Cadmium is a toxic heavy metal. Its wide use in industry is suspected to have widespread deleterious effects on human health through occupational and environmental exposure. The element contributes to pathogenesis of osteoporosis, non-hypertrophic emphysema, irreversible renal tubular injury, anemia, eosinophilia, anosmia and chronic rhinitis. In addition, cadmium is a potent human carcinogen and occupational exposure to the metal is associated with cancers of the lung, prostate, pancreas, and kidney . The biological half-life of cadmium in humans is estimated to be >25 years, thereby assuring the metal's accumulation in tissues during one's lifetime . Therefore, cadmium progressively accumulates in the human prostate with increasing age. Indeed, the prostate is an organ with one of the highest levels of cadmium [4, 5].
Multiple mechanisms of carcinogenesis for cadmium have been suggested. These include aberrant gene expression, inhibition of DNA damage repair, induction of oxidative stress, and inhibition of apoptosis . In contrast, some studies demonstrate that cadmium can inhibit the formation of chemically induced and spontaneously occurring tumors in animals when given at non-toxic concentrations [44, 45].
The present study provides the first evidence that cadmium induces depletion of XIAP in prostate caner cells at the post-transcriptional level via proteasome-mediated mechanisms. The inhibition or down-regulation of XIAP in cancer cells lowers the apoptotic threshold, thereby inducing cell death and/or enhancing the cytotoxic action of chemotherapeutic agents. Recent studies demonstrate that XIAP antagonist 1396-34 sensitizes PC-3 and DU-145 prostate cancer cells to chemotherapeutic agents and TRAIL . In accord with these reports, our data show that cadmium-mediated down-regulation of XIAP coincides with increased sensitivity of PC-3 prostate cancer cells to TNF-α-mediated apoptosis.
Cadmium induces generation of reactive oxygen species in target cells with subsequent mitochondrial damage . Mitochondrial collapse results in the release of various apoptogenic factors into the cytoplasm. These include IAP-binding proteins Smac/DIABLO and Omi/HtrA2 . Interestingly, both Smac/DIABLO and Omi/HtrA2 are capable of inducing caspase-independent degradation of IAPs including XIAP, cIAP1 and cIAP2 [49–51]. However, the potential involvement of Smac/DIABLO and Omi/HtrA2 in cadmium-mediated XIAP depletion could be excluded based on the findings that Smac/DIABLO selectively reduces the protein levels of cIAP1 and cIAP2 but not that of XIAP , whereas Omi/HtrA2 induces proteolytic cleavage of all IAPs (i.e. XIAP, cIAP1 and cIAP2) . Importantly, cadmium-mediated depletion of XIAP was selective, as cadmium had no effect on the levels of other members of the IAP family, namely cIAP1 and cIAP2 (Fig. 1A).
Selection of apoptosis-resistant cells is a potential mechanism for tumor progression. Results of our experiments reveal that pre-treatment with cadmium produces development of apoptosis-resistance in response to concomitant treatment with TNF-α and cadmium in prostate cancer cells (Fig. 5D). Development of apoptosis-resistance coincides with restoration of XIAP expression in cadmium-selected PC-3 cells. Interestingly, while some studies demonstrate that high levels of XIAP have an unfavorable prognosis in cancers of various tissue origins [21, 52], other data suggest that elevated XIAP levels are associated with a favorable clinical outcome [53, 54]. Likely, XIAP expression alone cannot serve as a predictive marker of chemoresistance. Given that tumorigenesis is a complex multifactorial process, expression levels and functional states of other critical pro- and anti-apoptotic molecules must be integrated for accurate prognostication. Recent studies by Seeger et al. demonstrate that the finely tuned balance between XIAP and its antagonists is critical in determining the clinical outcome in cancer patients .
A potential explanation for the development of cadmium-resistant phenotype in prostate cancer cells is increased expression of metallothioneins in response to cadmium treatment, resulting in the intracellular chelation of cadmium ions. Albrecht et al. demonstrated that exposure of the normal prostate cells to cadmium results in the rapid induction of the various metallothionein isoforms with eventual accumulation easily exceeding 10% of total cellular protein. Moreover, maximum accumulation of metallothioneins was detected on days 7-13 after the start of treatment .
Recent reports suggest that several metals may modulate XIAP integrity. For instance, elevated copper levels result in a conformational change in XIAP, which accelerates its degradation. Importantly, copper does not reduce XIAP mRNA expression . These data indicate that both cadmium and copper modulate XIAP expression at the post-transcriptional level. Despite these published reports in cell culture of other tissues, our experiments suggest that copper's effects on XIAP expression in prostate tissues are not as significant, since pre-treatment with copper had no effect on XIAP expression in prostate cancer cells (Fig. 4A).
Given cadmium's chemical similarity to zinc, a possibility exists that cadmium exchanges for zinc and leads to instability of the XIAP protein. The selective antagonism by zinc of the carcinogenic effect of cadmium suggests that zinc may act at a variety of important binding sites, including those that are potentially important for regulation of gene expression or of the enzyme's catalytic activity. Indeed, the ability of cadmium, to substitute zinc in the zinc finger domains and impair function of the wild-type zinc finger proteins has been demonstrated . Cadmium's substitution for zinc in the tumor suppressor protein, p53 alters p53 conformation and results in loss of DNA binding capacity and suppression of p53-dependent cell cycle arrest. Zinc supplementation, on the other hand, reactivates p53 and restores its tumor suppressive functions . Indeed, zinc and cadmium are the only two metal ions that appear to effect cellular XIAP levels. In contrast to cadmium, it is the depletion of zinc that leads to cellular reduction of XIAP . Nevertheless, in contrast to data available for p53, zinc supplementation in our hands failed to restore expression of XIAP in cells treated with cadmium (Fig. 4B).
XIAP is one of the NF-κB-regulated proteins . Nevertheless, our data suggest that suppression of XIAP expression by cadmium is likely an NF-κB-independent process. Our work shows that cadmium reduces XIAP levels even when the protein is expressed under an NF-κB-independent SV40 promoter. Instead, the mechanism for XIAP suppression may be proteasome-dependent.