No abstract

A variety of metal-binding compounds have been found to exert anti-cancer activity. We postulated that N-acetylcysteine (NAC), which is a membrane-permeable metal-binding compound, might have anti-cancer activity in the presence of metals. We found that NAC/Cu(II) significantly alters growth and induces apoptosis in human cancer lines, yet NAC/Zn(II) and NAC/Fe(III) do not. We further confirmed that this cytotoxicity of NAC/Cu(II) is attributed to reactive oxygen species (ROS). These findings indicate that the combination of Cu(II) and thiols generates cytotoxic ROS that induce apoptosis in cancer cells. They also indicate a fourth class of anti-neoplastic metal-binding compounds, the “ROS generator”.

No abstract

Docosahexaenoic acid (DHA; 22:6) is known to have anticancer activity, but its mechanisms of action remain to be further elucidated. We recently demonstrated that DHA downregulates superoxide dismutase (SOD) 1 gene expression, thereby weakening cellular antioxidant forces and enhancing cytotoxicity in various human cancer cells. The objective of this study was to investigate the mechanism of the inhibitory effect of DHA on SOD-1 gene expression in human cancer cells. A reporter gene assay indicated that DHA suppresses SOD-1 gene transcription in a time-and concentration-dependent manner in human cancer cells. Pretreatment with vitamin E did not block the inhibitory effect of DHA, indicating that this suppression does not depend on lipid peroxidation. The suppressive effect of DHA on SOD-1 gene transcription could be mimicked by the peroxisome proliferator-activator receptor (PPAR) ␣ ligand clofibrate but not the PPAR␥ ligand troglitazone, suggesting the involvement of PPAR␣ signaling. Deletion analysis of the key DNA binding elements in the SOD-1 gene promoter identified the distal hypoxia response element (HRE), but not the peroxisome proliferator response element or nuclear factor-B element, as essential for the suppressive effects of DHA. Coimmunoprecipitation confirmed that PPAR␣, but not PPAR␥, forms a complex with hypoxia-inducible factor (HIF)-2␣ in cancer cells. Chromatin immunoprecipitation analysis indicated that both DHA and clofibrate reduce HIF-2␣ binding to the HRE. Thus, we have identified the distal HRE in the SOD-1 gene promoter that mediates the suppression on the transcription of this gene by DHA, and we have demonstrated the involvement of PPAR␣ and HIF-2␣ signaling in this event.

Metal ionophores that transport copper or zinc into cells are toxic to cancer cells. Loading of metals into cells results in loss of protective intracellular thiols, which can contribute to cell death. We postulated that the metal-thiol interaction might also lead to the generation of cytotoxic compounds. We tested this hypothesis by adding N-acetylcysteine (NAC) to cancer cell lines in the presence of copper or zinc. NAC itself, at concentrations up to 10 mM, was not toxic to a variety of human cancer cell lines, including A2780 (ovarian), MCF-7 (breast), Panc-1 (pancreas), and A375 (melanoma). When added to cells in the presence of Cu++ (3-30 µM), NAC significantly enhanced the cytotoxicity induced by CuCl2 alone. The cytotoxicity of the NAC/copper combination was blocked by the copper-binding compounds tetrathiomolybdate and bathocuproinedisulfonic acid. NAC was not cytotoxic in the presence of zinc. This finding suggested that the redox activity of copper was an important determinant of cytotoxicity and raised the possibility that H2O2 was being generated. This was confirmed in vitro using a colorimetric assay (Biovision). The ability of other thiols to promote H2O2 generation when exposed to Cu++ was then examined. Reduced lipoic acid and L-cysteine generated higher level of H2O2 than NAC did; reduced glutathione was able to generate comparable levels of H2O2 as NAC, while oxidized glutathione generated significantly lower levels of H2O2; oxidized cysteine did not generate H2O2. To determine if H2O2 played a role in the observed cytotoxicity, catalase was added to cells treated with NAC/copper. Its addition completely blocked cell killing, indicating that significant amounts of extracellular H2O2 were being generated. These findings indicate that the interaction of copper with thiols leads to cytotoxic concentrations of H2O2. Thiols may differ in their reactivity with redox active compounds such as copper. Since the copper concentration of plasma is greater than that of tissue culture media, NAC's effects in vivo may differ from those seen in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4382.