* First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan;
National Cancer Institute at the National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709; and
Department of Health Science, Gunma University School of Medicine, Maebashi 371-8511, Japan
Received June 13, 2001; accepted September 12, 2001
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ABSTRACT |
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Key Words: cadmium; human; hepatoma; apoptosis; metallothionein; zinc; in vitro.
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INTRODUCTION |
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Recently, evidence has emerged that the process of carcinogenesis is often associated with alterations in apoptosis. In this regard, Cd induces apoptosis under some conditions (Achanzar et al., 2000; Habeebu et al., 1998
; Hart et al., 1999
) while it inhibits apoptosis in other cases (Shimada et al., 1998
; von Zglinicki et al., 1992
; Yuan et al., 2000
). For instance, Cd inhibits apoptosis induced by chromium, actinomycin D, and hygromycin B (Shimada et al., 1998
; Yuan et al., 2000
). Cd acts as an effective inhibitor of caspase-3 (Yuan et al., 2000
), a proapoptotic enzyme critical to dedication of cells to apoptosis (LaCasse et al., 1998
). Likewise, zinc (Zn) can be highly effective as a caspase-3 inhibitor (Perry et al., 1997
). In fact, it is suspected that free Zn may be a critical negative controlling factor for apoptosis (Chai et al., 1999
). Inhibition of apoptosis may allow a greater portion of genetically damaged cells to survive and escape normal cell population control mechanisms to go on to form tumors. On the other hand, Cd is also an effective inducer of apoptosis in vitro in a variety of human or rodent cell lines (Achanzar et al., 2000
; Hart et al., 1999
) and there is evidence of Cd-induced apoptosis in certain tissues with in vivo exposure (Habeebu et al., 1998
; Yan et al., 1997
). So it appears Cd can be both pro- and antiapoptotic, depending on the conditions. Likewise Zn, although often antiapoptotic, can also induce apoptosis (Hamatake et al., 2000
; Liang et al., 1999
; Souza et al., 1999
). For instance, Zn can induce apoptosis in human thyroid cell lines (Iitaka et al., 2001
), colorectal carcinoma cells (Souza et al., 1999
), Molt-4 cells (Hamatake et al., 2000
), and in human prostate cells (Liang et al., 1999
). So it is clear that both Cd and Zn can induce, as well as inhibit, apoptosis depending on the circumstances.
The metal-binding protein metallothionein (MT) is one important factor well known to alter Cd toxicity (Habeebu et al., 2000; Liu et al., 1995
, 2000
; Waalkes et al., 1999b
). In this regard, it is thought that MT typically mitigates Cd toxicity by sequestration of the metal, thereby rendering it toxicologically inert (Waalkes and Goering, 1990
). However, it is clear that, under most conditions, MT is a Zn-containing protein and that Cd binds to MT by the displacement of Zn (Waalkes, 1996
). In this regard there is evidence that Cd may inhibit apoptosis through displacement of cellular stores of Zn (Yuan et al., 2000
). It is also quite clear that MT-knockout animals show enhanced rates of apoptosis when exposed to Cd (Habeebu et al., 2000
), and various studies have found MT to be generally antiapoptotic (Levadoux-Martin et al., 2001
; Wang et al., 2001
). On the other hand, exposure to the Cd-MT complex can, itself, induce apoptosis under some circumstances (Hamada et al., 1996
; Ishido et al., 1998
). High levels of MT can also be associated with stimulation of apoptosis under some conditions (Liu et al., 2001
; Deng et al., 1998
) and apoptotic rate increases with increasing tumor immunoreactivity for MT in some tumors (Zhang and Takenaka, 1998
). Thus, MT appears to similarly have both pro- and antiapoptotic potential.
It is not clear how cellular MT levels would affect apoptosis induced by Cd, and there are several possible scenarios, including: (1) suppression of Cd-induced apoptosis by MT sequestration of Cd; (2) Cd displacement of Zn from MT, which in turn, stimulates or suppresses apoptosis; or (3) Cd-MT stimulation of apoptosis. We hypothesize that the presence of cellular MT will reduce Cd-induced apoptosis. Therefore, to test this hypothesis, the relationship between the amount of cellular MT and the toxic and apoptotic effects of Cd was investigated in 3 cell lines of varying relative endogenous MT levels, including Chang cells (high), HepG2 cells (intermediate) and PLC/PRF/5 cells (low). Effects of MT induction by Zn prior to Cd exposure were also studied.
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MATERIALS AND METHODS |
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Cell lines and culture conditions.
Two human hepatoma cell lines (PLC/PRF/5 and HepG2) and the Chang cell line (likely a Hela cell-derived line) were used. Chang cells, PLC/PRF/5 cells and HepG2 cells were purchased from ATCC (Manassas, VA). These cell lines were selected because in preliminary testing they showed wide variability of sensitivity to Cd and corresponding differences in basal MT levels that showed a 4-fold range. Cells were maintained in a 5% CO2 atmosphere, and cultured as monolayer in Dulbecco's modified Eagle's medium (DMEM), GIBCO-Life Technologies (Gaithersburg, MD), supplemented with 10% fetal bovine serum, 31 U/ml penicillin G and 50 µg/ml streptomycin.
Measurement of cytolethality.
The effect of Cd on cell viability was assessed by an MTT assay, which measures viable cells by assessing metabolic integrity (Mosmann, 1983). Cells were seeded in 96-well microtiter plates (Nunc, Naperville, IL) at a cell density of 2 x 104 cells/well in unaltered media. After 24 h, the medium was replaced with media containing various concentration of Cd, (0, 5, 10, 20, 40, 80, and 120 µM). Cells were exposed to Cd for designated periods of time (3, 6, 12, and 24 h). Cell survival rates were then assayed.
