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

PRX3 turnover promotes adduction of specific cysteine residues by thiostrepton in cells.

<p>(A) Reconstitution of the PRX3 catalytic cycle <i>in vitro</i> with purified components. (B) MM cells were treated with 5 μM TS for 18 hr or pre-incubated with 1 μM GV for 6 hr then treated with 5 μM TS (G/T) for 18 hr and immunoblotted for PRX3 after reducing SDS-PAGE. (C) MM cells were pre-incubated with 1 μM GV for the indicated times and then treated with 5 μM TS for 1 hr and cell lysates were immunoblotted for PRX3 after reducing SDS-PAGE. (D) Pre-incubation of MM cells with dimedone (Dim) for 6 hr blocked TS induced modification of PRX3. (E) HM Cells transfected with Flag-Tagged PRX3 expression plasmids were treated with 5 μM TS, lysates were collected at the indicated time points and TS induced modifications of PRX3 were visualized by immunoblotting with anti-PRX3 antibody after separation by reducing SDS-PAGE. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127310#pone.0127310.s001" target="_blank">S1 Fig</a>.</p

Adduction of PRX3 by TS correlates with cytotoxicity.

<p>(A) Human primary mesothelial, immortalized LP9 mesothelial, and HM and H2373 mesothelioma cell lines were incubated with 5 μM TS and lysates were collected at indicated time points over 24 hr. Formation of TS induced PRX3 dimers was visualized by reducing SDS-PAGE and immunoblotting with anti-PRX3 antibody. (B) Cell lines from (A) were incubated with indicated concentrations of TS (left) or GV (right) for 24 hr and total cell mass was determined by staining with crystal violet (Y axis values were normalized to untreated cells). The EC_<b>50</b> values for the indicated cell lines to TS or GV and the relative potency (REP) of TS and GV, as compared to primary mesothelial cells, are shown. (C) HM cells were treated with increasing concentrations of TS, GV, or TS + GV for 18 hr and extracts were resolved by reducing SDS-PAGE. PRX3 modification was assessed by immunoblotting as before. Note that GV accentuates modification of PRX3 by TS by blocking the activity of TRX2. (D) Extracts from cells treated as in panel C were resolved by non-reducing SDS-PAGE and PRX3 monomers (~23 kD) and disulfide-bonded dimers (~46 kD) were assessed by immunoblotting for PRX3. Note that GV markedly increased the level of disulfide-bonded PRX3 dimers, an indication of severe mitochondrial oxidative stress. (E) HMC2 and (F) HMC3 human primary mesothelial cell cultures were incubated with increasing doses of TS, GV or TS + GV for 18 hours, and assessed for PRX3 expression after reducing SDS-PAGE by immunoblotting. The formation of the modified species of PRX3 in response to TS was less evident in primary mesothelial cells.</p

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

shPRX3 cells are less sensitive to TS than WT MM cells.

<p>(A) PRX3 expression in cells treated with scrambled control (Sc) or PRX3 siRNA for 48 and 72 hr. (B) Cell morphology and density of cells as treated in panel A after 72 hr. (C) Cell number in HMshPRX3 cells as compared to controls over 4 days (n = 4, *** p < 0.001). (D and E) Transcript levels for PRX3 and FOXM1 in WT HM, HMshCtrl cells and HMshPRX3 cell lines (** p < 0.01, * p < 0.05, n.s. = not significant). (F) Cell lysates from HMshCtrl and HMshPRX3 cell lines incubated with 5 μM TS for 18 hr were examined for PRX3, FOXM1, and actin expression by immunoblotting after denaturing SDS-PAGE. (G) HMshCTRL and HMshPRX3 cells were treated with 5 μM TS, lysates were collected over time and examined for PRX3 by immunoblotting (ns = non-specific band). (H) Cell number in shPRX3/pZeo, shPRX3/CAT, and shPRX3/mCAT cells compared to HM controls measured over 4 days (n = 4), ** p < 0.01) (I) HM controls, shPRX3/pZeo, shPRX3/CAT, and shPRX3/mCAT cells were incubated with increasing concentrations of TS for 18 hr, and total cell mass was determined by staining with crystal violet (n = 4, ** statistically significant all groups compared to HM, p <0.01). Error bars represent SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127310#pone.0127310.s003" target="_blank">S3 Fig</a>.</p

TS and gentian violet reduce tumor volume in a SCID mouse xenograft model of MM.

<p>(A) Hematoxylin and eosin (H&E) stained tumor tissue isolated from the peritoneal cavity of a DMSO control Fox Chase SCID mouse 5 weeks after injection of human HM cells. MM tumors were of biphasic morphology with necrotic areas that stained with eosin (pink staining). Tumors often contained stromal tissue of mouse origin and locally invaded the pancreas, liver and omentum. (B-D) Representative H & E stained tumor tissue from mice treated with 50 mg/kg TS, 2 mg/kg GV, or 5 mg/kg TS plus 2 mg/kg GV, respectively, every other day for 21–25 days. Tumor architecture and morphology was similar between treated and untreated animals (scale bar = 0.5 mm). (E) Tumor volumes from animals treated with 5 mg/kg TS, 50 mg/kg TS, 2mg/kg GV, or 2 mg/kg GV plus 5 mg/kg TS are presented as percent of vehicle control (DMSO) (n = 4–6 animals per group, ** p < 0.01, *** p < 0.001). (F) Representative images of FOXM1 immunohistochemistry used for nuclear quantification of FOXM1 from indicated tumor tissue (scale bar = 100 μm). (G) Quantification of FOXM1 positive nuclei from indicated treated tumor sections expressed as relative to vehicle (Veh) (n = 5, * p < 0.05). (H) Immunoblot of PRX3 expression in HM tumor lysates after reducing SDS-PAGE. Error bars represent SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127310#pone.0127310.s005" target="_blank">S5 Fig</a>.</p