 1

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 407 volume of isopropyl alcohol. After incubation at -70 °C for 15 min, the DNA was collected by centrifugation (12000 rpm) and washed once with 70% alcohol. The DNA pellet was dissolved in TE buffer and analyzed electrophoretically on a 2% agarose gel. PI Exclusion Assay. The integrity of plasma membrane was assessed by determining the ability of cells to exclude PI. Cells were trypsinized, collected by centrifugation, washed once with PBS, and then resuspended in PBS containing 10 µg/mL PI. The cells were incubated at room temperature in the dark for 15 min. The levels of PI incorporation were determined by flow cytometry. Caspase-3 Activity Assay. Cells were lysed in 1% Triton X-100, 1% NP-40, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 2 mM PMSF and incubated on ice for 10 min. After centrifugation at 13000 rpm and 4 °C for 30 min, the supernatants were transferred to new tubes. Fifty micrograms of proteins from the supernatant was incubated in 100 µL of reaction buffer (10 mM HEPES, 2 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, and 10 mM DTT) containing 50 µM Ac-DEVD-AFC for 1 h at 37 °C. The AFC fluorescence was measured at excitation and emission wavelengths of 405 and 505 nm, respectively, with a fluorescence microplate reader (Wallac 1420 Multilabel Counter, Perkin-Elmer). Determination of ATP Content. The intracellular ATP level was determined using a luciferin-luciferase bioluminescence reaction. Cells were resuspended in ATP extraction buffer (20 mM glycine, 50 mM MgCl2, and 4 mM EDTA, pH 7.4), heated at 98 °C for 45 s, and then placed on ice. After centrifugation at 13000 rpm and 4 °C for 15 min, supernatants were transferred to new tubes. At the time of assay, 20 µL of supernatant was added to 100 µL of luciferin-luciferase solution (2 mg of firefly lantern extract in 1 mL of deionized water). Light emission was measured 20 s later with a Wallac 1420 Multilabel Counter (Perkin-Elmer). Determination of Intracellular ROS and Ca2+. Intracellular ROS and Ca2+ were measured with H2DCF-DA and Fluo-3-AM, respectively. Cells were treated with 4 µM Cd in the presence or absence of inhibitors for indicated time intervals. Five micromolar H2DCF-DA or 2 µM Fluo-3-AM was added to the cultures 30 min before cell harvest. Cellular fluorescence was measured using flow cytometry (FACScalibur, Becton-Dickinson) with excitation and emission wavelengths of 488 and 530 nm, respectively. Determination of Mitochondrial Membrane Potential (MMP). After treatment, cells were incubated with 5 µg/mL JC-1 for 30 min at 37 °C. Cells were then washed twice with PBS, harvested, and resuspended in PBS. The JC-1 fluorescence emissions were measured with flow cytometry (FACScalibur, Becton-Dickinson) at 530 (green fluorescence) and 590 nm (red fluorescence), with an excitation wavelength of 488 nm. JC-1 produced green fluorescence when accumulated in the cytoplasm and formed red fluorescent J aggregates in the mitochondria. A decrease in red fluorescence indicated a collapse of MMP. Calpain Activity Assay. Cells were lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 1 mM Na3VO4 and incubated on ice for 30 min. After centrifugation at 13000 rpm and 4 °C for 10 min, supernatants were transferred to new tubes. Ten microliters of protein extracts (30 µg) was incubated in 100 µL of 20 mM Tris-HCl, pH 7.5, 5% glycerol, 10 mM DTT, 0.05% BSA, 5 mM CaCl2, and 200 µM Suc-LY-AMC for 1 h at 37 °C. The AMC fluorescence was measured on a fluorescence microplate reader (Wallac 1420 Multilabel Counter, Perkin-Elmer) with excitation and emission wavelengths of 390 and 460 nm, respectively. Western Blot Analysis. Cells were lysed in an ice-cold buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% NP-40, 20% glycerol, 0.5 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 25 µg/mL leupeptin, and 10 µg/mL aprotinin at 4 °C for 30 min. After centrifugation, the supernatants were transferred to new tubes and the protein concentration was determined by a dyebinding assay with chemicals purchased from Bio-Rad. Proteins in the cell lysates (50 µg) were separated on a 12% SDSpolyacrylamide gel and then transferred electrophoretically onto a PVDF membrane (Amersham). The membrane was prehybridized in TBST buffer (20 mM Tris-HCl, pH 7.5, 1.5 M NaCl, and 0.05%

