Activation of multiple proteolysis systems contributes to acute cadmium cytotoxicity.

Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.

Topoisomerase II-mediated DNA cleavage and mutagenesis activated by nitric oxide underlie the inflammation-associated tumorigenesis.

AIMS: Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species (RNOS) have been suggested to play a role in tumor pathology, particularly those activities associated with chronic inflammation. Here, we address the impact of nitric oxide (NO) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II (TOP2). We also investigate the contribution of NO to the formation of skin melanoma in mice. RESULTS: Similar to the TOP2-targeting drug, etoposide (VP-16), the NO-donor, S-nitrosoglutathione (GSNO), induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene (DMBA)-initiated mice. To explore the mechanism(s) underlying this NO-induced tumorigenesis, we use a co-culture model system to demonstrate that inflamed macrophages with inducible NO synthase (iNOS) expression cause γ-H2AX activation, p53 phosphorylation, and chromosome DNA breaks in the target cells. Inhibitor experiments revealed that NO and TOP2 isozymes are responsible for the above described cellular phenotypes. Notably, NO, unlike VP-16, preferentially induces the formation of TOP2ß cleavable complexes (TOP2ßcc) in cells. Moreover, GSNO induced TOP2-dependent DNA sequence rearrangements and cytotoxicity. Furthermore, the incidences of GSNO- and VP-16-induced skin melanomas were also observed to be lower in the skin-specific top2ß-knockout mice. Our results suggest that TOP2 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation. INNOVATION AND CONCLUSIONS: We provide the first experimental evidence for the functional role of TOP2 in NO-caused DNA damage, mutagenesis, and carcinogenesis. Notably, these studies contribute to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation.