TS inhibits FOXM1 expression through a redox-dependent mechanism.

<p>A) HM cells were treated with the indicated concentration of TS for 18 hr (lanes 1–5), or were pre-incubated with 1 mM NAC for 16 hr prior to exposure to TS (lanes 6–10). Cell extracts were examined for FOXM1 expression by immunoblotting as before. B) The indicated cell lines were incubated with 2.5 µM TS, or 2.5 µM TS and 1 µM of the proteosome inhibitor MG132, for 18 hr and FOXM1 expression was examined by immunoblotting of cell extracts. C) The indicated cell lines were incubated for 16 hr with or without 10 µM MG132 as indicated and cell lysates were examined for FOXM1 expression by immunoblotting. D) HM cells were incubated with the indicated concentration of TS for 18 hr and levels of phospho-ERK1/2 (pERK1/2) and total ERK1/2 were assessed by immunoblotting. To test sensitivity of induction of phospho-ERK1/2 by TS to NAC, cells were pre-incubated with 1 mM NAC overnight and then exposed to 5.0 µM TS as before (lane 6). HM cells express significantly higher levels of ERK2 versus ERK1. E) HM cells were treated with 10 µM TS, with or without pre-incubation with 1 mM NAC, for 8 hr and examined by phase-contrast microscopy. Cell rounding, membrane retraction and other early morphological changes induced by TS were attenuated by pre-incubating HM cells with NAC.</p

Human MMs express FOXM1.

<p>A) Paraffin-embedded sections from human MM tissue specimens were examined for FOXM1 expression using immunohistochemistry; shown are representative results for the indicated tumor types at a 1∶3000 dilution of primary antibody, which accentuates nuclear localization of FOXM1. No signal for FOXM1 was observed in normal human liver. B) Nuclear expression of FOXM1 was scored for the indicated tumor types, using 0 =  no positive nuclei, 1 = <5% positive nuclei, 2 = >5% and <50% positive nuclei, and 3 = >50% positive nuclei. Significant levels of nuclear FOXM1 expression were observed in all MM tumor types. There was no significant difference in nuclear FOXM1 expression between mesothelioma tumor types. C) Total RNA was extracted from normal mesothelial tissue (N) or mesothelioma (T) from four patients and examined for FOXM1 mRNA expression relative to HPRT using qRT-PCR. Using the Student’s t-test, relative expression (RQ) for FOXM1 transcript in tumor tissue was increased as compared to normal mesothelium (p  = 0.0017).</p

GV potentiates the cytotoxicity of TS in MM cells.

<p>A) HM cells were plated in duplicate in 96 well plates, and treated with either increasing concentrations of GV or TS alone, or 1.0 µM TS with increasing concentrations of GV. After 4 days cells were stained with crystal violet and total cellular mass was assessed by absorbance of methanol-soluble dye at 540 nM. Using the four parameter non-linear regression model in Gen5 software (BioTek Instruments), 1 µM TS with increasing [GV] was estimated to be approximately 3.3-fold more potent than increasing concentrations of GV alone. B) HM cells were plated in 96 well dishes and treated with the indicated concentrations of TS and GV, or with 0.05 µM GV with increasing concentrations of TS. After 4 days total cell mass was quantified by staining with crystal violet as above. A constant concentration of 0.05 µM GV with increasing [TS] was estimated to be approximately 175 times more potent than increasing concentrations of TS alone.</p

Enhanced mitochondrial superoxide production by MM cells.

<p>A) Equivalent numbers of LP9 and HM cells were deprived of serum for 72 hr, then incubated in medium with either low serum (0.25% FBS) or high serum (10% FBS) and loaded with the fluorescent reporter hydro-Cy3. After 30 min relative fluorescence was measured in triplicate; data are expressed as relative fluorescence units (RFU) +/− the standard error of the mean. B) LP9 mesothelial cells (row b) and HM MM cells (row c) were incubated with nitroblue tetrazolium (NBT) and MitoTracker Deep Red and imaged by confocal microscopy. Colocalization of the NBT and MitoTracker Deep Red signals indicated that the majority of superoxide in LP9 and HM cells is derived from mitochondria. Row a represents images of LP9 cells without staining with NBT. C) H2373 and HM MM cells and LP9 controls were loaded with MitoSOX Red for 30 min and analyzed by flow cytometry for mitochondrial superoxide production. D) The indicated cell lines were transfected with an expression vector for mitochondrial roGFP, a genetically-encoded reporter that is responsive to mitochondrial redox status. The relative redox status of the different cell lines was examined by ratiometric live cell imaging as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Hanson1" target="_blank">[67]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Lohman1" target="_blank">[68]</a> Higher 400∶495 ratios are indicative of an increased oxidation state in cellular mitochondria. E) Total RNA was prepared from LP9, HM and H2373 cells and relative levels of expression of PRX3 and TR2 mRNA in comparison to HPRT were determined by qRT-PCR. Data are plotted as fold-increase in the MM cell lines as compared to LP9. F) Cell extracts were prepared from LP9, HM and H2373 cells and assayed for total cellular thioredoxin reductase (TR) activity. Data are expressed as arbitrary units (AU) per 25 µg cell extract.</p

Expression of FOXM1 in LP9 mesothelial cells and MM cells lines.