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Figure 1. Characterization of Cd-induced cell death. CHO K1 cells were treated with 4 µM Cd or irradiated with 25 J/m2 UV and incubated in medium for a further 24 h. (A) Cells were harvested and analyzed with flow cytometry. (B) DNA was extracted from the collected cells and displayed on a 2% agarose gel after electrophoretic separation. The DNA molecular weight marker (M) and a DNA sample from cells without any treatment (U) were included for comparison. (C) The caspase-3 activity of the treated cell was determined by measuring the cleavage product of a caspase substrate, Ac-DEVD-AFC. (D) Cells were treated with 4 µM Cd. At indicated time points, total proteins were extracted for Western blot analysis. The levels of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) proteins were examined. Actin was included as an internal control. (E) Cells were treated with 0-8 µM Cd for 24 h. The plasma membrane permeability was determined with the PI exclusion assay. (F) Cells were treated with 4 µM Cd for 12 and 24 h or irradiated with 25 J/m2 UV. After a further 24 h of incubation, the cellular ATP level was determined by a luciferin-luciferase bioluminescence method. Each value represents a mean ( SD of three samples. Asterisks (*) denote significant differences (p < 0.05) between untreated and UV-treated (C) or Cd-treated samples (E and F).

Tween-20) with 5% skim milk for 1 h, then was transferred to the same solution containing primary antibody, and was incubated overnight at 4 °C. After it was washed twice with the TBST buffer, the membrane was submerged in prehybridization buffer containing the horseradish peroxidase-conjugated secondary antibody for 1 h. The membrane was rinsed twice with TBS buffer, then developed by the ECL system, and exposed to X-ray film (Amersham). Transfection and Reporter Gene Assay. A pUC119 plasmid encoding the HA-tagged CAST (a generous gift from Dr. Maki, Nagoya University, Japan) was digested with XbaI and subcloned into pcDNA3. Transient transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 90-95% confluent cells were transfected with 8 µg of DNA in 2.5 mL of serum-free medium for 6 h at 37 °C. Then, 2.5 mL of medium containing 20% fetal bovine serum was added to the transfection mixture and cultured for an additional 24 h. After transfection, cells were subcultured for experimental use. For NF-κB transactivation activity measurement, NF-κB luciferase reporter plasmid (Strategene) was cotransfected with eGFP plasmid according to the method described above. After transfection, cells were replated in 24 well plates at a density of 6 × 104 per

well. After 24 h, cells were treated with chemicals for indicated time intervals. Cells were then lysed in 200 µL of 25 mM Trisphosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100. The cell lysate (20 µL) was mixed with an equal volume of luciferase assay reagent (Promega). Light emission was measured with a Wallac 1420 Multilabel Counter (Perkin-Elmer). Luminescence data were collected and normalized against eGFP values. Statistical Analysis. Means and SDs of samples (performed in triplicate) were calculated from the numerical data generated in this study. Significant differences between treatments were determined by Student’s t test.

Results Cd Induces Necrotic but not Apoptotic Cell Death in CHO K1 Cells. CHO K1 cells were treated with various concentrations of Cd for 24 h, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 was estimated to be 4.6 µM (data not shown), and 4 µM Cd was used in subsequent experiments.


Figure 2. Role of intracellular Ca2+ in Cd-induced necrosis. (A) Cells were treated with 4 µM Cd for 0-6 h, and 2 µM Fluo-3 AM was added 30 min before cell harvest. The Fluo-3 fluorescence was determined by flow cytometry. Each value represents a mean ( SD of three samples. (B) Cells were pretreated with 10 µM BAPTA-AM for 30 min and then exposed to 4 µM Cd for 6 h. The Fluo-3 fluorescence was determined with flow cytometry. (C) Cells were pretreated with 0-10 µM BAPTA-AM for 30 min and then exposed to 4 µM Cd for 24 h. Cd-induced necrosis was determined with the PI exclusion assay. Each value represents a mean ( SD of three samples. Asterisks (*) denote significant differences (p < 0.05) between untreated and Cdtreated (A) or BAPTA-AM-treated samples in the presence of Cd (B and C).

