Glutaredoxin attenuates glutathione levels via deglutathionylation of Otub1 and subsequent destabilization of system xC.

Glutathione (GSH) is a critical component of the cellular redox system that combats oxidative stress. The glutamate-cystine antiporter, system xC-, is a key player in GSH synthesis that allows for the uptake of cystine, the rate-limiting building block of GSH. It is unclear whether GSH or GSH-dependent protein oxidation [protein S-glutathionylation (PSSG)] regulates the activity of system xC-. We demonstrate that an environment of enhanced PSSG promotes GSH increases via a system xC--dependent mechanism. Absence of the deglutathionylase, glutaredoxin (GLRX), augmented SLC7A11 protein and led to significant increases of GSH content. S-glutathionylation of C23 or C204 of the deubiquitinase OTUB1 promoted interaction with the E2-conjugating enzyme UBCH5A, leading to diminished ubiquitination and proteasomal degradation of SLC7A11 and augmentation of GSH, effects that were reversed by GLRX. These findings demonstrate an intricate link between GLRX and GSH via S-glutathionylation of OTUB1 and system xC- and illuminate a previously unknown feed-forward regulatory mechanism whereby enhanced GSH protein oxidation augments cellular GSH.

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

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-1ß in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1ß 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-1ß-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1ß-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.

Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells.

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi-omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin-1 (Glrx), the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B-induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.

Dysregulation of Pyruvate Kinase M2 Promotes Inflammation in a Mouse Model of Obese Allergic Asthma.

Obesity is a risk factor for the development of asthma and represents a difficult-to-treat disease phenotype. Aerobic glycolysis is emerging as a key feature of asthma, and changes in glucose metabolism are linked to leukocyte activation and adaptation to oxidative stress. Dysregulation of PKM2 (pyruvate kinase M2), the enzyme that catalyzes the last step of glycolysis, contributes to house dust mite (HDM)-induced airway inflammation and remodeling in lean mice. It remains unclear whether glycolytic reprogramming and dysregulation of PKM2 also contribute to obese asthma. The goal of the present study was to elucidate the functional role of PKM2 in a murine model of obese allergic asthma. We evaluated the small molecule activator of PKM2, TEPP46, and assessed the role of PKM2 using conditional ablation of the Pkm2 allele from airway epithelial cells. In obese C57BL/6NJ mice, parameters indicative of glycolytic reprogramming remained unchanged in the absence of stimulation with HDM. Obese mice that were subjected to HDM showed evidence of glycolytic reprogramming, and treatment with TEPP46 diminished airway inflammation, whereas parameters of airway remodeling were unaffected. Epithelial ablation of Pkm2 decreased central airway resistance in both lean and obese allergic mice in addition to decreasing inflammatory cytokines in the lung tissue. Lastly, we highlight a novel role for PKM2 in the regulation of glutathione-dependent protein oxidation in the lung tissue of obese allergic mice via a putative IFN-γ-glutaredoxin1 pathway. Overall, targeting metabolism and protein oxidation may be a novel treatment strategy for obese allergic asthma.

Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite-Induced Allergic Airways Disease.

Asthma is a chronic disorder characterized by inflammation, mucus metaplasia, airway remodeling, and hyperresponsiveness. We recently showed that IL-1-induced glycolytic reprogramming contributes to allergic airway disease using a murine house dust mite model. Moreover, levels of pyruvate kinase M2 (PKM2) were increased in this model as well as in nasal epithelial cells from asthmatics as compared with healthy controls. Although the tetramer form of PKM2 converts phosphoenolpyruvate to pyruvate, the dimeric form of PKM2 has alternative, nonglycolysis functions as a transcriptional coactivator to enhance the transcription of several proinflammatory cytokines. In the current study, we examined the impact of PKM2 on the pathogenesis of house dust mite-induced allergic airways disease in C57BL/6NJ mice. We report, in this study, that activation of PKM2, using the small molecule activator, TEPP46, augmented PKM activity in lung tissues and attenuated airway eosinophils, mucus metaplasia, and subepithelial collagen. TEPP46 attenuated IL-1ß-mediated airway inflammation and expression of proinflammatory mediators. Exposure to TEPP46 strongly decreased the IL-1ß-mediated increases in thymic stromal lymphopoietin (TSLP) and GM-CSF in primary tracheal epithelial cells isolated from C57BL/6NJ mice. We also demonstrate that IL-1ß-mediated increases in nuclear phospho-STAT3 were decreased by TEPP46. Finally, STAT3 inhibition attenuated the IL-1ß-induced release of TSLP and GM-CSF, suggesting that the ability of PKM2 to phosphorylate STAT3 contributes to its proinflammatory function. Collectively, these results demonstrate that the glycolysis-inactive form of PKM2 plays a crucial role in the pathogenesis of allergic airways disease by increasing IL-1ß-induced proinflammatory signaling, in part, through phosphorylation of STAT3.