<p>A) FOXM1 expression was examined by immunoblotting of cell extracts; note that LP9 expresses a larger isoform of FOXM1 than the MM cell lines. B) Total RNA was extracted from the indicated cell lines in log phase growth, and FOXM1 mRNA expression relative to HPRT (RQ) was determined by qRT-PCR. C) Isoform-specific RT-PCR was used to examine expression of FOXM1A, FOXM1B, and FOXM1C mRNA in the indicated cell lines. HM cells expressed increased levels of the oncogenic isoforms B and C compared to LP9 controls while levels of isoform A were relatively similar. D) LP9 mesothelial cells and HM, HP-1 and H2373 MM cells were incubated in medium containing 10% FBS (H) or medium containing 0.25% FBS (L) for 48 hours, and cell extracts were examined for FOXM1 expression by immunoblotting. Note that MM cells continue to express FOXM1 in the absence of mitogenic stimulation. E) HM cells were transfected with control siRNA (ct) and siRNA directed toward all FOXM1 splice variants (si), and 24 hr later cell extracts were examined for expression of FOXM1 by immunoblotting. Specific knockdown of FOXM1 mRNA resulted in the loss of all immunoreactive FOXM1 bands, validating the antibodies used in this study.</p

The electrophoretic mobility of PRX3 is modified by TS.

<p>A) HM cells were treated with the indicated concentration of TS for 18 hr, and cell extracts were prepared, heated in SDS sample buffer at 95°C for 10 min, resolved by gel electrophoresis, and PRX3 expression was assessed by immunoblotting. Treatment of HM cells with increasing doses of TS caused the formation of a modified species of PRX3 that migrated with an apparent molecular weight of about 45–50 kD. B) Recombinant human PRX3 (rPRX3) was incubated with 10 µM TS in solution in vitro using conditions described by Chiu and colleagues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Chiu1" target="_blank">[43]</a>, and samples were resolved by gel electrophoresis and examined by immunoblotting. Pre-treatment of rPRX3 with sodium cyanoborohydride (NaCNB) accentuated the reactivity of TS (lane 2) whereas pre-incubation with NAC inhibited it (lane 5). Addition of TS and N-ethyl-maleimide (NEM) or NAC to rPRX3 treated with NaCNB resulted in multiple species of immunoreactive PRX3 (lanes 3 and 4), whereas treatment with NACNB alone (lane 7) had no effect. C) HM cells were treated with the indicated concentration of TS for 18 hr, and cell extracts were prepared in the absence of reducing agents as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039404#pone.0039404-Phalen1" target="_blank">[69]</a>. Samples (20 µg protein/lane) were resolved under denaturing but not reducing conditions, and the relative distribution of PRX3 monomers to dimers was assessed by immunoblotting.</p

Cooperative effects of GV and TS on the TR2/TRX2/PRX3 antioxidant network.

<p>Superoxide from the electron transport and chain and other sources is converted by MnSOD to H₂O₂, which is then metabolized by PRX3 to water. Disruption of PRX3 activity by covalent adduction of cysteine residues by TS increases mitochondrial oxidant levels, which repress FOXM1 expression through unknown mechanisms. Inhibition of TRX2 expression by GV potentiates the activity of TS, increasing its cytotoxicity. Combinatorial approaches to inactivating the TR2/TRX2/PRX3 antioxidant network provide an approach for disabling an important adaptive response in MM, and may enhance the sensitivity of MM to conventional chemotherapeutic drugs.</p

GV accentuates modification of PRX3 by TS.

<p>A) HM cells were treated with the indicated concentration of GV for 18 hr, cell extracts were prepared, and expression of TRX2 and TR2 was assessed by immunoblotting. Actin was used as a loading control. B) HM cells were treated with the indicated concentration of GV for 16 hr, cell extracts were prepared and examined for FOXM1 expression by immunoblotting. Actin was used as a loading control. C) HM cells were treated with the indicated concentration of TS, TS plus GV, or TS after pre-incubation with either BSO or NAC. Cell extracts were prepared and assessed for PRX3 expression by immunoblotting as before. Actin was used as a loading control. D) HM cells were treated with the indicated concentration of TS, GV or TS plus GV for 18 hr, loaded with MitoSOX Red for 40 min, and examined by flow cytometry. Data are expressed as the log of the relative fluorescent signal (FL2) versus the number of cells. NT indicates no treatment. E) HM cells were treated with TS, GV or TS plus GV at the indicated concentrations, and modification of PRX3 was examined by immunoblotting as before. Note that under these conditions the electrophoretic mobility of the entire cellular pool of PRX3 was modified in cells treated with TS plus GV.</p

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma

<div><p>Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.</p></div

A catalytic intermediate of PRX3 is a molecular target of thiostrepton.

<p>(A) Superposition of human Prx2 and bovine Prx3. The monomers of the Prx2 and Prx3 dimer are shown in blue/light blue and green/light green, respectively. The sulfur atoms of the Cys residues are shown as spheres. (B) Proximity of Cys residues. All Cys residues are conserved. The residue numbers indicated are for human Prx2/Prx3. Distances shown are in Angstroms. PDB codes 1QMV and 1ZYE. Note that the C-terminus of Prx3 is disordered and not shown. (C) Model for TS adduction of Prx3. During the PRX3 reaction cycle the formation of a disulfide bond at one catalytic dyad promotes local unfolding. We propose that this conformation change favors adduction of Cys108 and Cys229 in the neighboring catalytic center by TS, leading to an irreducible, crosslinked PRX3 dimer and loss of peroxidase activity. Pro-oxidant compounds such as gentian violet, Mito-CP or arsenic trioxide and oxidative stress increase the level of PRX3 disulfide-bonded dimers and promote adduction by TS. Dimedone attacks sulfenic acid moieties and blocks disulfide formation, thereby blocking modification of PRX3. Similarly, mutant forms of PRX3 lacking the peroxidatic or resolving cysteines are not targets of TS.</p