When cells were cultured in 4 µM Cd for 24 h, morphological changes, such as monolayer disruption and cell rounding, were observed. The cells were collected for flow cytometric analysis. As shown in Figure 1A, the cell cycle profile did not change after Cd treatment. However, cells that received 25 J/m2 UV irradiation have a significant increase in the sub-G1 fraction. Furthermore, an obvious DNA ladder was observed in UVirradiated but not in Cd-treated cells (Figure 1B). Because UV irradiation is known to induce apoptosis in CHO K1 cells (24), Cd treatment apparently did not cause the same effect. To further demonstrate that Cd did not induce apoptotic cell death, we measured the capase-3 activity after Cd treatment. Caspase-3 is activated during apoptosis. Cells were treated with 4 µM Cd for various time intervals, and the caspase-3 activity was determined. Figure 1C shows that caspase-3 activity was

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Figure 3. Role of calpains in Cd-induced necrosis. (A) Cells were treated with 4 µM Cd for 0-6 h. The calpain activity was determined by measuring the cleavage product of a calpain substrate, Suc-LYAMC. The asterisks (*) denote significant differences (p < 0.05) between untreated and Cd-treated samples. (B) Cells were pretreated with 0-100 µM calpeptin for 30 min and then exposed to 4 µM Cd for 24 h. Cd-induced necrosis was determined by the PI exclusion assay. The asterisks (*) denote significant differences (p < 0.05) between untreated and calpeptin-treated samples in the presence of Cd. (C) Transiently transfected cells of pcDNA3 (vector) and CAST were treated with 4 µM Cd for 24 h. Cd-induced necrosis was determined by the PI exclusion assay. Asterisks (*) denote significant differences (p < 0.05) between vector and CAST cells treated with Cd. Each value represents a mean ( SD of three samples.

not altered within 12 h of Cd treatment but increased 4 h after UV irradiation. Bax, a member of the pro-apoptotic Bcl-2 family, is involved in apoptosis. In contrast, Bcl-2 and Bcl-xL belong to the antiapoptotic Bcl-2 family. They associate with Bax to form heterodimers and neutralize its apoptotic effect. During apoptosis, the ratios of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family decide cell fate (25). We analyzed the expression of Bcl-2, Bcl-xL, and Bax during Cd exposure. Their expression did not change upon Cd challenge (Figure 1D). These results indicate again that Cd is not an apoptotic inducer in CHO K1 cells. Because Cd does not cause apoptosis, it is possible that Cd triggers necrosis in CHO K1 cells. Because plasma membrane disruption is a marker of necrosis, we examined the membrane integrity using the PI exclusion assay. As shown in Figure 1E,


Figure 4. Role of ROS in Cd-induced necrosis. (A) Cells were pretreated with 150 µM BHA for 30 min and then exposed to 4 µM Cd for 24 h. The cellular ROS level was measured by DCF fluorescence with flow cytometry. (B) Cells were pretreated with 150 µM BHA for 30 min and then exposed to 4 µM Cd for 24 h. Cd-induced necrosis was determined by the PI exclusion assay. (C) Cells were pretreated with 10 µM BAPTA-AM or 100 µM calpeptin for 30 min and then exposed to 4 µM Cd for 12 h. The cellular ROS level was measured by DCF fluorescence with flow cytometry. Each value represents a mean ( SD of three samples. Asterisks (*) denote significant differences (p < 0.05) between Cd-treated samples in the absence and presence of BHA (A and B), BAPTA-AM, or calpeptin (C).

the proportion of PI positive (necrotic) cells increases in a dosedependent manner after Cd exposure. Energy depletion occurs during necrosis but not apoptosis (26). We estimated the intracellular ATP level of Cd-treated cells. The cellular ATP level reduced to 58 and 34% of that of the untreated cells after 12 and 24 h of Cd administration, respectively. On the other hand, the ATP level in CHO K1 cells did not alter after UV irradiation since apoptosis is an energydependent process (Figure 1F). These results demonstrate clearly that Cd triggers necrotic cell death in CHO K1 cells. Role of Intracellular Ca2+. Intracellular Ca2+ homeostasis plays an important role in maintaining normal cellular functions. Disturbance of intracellular Ca2+ homeostasis is a general cause of apoptosis (27). It has also been shown that Ca2+ overload is critical to Cd-induced apoptosis in mouse thymocytes (14). To elucidate the possible roles of intracellular Ca2+ on Cd-induced

