Apoptosis Induced by Cadmium in Human Lymphoma U937 Cells through Ca2+-calpain and Caspase-Mitochondria- dependent Pathways*

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Δψm) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-xL and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 μm), a voltage-dependent Ca2+ channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 μm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP3R) but not type 2 or type 3 IP3R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP3R or of calpain inhibitors on the loss of Δψm were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca2+-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Recently, several reports have shown that cadmium can induce apoptosis of many tissues and cells both in vivo and in vitro, such as the cells of the respiratory system (3), the testis (4 -6), the kidney (7,8), the liver (9), and the immune system (10,11), etc. This evidence indicates that apoptosis probably plays a very important role in acute and chronic intoxication with cadmium. Further research on this aspect of cadmium would have significance in the prevention and cure of the diseases induced by cadmium.
Apoptosis is a fundamental form of cell death, and it plays an essential role in the development and homeostasis of multicellular organisms. Apoptosis disorders are associated with many diseases, such as cancer, autoimmune disorders, neurodegenerative disease, toxin-induced disease, etc. (12). In the apoptotic process, caspases, a family of asparate proteases, lie in a pivotal position (13). Two independent pathways of apical caspases activation, receptor-intermediated caspase 8 and mitochondria-cytochrome c intermediated caspase 9 activation, converge on the activation of executing caspases, key substrate cleavage, and apoptotic death (14).
Intracellular Ca 2ϩ homeostasis is very important in maintaining the normal function of the cell. Increases and decreases in calcium ion are possible causes of apoptosis (15,16). The radius of Cd 2ϩ , a common form of free cadmium in the body, is very similar to that of Ca 2ϩ (0.099 and 0.097 nm, respectively). Cd 2ϩ not only competitively inhibits the influx of Ca 2ϩ (17) but also causes the increase of [Ca 2ϩ ] i through inhibiting Ca 2ϩ -ATPase on the membrane of the depot of calcium (18). In addition, cadmium can activate or inhibit some calcium-related enzymes instead of Ca 2ϩ (19). Thus, investigation on the role of interactions of calcium ions in cadmium-induced apoptosis is important for understanding the mechanism of toxicity of cadmium.
In this study, the molecular mechanism of apoptosis induced by cadmium in human histiocytic lymphoma U937 cells, which possess the Ca 2ϩ /Mg 2ϩ -dependent endonuclease (20), was investigated. We herein show that two independent pathways, the Ca 2ϩ -calpain pathway and the caspase-mitochondria pathway, are probably involved in the regulation of cadmium-induced apoptosis.

MATERIALS AND METHODS
Reagent-Cadmium chloride, verapamil, 3,3Ј-dihexyloxacarbocyanine iodide (DiOC 6 (3)), 1 dichlorodihydrofluorescein diacetate, and dihy-* This work was supported by the Nishiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Culture and Treatment-U937 cells obtained from the Japanese Cancer Resource Bank were grown in RPMI 1640 culture medium containing 10% heat-inactivated fetal bovine serum. Apoptosis in U937 cells was induced with cadmium chloride. Some cells were pretreated with caspase inhibitors (100 M) or calpain inhibitor I and II (20 and 50 M) for 2 h prior to exposure to cadmium.
Assessment of Apoptosis-Quantitative DNA fragmentation assay was carried out according to the method of Sellins and Cohen (21). The formation of DNA ladder was examined in 1.2% agarose gel electrophoresis (22). Cell morphology was examined by Giemsa staining. Phosphatidylserine (PS) externalization of apoptosis was determined by two-color analysis of Annexin V/FITC binding and propidium iodide (PI) uptake using flow cytometry (EPICS XL TM Coulter, Beckman) according to the instructions of the manufacturer.
Change in Ca 2ϩ /Cd 2ϩ Intracellular Concentration-After treatment for 6 h, cells were collected by centrifugation and washed with Ca 2ϩ -free HEPES-buffered Ringer solution (HR; pH 7.4, 118 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl 2 , 1.0 mM Na 2 HPO 4 , 5.5 mM glucose, and 10 mM HEPES). The buffer was supplemented with 0.2% bovine serum albumin Fraction V, 2% minimal Eagle's essential amino acids, and 2 mM L-glutamine. About 3 ϫ 10 5 cells in 1 ml of HR were loaded with Fura-2/AM for 30 min at 37°C. Then an aliquot of cell suspension (10 l) was transferred to a glass bottom dish coated with Cell-Tak and left for 10 min. After addition of HR to the chamber, digital imaging by Fura-2 fluorescence (ratio of 340 nm/380 nm at 510 nm) was carried out with an inverted microscope (TE300; Nikon) and a digital image processor (Argus 50 CA; Hamamatsu Photonics, Japan) (23).
