Asbestos modulates thioredoxin-thioredoxin interacting protein interaction to regulate inflammasome activation

BACKGROUND: Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes. METHODS: The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription. RESULTS: Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome. CONCLUSION: Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM

Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin

<p>Abstract</p> <p>Background</p> <p>Malignant mesotheliomas (MM) have a poor prognosis, largely because of their chemoresistance to anti-cancer drugs such as doxorubicin (Dox). Here we show using human MM lines that Dox activates extracellular signal-regulated kinases (ERK1 and 2), causally linked to increased expression of ABC transporter genes, decreased accumulation of Dox, and enhanced MM growth. Using the MEK1/2 inhibitor, U0126 and stably transfected shERK1 and shERK2 MM cell lines, we show that inhibition of both ERK1 and 2 sensitizes MM cells to Dox.</p> <p>Results</p> <p>U0126 significantly modulated endogenous expression of several important drug resistance (<it>BCL2, ABCB1, ABCC3</it>), prosurvival (<it>BCL2</it>), DNA repair (<it>BRCA1, BRCA2</it>), hormone receptor (<it>AR, ESR2, PPARγ</it>) and drug metabolism (<it>CYP3A4</it>) genes newly identified in MM cells. In comparison to shControl lines, MM cell lines stably transfected with shERK1 or shERK2 exhibited significant increases in intracellular accumulation of Dox and decreases in cell viability. Affymetrix microarray analysis on stable shERK1 and shERK2 MM lines showed more than 2-fold inhibition (p ≤ 0.05) of expression of ATP binding cassette genes (<it>ABCG1, ABCA5, ABCA2, MDR/TAP, ABCA1, ABCA8, ABCC2</it>) in comparison to shControl lines. Moreover, injection of human MM lines into SCID mice showed that stable shERK1 or shERK2 lines had significantly slower tumor growth rates in comparison to shControl lines after Dox treatment.</p> <p>Conclusions</p> <p>These studies suggest that blocking ERK1 and 2, which play critical roles in multi-drug resistance and survival, may be beneficial in combination with chemotherapeutic drugs in the treatment of MMs and other tumors.</p

CREB-Induced Inflammation Is Important for Malignant Mesothelioma Growth

Malignant mesothelioma (MM) is an aggressive tumor with no treatment regimen. Previously we have demonstrated that cyclic AMP response element binding protein (CREB) is constitutively activated in MM tumor cells and tissues and plays an important role in MM pathogenesis. To understand the role of CREB in MM tumor growth, we generated CREB-inhibited MM cell lines and performed in vitro and in vivo experiments. In vitro experiments demonstrated that CREB inhibition results in significant attenuation of proliferation and drug resistance of MM cells. CREB-silenced MM cells were then injected into severe combined immunodeficiency mice, and tumor growth in s.c. and i.p. models of MM was followed. We observed significant inhibition in MM tumor growth in both s.c. and i.p. models and the presence of a chemotherapeutic drug, doxorubicin, further inhibited MM tumor growth in the i.p. model. Peritoneal lavage fluids from CREB-inhibited tumor-bearing mice showed a significantly reduced total cell number, differential cell counts, and pro-inflammatory cytokines and chemokines (IL-6, IL-8, regulated on activation normal T cell expressed and secreted, monocyte chemotactic protein-1, and vascular endothelial growth factor). In vitro studies showed that asbestos-induced inflammasome/inflammation activation in mesothelial cells was CREB dependent, further supporting the role of CREB in inflammation-induced MM pathogenesis. In conclusion, our data demonstrate the involvement of CREB in the regulation of MM pathogenesis by regulation of inflammation

Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells

<p>Abstract</p> <p>Background</p> <p>Exposures to an amphibole fiber in Libby, Montana cause increases in malignant mesothelioma (MM), a tumor of the pleural and peritoneal cavities with a poor prognosis. Affymetrix microarray/GeneSifter analysis was used to determine alterations in gene expression of a human mesothelial cell line (LP9/TERT-1) by a non-toxic concentration (15×10⁶μm²/cm²) of unprocessed Libby six-mix and negative (glass beads) and positive (crocidolite asbestos) controls. Because manganese superoxide dismutase (MnSOD; SOD2) was the only gene upregulated significantly (p < 0.05) at both 8 and 24 h, we measured SOD protein and activity, oxidative stress and glutathione (GSH) levels to better understand oxidative events after exposure to non-toxic (15×10⁶μm²/cm²) and toxic concentrations (75×10⁶μm²/cm²) of Libby six-mix.</p> <p>Results</p> <p>Exposure to 15×10⁶μm²/cm²Libby six-mix elicited significant (p < 0.05) upregulation of one gene (<it>SOD2</it>; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in <it>SOD2</it>. Increased levels of SOD2 mRNA at 24 h were also confirmed in HKNM-2 normal human pleural mesothelial cells by qRT-PCR. SOD2 protein levels were increased at toxic concentrations (75×10⁶μm²/cm²) of Libby six-mix at 24 h. In addition, levels of copper-zinc superoxide dismutase (Cu/ZnSOD; SOD1) protein were increased at 24 h in all mineral groups. A dose-related increase in SOD2 activity was observed, although total SOD activity remained unchanged. Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence staining and flow cytometry revealed a dose- and time-dependent increase in reactive oxygen species (ROS) production by LP9/TERT-1 cells exposed to Libby six-mix. Both Libby six-mix and crocidolite asbestos at 75×10⁶μm²/cm²caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (<it>HO-1</it>) in LP9/TERT-1 and HKNM-2 cells.</p> <p>Conclusions</p> <p>Libby six-mix causes multiple gene expression changes in LP9/TERT-1 human mesothelial cells, as well as increases in SOD2, increased production of oxidants, and transient decreases in intracellular GSH. These events are not observed at equal surface area concentrations of nontoxic glass beads. Results support a mechanistic basis for the importance of SOD2 in proliferation and apoptosis of mesothelial cells and its potential use as a biomarker of early responses to mesotheliomagenic minerals.</p

Glutathione-S-transferase P promotes glycolysis in asthma in association with oxidation of pyruvate kinase M2

Background: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease

Asbestos-Induced Mesothelial to Fibroblastic Transition Is Modulated by the Inflammasome

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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

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 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