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necrotic cell death, we analyzed the intracellular Ca2+ level by flow cytometry. Figure 2A shows that Ca2+ level increases and persists time-dependently over a 6 h Cd treatment period. In fact, this increase can be detected within 10 min of Cd exposure (data not shown). Using an intracellular Ca2+ chelator (BAPTAAM), the increase in Ca2+ could be efficiently inhibited (Figure 2B). The correlation of Ca2+ overload and Cd-induced cell damage was then investigated. Cells were treated with 4 µM Cd for 24 h in the presence of various concentrations of BAPTA-AM, and cell death was quantified by the PI exclusion assay. As shown in Figure 2C, BAPTA-AM rescued a significant amount of cells from Cd-induced cell death. These findings suggest that intracellular Ca2+ plays a major role in causing Cd-induced cell death. Role of Calpains. Ca2+ is a second messenger that activates several signaling proteins and regulates a variety of cellular functions. Calpains are cysteine proteases and are among the downstream signaling targets activated by Ca2+ (15). Calpains mediate oncosis (necrotic cell death) in different cells (28). Accordingly, calpains might participate in Cd-induced necrosis. As shown in Figure 3A, calpain activity is elevated within 30 min and remains high at 6 h after Cd treatment. The addition of calpeptin, a calpain inhibitor, inhibited the Cd-induced cell death (Figure 3B). To further confirm the role of calpain in Cd-induced cell death, a gene encoding an endogenous calpain inhibitor, CAST, was transfected into CHO K1 cells. As shown in Figure 3C, the expression of CAST partially reduced the Cdinduced cell death. These results suggest that calpain activation is involved in Cd-induced necrosis. Role of ROS. ROS produced by Cd treatment is one of the critical factors that cause cell damage. We have shown previously that Cd increases cellular ROS levels in CHO K1 cells (29). It is not clear whether the produced ROS are involved in Cd-induced necrosis. A three-fold increase in ROS was observed in cells treated with 4 µM Cd for 24 h. This increase was effectively diminished by an antioxidant, BHA (Figure 4A). To investigate whether ROS is associated with Cd-induced necrosis, the PI exclusion assay was employed to estimate cell damage. As shown in Figure 4B, in the presence of 150 µM BHA, PI positive cells were reduced from 44 to 30%. These results indicate that ROS generation is crucial for Cd-triggered necrosis. The ROS level elevates 4-8 h after Cd exposure (29). This is a late response as compared to that of the time for Ca2+ elevation and calpain activation (within 1 h after exposure). We speculated that Ca2+ overload and calpain activation were upstream signals for ROS production. To clarify this possibility, cells were treated with 4 µM Cd for 12 h in the presence of 10 µM BAPTA-AM or 100 µM calpeptin, and the ROS level was examined. Figure 4C shows that only BAPTA-AM effectively reduced the Cd-induced ROS elevation. Calpeptin alone caused an increase in ROS, which is similar to that reported for Swiss 3T3 fibroblasts (30). However, calpeptin treatment did not further enhance the Cd-induced ROS level. Similarly, cells transfected with CAST gene did not alter the Cd-induced ROS production (data not shown). This finding suggests that Cd induces ROS production through a Ca2+-dependent but calpainindependent pathway. Role of MPT. MPT is also known as mitochondrial depolarization and plays critical roles in the induction of apoptosis and necrosis (18). To evaluate the roles of MPT in Cd-induced necrosis, we first measured the extent of MPT in cells after Cd treatment. The MPT was detected by the collapse of MMP using the cationic dye, JC-1. The MMP of cells was unaltered within 12 h of Cd exposure (data not shown). However, 56% of the