Mitochondrial Trans-membrane Potential (⌬ m ) Assay-After treatment with cadmium, cells were stained with 40 nM DiOC 6 (3) in 1 ml of phosphate-buffered saline and 1% fetal bovine serum for 15 min at 37°C, then washed twice with phosphate-buffered saline, and harvested for flow cytometry (24).
Western Blot Analyses of Proteins-Control and Cd 2ϩ -treated cells were collected and washed with cold phosphate-buffered saline. Cells were lysed at a density of 10 6 cells/50 l of lysis buffer (1 M Tris-HCl, 5 M NaCl, 1% Nonidet P-40 (v/v), 1% sodium deoxycholate, 0.05% SDS, 1 mM phenylmethylsulfonyl fluoride) for 20 min. After brief sonication, the lysates were centrifuged at 12,000 rpm for 10 min at 4°C, and protein content in the supernatant was measured using a Bio-Rad protein system. Western blot analyses of Bcl-2, Bcl-x L , Bax, Bid, Bad, IP 3 Rs 1, 2, and 3, and ␤-actin were performed using specific polyclonal or monoclonal antibodies (see above), as described previously (25).

Induction of Apoptosis by Cadmium-
The results of examinations of DNA fragmentation, DNA ladder formation, observation of cell morphology, and PS externalization consistently revealed that cadmium induced apoptosis in U937 cells. Nonrandom DNA fragmentation has been regarded as one of the typical end points of apoptosis (26). Fig. 1A illustrates that DNA fragmentation induced by cadmium was concentrationdependent and time-dependent; DNA fragmentation increased with increasing concentrations of cadmium up to 100 M after 6, 12, and 24 h of exposure to cadmium but decreased beyond 100 M. However, no DNA fragmentation increased after 3 h of treatment. Furthermore, DNA fragmentation culminated 12 h after treatment with cadmium. Thus, 100 M cadmium and 12 h of treatment were often used in this study. After cadmium treatment for 12 h, observation of DNA ladder formation and morphological changes supported the evidence of DNA fragmentation (Figs. 1B and 2, C and D). The observation of cell morphology illustrated that cadmium-treated cells underwent prominent cytoplasmic aggregation, nucleatic condensation, and fragmentation, which were typical signs of apoptotic features. Morphological change (including PI staining) showed that at concentrations of more than 100 M, the number of necrotic cells apparently increased (data not shown).
PS externalization is an early symbol of apoptosis (27). Flow cytometry using Annexin V/FITC and PI double staining revealed that after exposure to cadmium, cells with externalized PS significantly increased depending on cadmium concentration and exposure time. At 6 h, early apoptotic cells (only Annexin V/FITC staining) were predominantly observed; as time progressed or as the concentration increased, secondary necrotic cells in a later stage of apoptosis (double Annexin/ FITC V and PI staining) were in the main position. These results indicate that at later stages of apoptosis, the plasma membrane was damaged by cadmium (Fig. 2, A and B).
Elevation of Ca 2ϩ /Cd 2ϩ Concentration in Cells-To eluci- date the effect of the interaction between Ca 2ϩ and Cd 2ϩ in apoptosis induced by cadmium, we used Fura-2, which is derived from Fura-2/AM through esterase in cells, to determine the change in ion concentration of the cells. Fluorescence in cells treated for 6 h with cadmium became much stronger than that in control cells, and this change showed a dose-effect relationship. Unfortunately, the excitation response of Fura-2 to Cd 2ϩ is almost an exact match to that of Fura-2 to Ca 2ϩ . Therefore, it is difficult to distinguish Ca 2ϩ from Cd 2ϩ (28).
