Chemotherapeutic drugs cause increased release of pro-inflammatory molecules from MM cells.

<p>(A) Hmeso MM cells treated with either Dox or cisplatin caused increased release of HMGB1, FGF2, IL-1β and IL-18 as measured by Western blot analysis or ELISA. No loading controls were included as analysis was performed in cell culture medium. (B) H2373 MM cells also showed increased release of various pro-inflammatory molecules in response to drugs. IL-6 levels were either decreased (H2373) or not significantly changed (Hmeso) by drug treatments. *p≤0.05 as compared to untreated controls (0).</p

MM cells and tumors have low levels of NLRP3 protein and caspase-1 activity.

<p>A. Seven human MM cell lines were assessed for NLRP3 steady-state mRNA levels and compared with human mesothelial cell line (LP9), *p≤0.05 as compared to MM cell lines; B. NLRP3 protein levels in 4 human MM cell lines as compared to LP9 (red-NLRP3 protein, green-nucleus); C. NLRP3 mRNA levels in human MM tumor tissues (T) and normal counter parts (N); D. NLRP3 protein levels in human MM tumor tissue arrays as compared to normal lung (red-NLRP3 protein, green-nucleus); E. Caspase-1 activity in human MM cell lines as compared to LP9, *p≤0.05 as compared to MM cell lines. Scale bar = 50 microns.</p

Combination of IL-1R antagonist and cisplatin attenuated MM tumor weight/volume in an intraperitoneal xenograft mouse model.

<p>SCID mice injected with Hmeso MM cells received either cisplatin (2mg/kg, ip, 1X/week for 2 weeks), Anakinra (92 mg/kg, ip, 2x daily for 3 weeks), Cisplatin and Anakinra together or saline. (A, B) tumor weights and volumes were drastically reduced in combination group. (C) Total cell count was not significantly affected by combination treatment. (D) Neutrophil counts were significantly lower in cisplatin and combination group. *p≤0.05 as compared to saline treated group; †p≤0.05 as compared to cisplatin alone (by Student’s‘t’ test).</p

Chemotherapeutic drugs cause priming and activation of the NLRP3 inflammasome in MM cell lines.

<p>Treatment of Hmeso MM cells with Dox or cisplatin resulted in increased NLRP3 protein levels (A) and activation of inflammasome (B). Activation was measured by increased caspase-1 p20 release into the medium. *p≤0.05 as compared to untreated controls (0). (C) Cisplatin increased the protein levels of NLRP3 in H2373 MM cells and both Dox and cisplatin caused activation of caspase-1 in H2373 cells as measured by caspase-1 p20 release into the medium (D). *p≤0.05 as compared to untreated controls.</p

Chemotherapeutic drugs cause pyroptosis in MM cell lines.

<p>MTS assay performed to measure cell viability on Hmeso or H2373 MM cells in presence of Dox or cisplatin with and without specific caspase-1 inhibitor (A, B) or pan caspase inhibitor (C, D). The effect of these inhibitors on drug-induced caspase-1 activity was also performed in both MM cell lines (E, F). *p≤0.05 as compared to untreated controls (0); †p≤0.05 as compared to similar treatment without caspase- inhibitor, ‡ p≤0.05 as compared to low dose caspase inhibitor.</p

Chemotherapeutic drugs regulate PYCARD/ASC and pro-caspase levels in MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM) for 24 or 48 h and steady-state PYCARD/ASC or pro-caspase-1 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</p

Chemotherapeutic drugs increase NLRP3 levels in human MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM,) for 24 or 48 h and steady-state NLRP3 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</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

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

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