Figure 5. Role of MPT in Cd-induced necrosis. (A) Cells were pretreated with 10 µM CsA for 30 min and then exposed to 4 µM Cd for 18 h. The collapse of MMP was measured by the decrease in red fluorescence of the JC-1 with flow cytometry. (B) Cells were pretreated with 0-10 µM CsA for 30 min and then exposed to 4 µM Cd for 24 h. Cd-induced necrosis was determined by the PI exclusion assay. (C) Cells were pretreated with 10 µM BAPTA-AM, 100 µM calpeptin, or 150 µM BHA for 30 min and then exposed to 4 µM Cd for 18 h. The collapse of MMP was measured by the decrease in red fluorescence of the JC-1 with flow cytometry. (D) Cells were pretreated with 10 µM CsA for 30 min and then exposed to 4 µM Cd for 12 h. The cellular ROS level was measured by DCF fluorescence with flow cytometry. Asterisks (*) denote significant differences (p < 0.05) between untreated and CsA (A and B) or BAPTA-AM-treated (C) samples in the presence of Cd. Each value represents a mean ( SD of three samples.

cells were depolarized after 18 h of Cd treatment. This depolarizing effect was significantly inhibited by CsA, a MPT inhibitor (Figure 5A). To investigate whether MPT was associated with Cd-induced necrosis, we estimated the extent of cell damage with the PI exclusion assay. As shown in Figure 5B, 5 and 10 µM CsA reduced PI-positive cells from 44 to 37 and 22%, respectively. We then correlated Ca2+ overload, calpain activation, and ROS with mitochondrial depolarization. As shown in Figure 5C, Cd-induced MPT was inhibited by BAPTA-AM and calpeptin but not by BHA. This observation indicates that Cd induced MPT through a Ca2+/calpain-dependent pathway but not through the elevation of ROS. Furthermore, mitochondrial depolarization did not elevate ROS since the MPT inhibitor (CsA) could not prohibit the Cd-induced ROS generation (Figure 5D). Role of NF-KB. Cd interferes NF-κB activity in rat kidney proximal tubule cells (31), murine macrophages (32), and cellfree system (33). To explore the role of NF-κB in Cd-induced necrosis, cells transiently transected with NF-κB luciferase reporter plasmid were treated with 4 µM Cd for 6, 12, and 24 h. The basal NF-κB activity was reduced by 20% 6 h after Cd exposure and dropped further with time (Figure 6A). The NFκB activity was down-regulated to 30% of that of the untreated cells at 24 h. At this time point, the cellular levels of cellular Bcl-2 and Bcl-xL (two target genes of NF-κB) were not affected (Figure 1D) despite the decrease in NF-κB activity. To investigate which factor(s) down-regulates the NF-κB activity, we treated the cells with Cd in the presence of BAPTA-

AM, calpeptin, BHA, or CsA. Cellular NF-κB activity was determined by the reporter gene assay. Figure 6B shows that BAPTA-AM and BHA effectively restore the NF-κB activity after Cd treatment. However, calpeptin and CsA treatments have no such effect. These results indicate that Cd suppresses basal NF-κB activity through a Ca2+/ROS-dependent but calpain/ MPT-independent pathway. To affirm that NF-κB plays a role in Cd-induced necrosis, we altered the NF-κB activity and then investigated the subsequent effect. An NF-κB activator, PMA, was used to restore the NF-κB activity after Cd treatment. As demonstrated by the luciferase reporter gene assay (Figure 6C), the NF-κB activity of Cd-treated cells increased dose-dependently with the addition of PMA. Cells were then treated with 4 µM Cd in the presence of various concentrations of PMA for 24 h, and cell damage was determined by the PI exclusion assay. Figure 6D shows that increasing the NF-κB activity can attenuate the Cdinduced necrosis. This finding indicates that suppression of basal NF-κB activity correlates with Cd-induced necrosis. Cooperation of ROS- and Calpain-Dependent Pathways on Cd-Induced Necrosis. Because calpain activation and ROS production are two independent pathways downstream of Ca2+ elevation during Cd-induced necrosis, we further analyzed whether ROS and calpains exert cooperative effects on Cdinduced necrosis. As shown in Figure 7A, calpeptin and BHA can reduce approximately 25 and 40% of the Cd-induced cell damage, respectively. When combining both calpeptin and BHA, the damage can be reduced to 60% of that of the Cd-treated cells. The same effect can also be observed in treating CAST-