Verapamil, a voltage-dependent Ca 2ϩ channel inhibitor (10), reduced fluorescence of Fura-2 in cells; moreover, 70 M verapamil delayed DNA fragmentation induced by cadmium. These results indicated that cadmium probably entered into U937 cell via voltage-dependent calcium channels, and verapamil inhibited this passage, resulting in delayed DNA fragmentation (Fig. 3). After combined treatment of cadmium and verapamil, the increased fluorescence (cells indicating a ratio higher than 1.6) appeared to indicate intracellular Ca 2ϩ release induced by cadmium, because verapamil blocks both Ca 2ϩ and Cd 2ϩ entry into the cells.
To further determine the contribution of [Ca 2ϩ ] i to the increase in ion concentration, we used a cell-permeable, specific Cd 2ϩ chelator (TPEN, Cd 2ϩ K a ϭ 10 16.33 ; Ca 2ϩ K a ϭ 10 4.47 ), and fluorescence in cells significantly decreased, but it did not completely reverse the fluorescence of Fura-2 in cells, even though the concentration of TPEN increased to 100 M. These results suggest that in addition to augmentation of [Cd 2ϩ ] i , [Ca 2ϩ ] i was probably elevated (Fig. 4A). (18), the mechanism of [Ca 2ϩ ] i increase in cells exposed to cadmium has not been elucidated. In some apoptotic systems, elevation of [Ca 2ϩ ] i was mediated through IP 3 Rs (25,29). In our study, therefore, the expression of IP 3 R subtypes was investigated at 12 h after cadmium treatment (Fig. 4B). The expression of IP 3 R1 was apparently enhanced in a dose-dependent manner, but IP 3 R2 and IP 3 R3 were not detected. These results demonstrated that, probably via the IP 3 R1 pathway, cadmium induced the release of calcium into the cytoplasm from its intracellular depot, such as endoplasmic reticulum. In addition, caspase inhibitor (Z-VAD-FMK) did not affect the expression of IP 3 R1.

IP 3 R Expression and Effects of Calpain Inhibitor-Because cadmium competitively inhibits Ca 2ϩ influx
Calcium ion can act on multiple targets to trigger apoptosis (14). Recently, calpain, calcium-dependent protease has been considered as a possible target through which elevated calcium triggers apoptosis (30). Calpain inhibitor I significantly reduced DNA fragmentation by 40% at low concentrations, and there was little difference between 20 and 50 M. However, calpain inhibitor II was not effective until its concentration reached 50 M. The results, therefore, indicate that calpain may take part in the Ca 2ϩ -dependent pathway of apoptosis induced by cadmium (see Fig. 6A).
Loss of Mitochondrial Membrane Potential Induced by Cadmium-In many systems, apoptosis is associated with the loss of mitochondrial inner membrane potential (⌬ m ), which may be regarded as a limiting factor in the apoptotic pathway (31). To observe the change in ⌬ m in cells exposed to cadmium, DiOC 6 (3), a mitochondria-specific and voltage-dependent dye, was employed. After U937 cells were exposed to cadmium for 12 h, ⌬ m was significantly reduced in a dose-dependent manner (Fig. 5C). However, at 3 and 6 h, the loss of ⌬ m was indiscernible. Although it has been reported that cadmium was able to cause the loss of ⌬ m via oxidant injury (32,33), no significant increase in intracellular oxidants, such as hydrogen peroxide or superoxide, in the cadmium-treated cells was observed by flow cytometry with dichlorodihydrofluorescein diacetate and dihydroethidium in our study (data not shown).

Inhibition of Caspase Inhibitors in Cadmium-induced Apo-
ptosis-Because of the loss of mitochondrial membrane potential in the cadmium-treated cells, it was speculated that caspases may play an essential role in the process of cadmiumtriggered apoptosis. First, Z-VAD-FMK (caspase inhibitor I) and Boc-D-FMK (caspase inhibitor III), two kinds of broad spectrum, irreversible caspase inhibitors, were used. Z-VAD-FMK almost completely inhibited cadmium-induced DNA fragmentation (Fig. 6B); Boc-D-FMK also showed a similar result (data not shown). Moreover, Z-VAD-FMK also inhibited the loss of ⌬ m . These results suggest that cadmium probably triggers apoptosis of U937 cells via a caspase-dependent pathway.
Expression of Apoptosis-related Protein-Cleavage of Bid is important for the release of cytochrome c from mitochondria in CD95-induced apoptosis (34). Bid was present as a ϳ26-kDa protein in U937 cells (Fig. 7A). The proform of Bid slowly decreased depending on the cadmium concentration. At the same time, caspase inhibitor (Z-VAD-FMK) and caspase-8 in-hibitor (Z-IETD-FMK) both inhibited the cleavage of Bid induced by 100 M cadmium.