MT quantitation.
MT concentrations were estimated by the Cd-hemoglobin radioassay method as described by Eaton and Toal (1982). MT was measured in untreated cells or in cells treated with Cd or Zn, at the designated concentrations, for 24 h prior to evaluation. Endogenous Zn and Cd content of MT was determined by using this assay in the absence of exogenously added Cd, thus allowing direct measurement of the Cd or Zn compliment in MT by using atomic absorption spectrometry.
Assessment of apoptosis by agarose gel electrophoresis.
Cells (4 x 106) were incubated with 0, 10, or 40 µM of Cd for 16 h. Attached and unattached cells were harvested and DNA was extracted with 0.5% triton-X100 cell lysis buffer. Small molecular weight DNA was obtained in the supernatant after centrifugation. DNA was electrophoresed on 2% agarose gel to detect the laddering characteristic of apoptotic DNA degradation.
Quantitation of apoptosis by DNA fragmentation detection assay (ELISA).
Quantitative determination of cytoplasmic histone-DNA fragments indicative of apoptosis was performed by enzyme-linked immunosorbent assay (ELISA) using the Cell Death Detection ELISA kit (Roche, Indianapolis, IN). The assay is based on a quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively, which allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Cells were seeded in a 96-well plate (1 x 104/well) and treated with designated concentrations of Cd for 12 to 16 h. Floating cells were first pelleted by centrifugation, and then both adherent and floating cells were lysed and incubated with mouse monoclonal antihistone-biotin antibody and mouse monoclonal anti-DNA-peroxidase in streptavidin-coated microtiter plates. Unbound antibodies were washed out. The amount of nucleosome was determined quantitatively by evaluating peroxidase activity photometrically with 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) as substrate.
Statistics.
Data are given as the mean ± SEM unless otherwise noted. Results were analyzed by Student's t-test or Dunnett's t-test after ANOVA as appropriate. Correlation between apoptotic rates and basal or induced-MT concentrations was tested by calculating Pearson's correlation coefficient, r. The level of significance was set at p < 0.05 in all cases.
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RESULTS |
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DISCUSSION |
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The sequestration of Cd by MT usually occurs through the displacement of bound Zn (Waalkes, 1996), and this was observed in the present study, at least in the HepG2 cells. Zn can be proapoptotic in many cases (Hamatake et al., 2000
; Liang et al., 1999
; Souza et al., 1999
). Thus, MT sequestration of Cd and consequential Zn release might enhance apoptosis through Zn-mediated pathways. In fact, Liu et al. (2001) have shown that metal released from MT can go on to induce apoptosis, using copper-MT and a nitric oxide producing agent to release the MT bound metal, clearly showing the plausibility of such a scenario with Zn. However, the present results indicate this is probably not the case with released Zn, as the Zn released from MT during Cd sequestration did not appear to enhance apoptosis, at least not in the 3 cell lines used in this study. The role that Zn released from MT might play in stimulating apoptosis in other cell lines remains to be determined, but in the present series of experiments, release of Zn from this pool did not appear to substantially increase apoptosis.
Exposure to the Cd-MT complex can induce apoptosis in some instances (Hamatake et al., 1996; Ishido et al., 1998; 1999
; Liu et al., 1998
). In in vivo systems, Cd-MT exposure induces acute nephropathy and renal apoptosis (Ishido et al., 1998
, 1999
; Liu et al., 1998
). However, it is thought that, in these cases, MT acts largely as a carrier for Cd that causes high levels to be deposited in specific renal cells (Liu et al., 1998
). It appears the Cd-MT complex is taken up by renal cells, but it is then degraded to release locally high levels of Cd (Liu et al., 1998
). It is the released Cd that likely induces the apoptosis, as exposure of renal cells to the Cd-MT complex in vitro does not produce apoptosis, pointing toward the Cd ion as the causative factor (Ishido et al., 1999
). Indeed, the differential induction of Cd-MT seen in the cell lines used in the present study was not correlated with increased apoptosis. In fact, the inverse relationship occurred. Thus it appears likely that the intracellular Cd-MT complex does not stimulate apoptosis.
The availability of MT probably dictates Cd-induced apoptosis in a variety of circumstances. In this regard, hepatocellular tumors (Waalkes et al., 1991, 1993
) and cell lines derived from hepatocellular tumors (present study) appear particularly sensitive to Cd. There appears to be a down-regulation of MT expression in human and murine liver tumors, while surrounding normal tissue has much higher MT levels (Ghoshal et al., 2000
; Onosaka et al., 1986
; Waalkes et al., 1996
). Similar results are seen with murine lung tumors, which poorly express MT (Waalkes et al., 1991
, 1993
, 1996
). The poor expression of MT appears to account for relative sensitivity of the hepatocellular carcinoma cell lines to Cd-induced apoptosis in the present study. In this regard, Cd can effectively and selectively destroy liver and lung tumors in mice (Waalkes et al., 1991
, 1993
), and inhibit growth and metastasis of human lung carcinoma xenografts implanted into mice (Waalkes and Diwan, 1999
). Thus, the antitumor effects of Cd appear to be dictated by MT expression in tumors (Waalkes et al., 1991
, 1993
, 1996
) and the present results show hepatoma-derived tumor cells appear more sensitive to Cd-induced apoptosis. Induction of tumor cell apoptosis is a very common mode of action for cancer chemotherapeutics (Martin and Green, 1994
).
In summary, MT appears to mitigate Cd-induced apoptosis regardless of the precise circumstances. The relative expression of MT may well be predictive of the ability of Cd to kill cells, including tumor cells, through stimulation of apoptosis.
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ACKNOWLEDGMENTS |
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NOTES |
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