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Figure 6. Role of NF-κB in Cd-induced necrosis. (A) Cells were transfected with NF-κB reporter plasmid and replated 24 h later. After replating, cells were treated with 4 µM Cd for 0-24 h. The relative NF-κB activity was measured as described in the Materials and Methods. Asterisks (*) denote significant differences (p < 0.05) between untreated and Cd-treated samples. (B) Cells were pretreated with 10 µM BAPTA-AM, 100 µM calpeptin, 10 µM CsA, or 150 µM BHA for 30 min and were then exposed to 4 µM Cd for 12 h. The relative NF-κB activity was measured. Asterisks (*) denote significant differences (p < 0.05) between untreated and BAPTA-AM- or calpeptin-treated samples in the presence of Cd. (C) Cells were transfected with NF-κB reporter plasmid and replated 24 h later. After replating, cells were pretreated with 0-6 µM PMA for 30 min and then exposed to 4 µM Cd for 12 h. The relative NF-κB activity was measured. (D) Cells were pretreated with 0-6 µM PMA for 30 min and then exposed to 4 µM Cd for 24 h. Cd-induced necrosis was determined with the PI exclusion assay. Asterisks (*) denote significant differences between untreated and PMA-treated samples in the presence of Cd. Each value represents a mean ( SD of three samples.

overexpressed cells with BHA (Figure 7B). Cells transfected with CAST were more resistant to Cd-induced necrosis. With the addition of 100 µM BHA, the necrotic fraction in CASTtransfected cells was further reduced. The damage is approximately 65% lower than that of vector-transfected cells treated with Cd. Cd treatment changes the cellular morphology. Because ROS and calpain are involved in the Cd-induced cell damage, we examined whether their inhibitors could restore the Cd-induced morphological changes. As shown in Figure 7C, cells rounded up or showed an irregular shape with Cd treatment, and the morphological changes were partly restored when BAPTA, calpeptin, or BHA was administered. The cells extended better when both calpeptin and BHA were added together. These observations further support the notion that calpain/MPT and ROS/NF-κB cooperatively regulate the Cd-induced, Ca2+dependent necrosis.

Discussion A variety of toxicants can induce both apoptosis and necrosis in the same or different cell types depending on the circumstances such as the apoptosis/necrosis threshold of the particular cell type, the metabolic state of the cell, exposure to other stresses, preconditioning, etc. (34). Usually, toxicants induce apoptosis at a lower dosage but effectuate necrosis at a higher dosage (35). It has been reported that Cd can engender both apoptosis and necrosis in the same cell line (11). The majority of studies has been focused on the mechanism of Cd-induced apoptosis. The mechanism of Cd-induced necrotic cell death

has not been investigated because it is difficult to dissect the contributing factors when both types of cell death are present. In the work showed herein, Cd induces only necrosis in CHO K1 cells. This unique characteristic allows us to investigate the molecular mechanism of nonapoptotic cell death induced by Cd. Recently, Banfalvi et al. (36) reported that Cd (1 µM) could induce apoptotic-like changes in the chromatin structure of CHO cells but no biochemical evidence of apoptotic induction has been provided. We could not find a sign of apoptosis in cells treated with Cd at or lower than 1 µM (29). Similarly, Shimada et al. (37) reported that 1 µM Cd did not induce apoptosis in CHO cells. We have shown previously that Cd (1 µM) arrests cell cycle at the G2/M phase with a marked Cdk1 activation (29). Cdk1 activation is required for mitosis, which shares a number of morphological and biochemical features with apoptosis (38). We speculate that dysregulation of Cdk1 activity by Cd (1 µM) changes the chromatin structure of CHO K1 cells. Cd competitively inhibits Ca2+ influx (39). The elevation of cellular Ca2+ during Cd exposure is attributed to the release of Ca2+ from endogenous Ca2+ storage depot (i.e., endoplasmic reticulum, ER). The release of Ca2+ from the ER can occur through the inositol 1,4,5-triphosphate receptor (IP3R) and/or the ryanodine receptor (RyR) on the ER membrane. Both IP3R and RyR play important roles in Ca2+-mediated apoptosis and necrosis (40). An increase of IP3R1, but not IP3R2 or IP3R3, expression has been found in human lymphoma (U937) cells during Cd-induced apoptosis (41). Cd might induce the release of Ca2+ from ER via the IP3R1 pathway. Notably, a study has shown that sustained depletion of Ca2+ storage in the ER by