A member of the Bcl-2 family, Bcl-x L , significantly decreased in a concentration-dependent manner, and at the same time, Bcl-x S , which is the cleaving product of Bcl-x L, slowly appeared as Bcl-x L was reduced . It has been reported that Bcl-x L and Bcl-x S have completely contrary effects; the former inhibits but the latter promotes apoptosis (35) (Fig. 7B). This evidence is consistent with our results. Caspase inhibitor (Z-VAD-FMK) and caspase-8 inhibitor (Z-IETD-FMK) did not affect the expression of Bcl-x L . In contrast, no change in the expression of Bcl-2/Bax proteins was apparent in cadmium-induced apoptosis in U937 cells (Fig. 7C), although Bcl-2/Bax has been regarded as the regulator of the release of cytochrome c in apo- ptosis (36). Bad expression was not detectable in U937 cells treated with cadmium, although Bcl-x L can counteract the effect of Bax (37) and be sequestered by Bad in cytosol (38). DISCUSSION The present study shows that cadmium is able to induce apoptosis in U937 cells, and in this process, there may be two different and independent pathways for inducing apoptosis by cadmium. One is the Ca 2ϩ -calpain-dependent pathway, and the other is the caspase-dependent pathway.
The Ca 2ϩ -Calpain-dependent Pathway-In recent years, the effect of IP 3 R in apoptosis has been the focus of attention. The elevation of and selective expression of IP 3 R subtype involved in apoptosis are due to many stimuli, such as anti-immunoglobulin M, dexamethasone (29), irradiation (25), B-cell antigen receptor (39), etc. On the other hand, cells with failure in Ca 2ϩ elevation owing to deficient IP 3 R1 or IP 3 R2 are resistant to apoptosis by dexamethasone, irradiation, or Fas ligand in U937 cells (40). Our study reveals that cadmium is able to up-regulate the expression of IP 3 R1 but not IP 3 R2 or IP 3 R3 in U937 cells. It has been reported that a majority of IP 3 R1 was distributed on the surface of endoplasmic reticulum (41). Hence, because of competitive inhibition of Ca 2ϩ influx via the calcium channel and the expression of IP 3 R1 in U937 cells, the elevation of calcium ion caused by cadmium possibly derives from endoplasmic reticulum. Verapamil delayed cadmium-induced DNA fragmentation, probably because it inhibited the Cd 2ϩ influx via the Ca 2ϩ channel so that the IP 3 R1 up-regulation was extended. Of course, the possibility should not be excluded that cadmium in cells may directly mobilize Ca 2ϩ or enhance Ca 2ϩ mobilization, because cadmium can inhibit all types of Ca 2ϩ -ATPase (19,42,43).
Calpain is a family of Ca 2ϩ -dependent cystein proteases whose members are expressed ubiquitously (44). Calpain has been reported to be involved in several models of apoptosis and to take effect as a target of Ca 2ϩ -dependent activation (45,46). Presumably, calpain plays an essential role in apoptosis because of cadmium, because calpain inhibitors inhibited the DNA fragmentation induced by cadmium in our study. Furthermore, calpain inhibitor I was more potent than calpain inhibitor II. In addition, in addition to calpain, calcium ions are able to activate other targets to trigger apoptosis, such as Ca 2ϩ /Mg 2ϩ -dependent endonuclease, whose involvement has been shown in UV-induced apoptosis in U937 cells.
On the other hand, the other effects of cadmium in cells should be considered. Cd 2ϩ not only causes Ca 2ϩ elevation but also activates some calcium-related enzymes, for example, protein kinase C (47), mitogen-activated protein kinase (48), calmodulin-dependent kinase (49), etc. Hence, further research is necessary to determine whether cadmium can directly activate apoptotic proteases (calpain, calcium-dependent endonuclease, etc.) instead of calcium.
