Kheng Newick

Peroxiredoxin 3 Is a Redox-Dependent Target of Thiostrepton in Malignant Mesothelioma Cells

Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.

Non-specific DNA binding interferes with the efficient excision of oxidative lesions from chromatin by the human DNA glycosylase, NEIL1

Although DNA in eukaryotes is packaged in nucleosomes, it remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway, which begins with the excision of damaged bases by DNA glycosylases. We reported recently that the human DNA glycosylase hNTH1 (human Endonuclease III), a member of the HhH GpG superfamily of glycosylases, can excise thymine glycol lesions from nucleosomes without requiring or inducing nucleosome disruption; optimally oriented lesions are excised with an efficiency approaching that seen for naked DNA [1]. To determine if this property is shared by human DNA glycoylases in the Fpg/Nei family, we investigated the activity of NEIL1 on defined nucleosome substrates. We report here that the cellular concentrations and apparent k(cat)/K(M) ratios for hNTH1 and NEIL1 are similar. Additionally, after adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities toward nucleosome substrates. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. As a result, hNTH1 is able to excise both accessible and sterically occluded lesions from nucleosomes at physiological concentrations, while the high non-specific DNA affinity of NEIL1 would likely hinder its ability to process sterically occluded lesions in cells. These results suggest that, in vivo, NEIL1 functions either at nucleosome-free regions (such as those near replication forks) or with cofactors that limit its non-specific binding to DNA.

Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial‐derived oxidants and expression of FOXM1, a redox‐responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito‐carboxy‐proxyl (MCP) and Mito‐TEMPOL (MT) caused dose‐dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox‐responsive green fluorescent protein targeted to mitochondria (mito‐roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi‐1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM. J. Cell. Physiol. 228: 835–845, 2013.

Abstract LB-28: Regulation of FOXM1 by NADPH oxidases in malignant mesothelioma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnMalignant mesothelioma (MM) is linked to the occupational exposure to asbestos with an estimated median survival of less than two years; effective therapy is lacking. Due to long latency periods, incidence is expected to rise in regions where asbestos use has been banned, as well as where worker protection is lacking. MM cells exhibit changes in oxidant and energy metabolism, with elevated expression of the NADPH oxidase NOX4. We hypothesize that NOX4 produces reactive oxygen species (ROS) that promote uncontrolled growth through redox-dependent signaling to core cell cycle factors such as FOXM1 and FOXO3a, transcription factors involved in responses in oxidative stress and cell cycle progression. Targeted microarrays were used to assess the expression levels of 96 genes involving redox metabolism in four human MM cell lines and one control mesothelial line (LP9) immortalized with hTERT. Gene expression profiles from MM cell lines indicate tumorigenesis commonly results in elevated expression of NOX activity and adaptation to constitutive production of ROS, including up-regulation of antioxidant enzymes and cytoprotective factors. mRNA for FOXM1, a transcription factor that regulates the G2/M transition in the cell cycle, was up-regulated in all four MM cell lines, and FOXM1 continued to be expressed in MM cells, but not LP9 cells, in the absence of growth factors. Inhibitors of NOX activity markedly reduced expression of FOXM1, which correlated with dose-dependent effects on MM cell proliferation and viability. In the HM MM cell line, thiostrepton (TS), a thiazole antibiotic that inhibits the expression of FOXM1 via an unknown mechanism, increased the rate of oxidation of DCF, hyper-activated ERK1/2, and showed dose-dependent effects on MM cell viability in vitro and MM tumor growth in Fox Chase SCID mice. N-acetyl-L-cysteine blocked activation of ERK1/2 and cell death by TS, indicating the cytotoxic effects of TS in MM cells may be dependent on induction of intolerable levels of oxidative stress. In support of this possibility, inhibitors of p38 MAPK accentuated the cytotoxic effects of TS on cell viability. Our studies indicate that altered oxidant metabolism that supports expression of cell cycle factors such as FOXM1 is a common property of MM cells, and suggest that either inhibiting or accentuating the production of ROS in MM cells may be useful therapeutic approaches in the treatment of MM.nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation 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 LB-28.

Using BioTek's Synergy™ HT Reader to Measure Reactive Oxygen Species (ROS) Generation in Stimulated Cells

The investigation of biological agents that can stimulate or inhibit cell growth is of great interest to cellular biologists. One such measure is the formation of Reactive Oxygen Species (ROS) as a result of cell stimulation. While there are numerous methodologies to assess ROS species, the use of fluorescent probes has become the preferred method. Here we describe the measurement of DCF fluorescence using a SynergyTM HT to detect and analyze the production of ROS as a result of cell growth stimulation.