Figure 7. Cooperactive effect of calpain inhibitor and ROS scavenger in attenuating Cd-induced necrosis and cellular morphological changes. (A) Cells were pretreated with 50 µM calpeptin and/or 100 µM BHA for 30 min and then exposed to 4 µM Cd for 24 h. PI uptake was measured by flow cytometry. Each value represents a mean ( SD of three samples. * and # denote significant differences (p < 0.05) between untreated and calpeptin- or BHA-treated samples in the presence of Cd and between the paired samples, respectively. (B) Cells were transfected with the pcDNA3 (vector) or the CAST gene and replated 24 h later. After replating, cells were pretreated with 100 µM BHA before exposure to 4 µM Cd for 24 h. Cd-induced necrosis was determined with the PI exclusion assay. Each value represents a mean ( SD of three samples. Asterisks (*) denote values obtained that differ significantly (p < 0.05) from cells transfected with vector only. Significant differences (p < 0.05) between the paired samples are denoted with #. (C) Cells were pretreated with 10 M BAPTA, 50 µM calpeptin, 100 µM BHA, or both calpeptin and BHA for 30 min and then exposed to 4 µM Cd for 24 h. The cellular morphology was photographed. Original magnification, 200×.

caffeine and ryanodine leads CHO cells to apoptosis while RyR was stably overexpressed. However, coexpression of Bcl-xL, an anti-apoptotic protein in RyR stably expressed CHO cells, causes necrosis (42). Intracellular buffering of Ca2+ by BAPTAAM fails to inhibit apoptosis but does inhibit necrotic cell death (42). Results from these studies suggest cytosolic Ca2+ overload might be important in the execution phase of necrotic death while sustained depletion of Ca2+ stores in ER leads to apoptosis. Because Cd elevates cytosolic Ca2+ in CHO K1 cells (Figure 2), it possibly directs cells toward necrosis. Apparently, participation of Ca2+ and Ca2+-dependent signaling in necrotic cell death is a mechanism conserved from nematodes to primates. Hyperactivation of the C. elegans MEC-4 Na+ channel of the DEG/ENaC superfamily (MEC4d) stimulates the influx of Ca2+ and induces Ca2+ release from ER with consequential neuronal necrosis. Four genes have been identified for necrotic initiation: calreticulin, an intralumenal ER Ca2+-binding protein; calnexin, an ER Ca2+-binding chaperone; IP3R; and RyR (3). Further investigation proves that calpains are important effectors in this process (4). Numerous lines of evidence demonstrate that calpains are involved in necrotic cell death in various mammalian models (28). We also showed in this study the involvement of calpain in Cd-induced cell necrosis. Under normal conditions, ROS is mainly generated in mitochondria and is rapidly scavenged by cellular antioxidants

(43). Generally, superoxide dismutase (SOD), catalase, glutathione peroxidase (GP), and various isoforms of peroxiredoxin are involved in the removal of ROS. GP requires reduced glutathione (GSH) that is converted to the oxidized form (GSSG) during H2O2 elimination. GSSG is then recycled to GSH by glutathione reductase (GR). GSH and other thiol-containing proteins are important in cellular defense against Cd toxicity. The GSH redox cycle also plays a key role in the detoxification of ROS that are generated by Cd (8). GSH depletion by L-buthionine[S,R]sulfoximine can switch Cd-induced apoptosis to necrosis (11). It is proposed in the same study that prolonged oxidative stress promotes necrosis. Consistently, Cd causes prolonged ROS generation and necrosis in this study (Figure 4). Furthermore, Cd (4 µM for 4 h) decreases the cellular GSH level and the enzymatic activities of GR and GP (44). It has also been shown that an increase of oxidative stress inhibits caspase activation and apoptosis and triggers necrosis (45). Possibly, Cd reduces the incidence of apoptosis in CHO K1 cells by depleting the cellular GSH and prolonging the generation of ROS, and consequently leads cells to necrosis. MPT is crucial to apoptosis and necrosis, depending on the bioenergetic condition (18). Consistently, we observed ATP depletion (Figure 1F) and MMP collapse caused by MPT in Cd-treated cells (Figure 5A). Calpain-like activity in mitochondria has been proved to be associated with mitochondrial dysfunction (46, 47). Our results also indicate that calpains


Figure 8. Model of the roles of Ca2+, calpains, ROS, and NF-κB on Cd-induced necrotic cell death in CHO K1 cells.