The Caspase-dependent Pathway-The caspase family is divided into two groups: one group, derived from procaspase with long predomains (caspase-2, -8, -9, and -10), is called "initiator" or "upstream" caspases, and the other, which is derived from precursor with short predomains is called "effector" or "downstream" caspases (caspase-3, -6, -7, and -14) (50). In our study, Z-VAD-FMK (broad spectrum inhibitor) inhibited DNA fragmentation and PS externalization induced by cadmium, suggesting that caspase may be involved in the process of cadmium-triggered apoptosis. Furthermore, caspase-8 and -3 inhibitors also inhibit cadmium-induced DNA fragmentation, but caspase-2, -6, -7, and -9 inhibitors do not. Therefore, we hypothesize that probably caspase-8 is the most apical caspase in cadmium-induced apoptosis, and finally signal converges to caspase-3. According to previous reports, caspase-8 not only directly cleaves and activates caspase-3 (51) but also indirectly activates caspase-3 by inducing cytochrome c release (52). According to observations, the loss of ⌬ m because of cadmium seems to support this evidence.
Bid, a proapoptotic Bcl-2 family member containing BH 3 domain, can be cleaved by caspase-8, and the cleaved Bid, the carboxyl-terminal fragment, translocates to mitochondria to induce the release of cytochrome c, which is 500 times more numerous than Bax (53). In our study, the decrease in the proform of Bid means that it is cleaved in cadmium-induced apoptosis, and Z-VAD-FMK and caspase-8 inhibitor block this cleavage. This suggests that the cleavage of Bid is caspase-8-dependent.
Besides Bid, other Bcl-2 family members are associated with the release of cytochrome c. For example, Bax promotes cyto- chrome c release, but Bcl-2 and Bcl-x L counteract the effect of Bax and inhibit the release of cytochrome c (37,54). Moreover, Bcl-x L can itself bind to cytochrome c and Apaf-1 to prevent apoptosis (55,56). In cadmium-induced apoptosis, the change in Bcl-2 and Bax is not apparent, but the level of Bcl-x L apparently decreases in a concentration-dependent manner, and Bclx S , which is the product of Bcl-x L cleavage and which promotes apoptosis (36), increases along with the decrease in Bcl-x L . Bad, another Bcl-2 family members that sequesters Bcl-x L , was not detected in U937 cells. Taken together, these results indicate that Bid and Bcl-x L are possibly involved in the caspasedependent pathway of cadmium-induced apoptosis, resulting in cytochrome c release and enhancement of the apoptotic process.
Although no reports have been published showing the activation of caspase-8 induced by cadmium, it is speculated that cadmium may activate caspase-8 by elevating the expression of Fas ligand. This is not only because caspase-8 is a key component of the Fas/APO 1 death receptor-triggered apoptosis pathway (53), but also because many other apoptotic pathways initiated by distinct stimuli require Fas engagement. For instance, cell apoptosis triggered by anticancer drugs (57)(58)(59), irradiation (60) and ceramide (61) is mediated by up-regulation of Fas L and its interaction with Fas. Of course, we cannot exclude the possibility that cadmium activates caspase-8 via a pathway independent of Fas L, such as activated Lck (62). Further study is necessary to elucidate the mechanism.
In summary, possibly via two different pathways, cadmium induces apoptosis in U937 cells (Fig. 8). In addition, because caspase degrades calpastatin, which is an endogenous inhibitor of calpain (63), and promotes the Ca 2ϩ -calpain pathway and because the Bcl-2 family and Ca 2ϩ act on each other (64), they may complement each other. FIG. 8. Scheme of the cell signaling pathway mediating the Cd 2؉ -induced apoptosis. Cd 2ϩ enters into U937 cells through the voltage-dependent Ca 2ϩ channel and up-regulates IP 3 R1 expression, and then Ca 2ϩ release from endoplasmic reticulum (ER) is induced. Ca 2ϩ activates calpain and induces DNA fragmentation and apoptosis. On the other hand, cadmium can possibly activate caspase-8 to induce apoptosis. Caspase-8 may directly activate the effector caspases (caspase-3 and -7) responsible for many of the biochemical and morphological changes in apoptosis. Alternatively, cadmium can cause loss of the ⌬ m , cleavage of Bid and Bcl-x L , and increase of Bcl-x S . These factors are probably responsible for the release of cytochrome c and activation of caspase-9, with subsequent activation of effector caspases to induce apoptosis. Therefore, two independent pathways, the Ca 2ϩcalpain pathway and the caspase-mitochondria pathway, may be involved in the regulation of cadmium-induced apoptosis.