mediate the collapse of MMP. However, it is not clear whether cytosolic or mitochondrial calpain activity is required for the mitochondrial dysfunction. We have overexpressed the endogenous calpain inhibitor (CAST), which locates predominantly in cytosol (48), and could not inhibit the loss of MMP (data not shown). We speculate that Cd may activate both cytosolic and mitochondrial calpains but that mitochondrial depolarization is caused only by the mitochondrial pool of calpains. Oh et al. (49). have shown that Cd induced the activation of a mitochondrial 30 kDa small subunit of calpain, which may cleave the full-length Bax into a 18 kDa fragment in mitochondria and cause apoptosis. The finding may differentiate the regulation between Cd-induced apoptosis and necrosis because the integrity of Bax was unaltered in this study (Figure 1D). The fact that Cd activates calpain activity in mitochondrial fraction may explain the reason why expression of CAST has a marginal effect in inhibiting Cd-induced cell damage while addition of calpeptin exhibits a more significant inhibitory effect (Figures 3 and 7). Because CAST presents mainly in cytosol (48), the expressed proteins have less accessibility to the mitochondrial pool of calpains. However, calpeptin is a low molecular weight chemical, which can diffuse into mitochondria and exerts its effect. The effects of Cd on NF-κB were rarely investigated. In rat kidney proximal tubule cells, Cd induces NF-κB activation that increases the expression of the multidrug resistance P-glycoprotein (mdr1) via the ROS-dependent pathway. Mdr1 then provides an anti-apoptotic function (31). In murine macrophages, Cd induces NF-κB expression and cell proliferation through Ca2+-dependent signaling (32). These studies suggest a protective role of NF-κB in Cd toxicity. In our study, the basal NFκB activity was down-regulated upon Cd exposure (Figure 6A). Reversion of NF-κB activity by BAPTA-AM, BHA, and PMA reduced Cd-induced necrosis. These results indicate that basal NF-κB activity protects cells from Cd toxicity. Moreover, murine embryonic fibroblasts (MEFs) deficient in tumor necrosis factor (TNF) receptor-associated factor 2 and 5 (TRAF2/5) or p65 NF-κB subunit accumulate ROS after TNF stimulation (50). The ROS subsequently leads to cell death with necrotic and apoptotic morphological changes. Necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant (BHA) (50). Importantly, TNF does not induce ROS accumulation in wildtype MEFs. Thus, we propose that under defect of NF-κB signaling, necrotic cell death tends to be induced through ROS accumulation. ROS has been proposed to participate in cell type-specific activation of NF-κB (51). Evidence also shows that ROS released from NADPH oxidase do not mediate NF-κB activation (52). In this study, ROS produced by Cd did not activate but reduced the basal activity of NF-κB. This reduction was

Yang et al.

effectively inhibited by BHA (Figure 6). Our results suggest that ROS does not activate NF-κB in CHO K1 cells. Recently, Xie and Shaikh have shown that Cd-induced apoptosis involves suppression of NF-κB activity, which is mediated by oxidative stress (53). It has also been reported that manganese superoxide dismutase (MnSOD) activity is inhibited in rat liver and kidney 6 h after injection of Cd (2.5 mg/kg body weight) (54). MnSOD is the primary enzyme that protects cells from oxidative stress, and its expression is regulated by NF-κB (55). Possibly, Cd down-regulates the expression of MnSOD by inhibiting the basal activity of NF-κB and subsequently enhances ROS generation by a feedback mechanism. Results of our study can be concluded and summarized in Figure 8. Cd triggers necrosis but not apoptosis in CHO K1 cells. Ca2+ overload plays a critical role in initiating the necrotic cell death. Ca2+ subsequently activates calpain, a cysteine protease, and mediates ROS generation independently. Calpain activation further causes the mitochondrial depolarization. ROS suppresses basal NF-κB activity, which may influence the balance between cell survival and death. Both calpain- and ROSdependent pathways exert a cooperative effect to induce necrosis. Acknowledgment. This work was supported by Grants NSC93-2311-B-007-008 and NSC94-2311-B-007-004 from the National Science Council, Taiwan, Republic of China. We thank Dr. M. F. Tam for critical reading of the manuscript.

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Cadmium Induces Ca2+-Dependent Necrotic Cell Death through

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