Mouse serum exosomal proteomic signature in response to asbestos exposure.

Asbestos-induced diseases like fibrosis and mesothelioma are very aggressive, without any treatment options. These diseases are diagnosed only at the terminal stages due to lack of early stage biomarkers. The recent discovery of exosomes as circulating biomarkers led us to look for exosomal biomarkers of asbestos exposure in mouse blood. In our model, mice were exposed to asbestos as a single bolus dose by oropharyngeal aspiration. Fifty-six days later blood was collected, exosomes were isolated from plasma and characterized and subjected to proteomic analysis using Tandem Mass Tag labeling. We identified many proteins, some of which were more abundant in asbestos exposed mouse serum exosomes, and three selected proteins were validated by immunoblotting. Our study is the first to show that serum exosomal proteomic signatures can reveal some important proteins relevant to asbestos exposure that have the potential to be validated as candidate biomarkers. We hope to extrapolate the positive findings of this study to humans in future studies.

Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells.

Asbestos exposure is a determinate cause of many diseases, such as mesothelioma, fibrosis, and lung cancer, and poses a major human health hazard. At this time, there are no identified biomarkers to demarcate asbestos exposure before the presentation of disease and symptoms, and there is only limited understanding of the underlying biology that governs asbestos-induced disease. In our study, we used exosomes, 30-140 nm extracellular vesicles, to gain insight into these knowledge gaps. As inhaled asbestos is first encountered by lung epithelial cells and macrophages, we hypothesize that asbestos-exposed cells secrete exosomes with signature proteomic cargo that can alter the gene expression of mesothelial cells, contributing to disease outcomes like mesothelioma. In the present study using lung epithelial cells (BEAS2B) and macrophages (THP-1), we first show that asbestos exposure causes changes in abundance of some proteins in the exosomes secreted from these cells. Furthermore, exposure of human mesothelial cells (HPM3) to these exosomes resulted in gene expression changes related to epithelial-to-mesenchymal transition and other cancer-related genes. This is the first report to indicate that asbestos-exposed cells secrete exosomes with differentially abundant proteins and that those exosomes have a gene-altering effect on mesothelial cells.-Munson, P., Lam, Y.-W., Dragon, J. MacPherson, M., Shukla, A. Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells.

Extracellular signal regulated kinase 5 and inflammasome in progression of mesothelioma.

Malignant mesothelioma is an aggressive cancer in desperate need of treatment. We have previously shown that extracellular signaling regulated kinase 5 (ERK5) plays an important role in mesothelioma pathogenesis using ERK5 silenced human mesothelioma cells exhibiting significantly reduced tumor growth in immunocompromised mice. Here, we used a specific ERK 5 inhibitor, XMD8-92 in various in vitro and in vivo models to demonstrate that inhibition of ERK5 can slow down mesothelioma tumorigenesis. First, we show a dose dependent toxicity of XMD8-92 to 2 human mesothelioma cell lines growing as a monolayer. We also demonstrate the inhibition of ERK5 phosphorylation in various human mesothelioma cell lines by XMD8-92. We further confirmed the toxicity of XMD8-92 towards mesothelioma cell lines grown as spheroids in a 3-D model as well as in intraperitoneal (immune-competent) and intrapleural (immune-deficient) mouse models with and without chemotherapeutic drugs. To ascertain the mechanism, we explored the role of the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in the process. We found XMD8-92 attenuated naïve and chemotherapeutic-induced inflammasome priming and activation in mesothelioma cells. It can thus be concluded that ERK5 inhibition attenuates mesothelioma tumor growth and this phenomenon in part is regulated by the inflammasome.

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

Despite the causal relationship established between malignant mesothelioma (MM) and asbestos exposure, the exact mechanism by which asbestos induces this neoplasm and other asbestos-related diseases is still not well understood. MM is characterized by chronic inflammation, which is believed to play an intrinsic role in the origin of this disease. We recently found that asbestos activates the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in a protracted manner, leading to an up-regulation of IL-1ß and IL-18 production in human mesothelial cells. Combined with biopersistence of asbestos fibers, we hypothesize that this creates an environment of chronic IL-1ß signaling in human mesothelial cells, which may promote mesothelial to fibroblastic transition (MFT) in an NLRP3-dependent manner. Using a series of experiments, we found that asbestos induces a fibroblastic transition of mesothelial cells with a gain of mesenchymal markers (vimentin and N-cadherin), whereas epithelial markers, such as E-cadherin, are down-regulated. Use of siRNA against NLRP3, recombinant IL-1ß, and IL-1 receptor antagonist confirmed the role of NLRP3 inflammasome-dependent IL-1ß in the process. In vivo studies using wild-type and various inflammasome component knockout mice also revealed the process of asbestos-induced mesothelial to fibroblastic transition and its amelioration in caspase-1 knockout mice. Taken together, our data are the first to suggest that asbestos induces mesothelial to fibroblastic transition in an inflammasome-dependent manner.

Actin polymerization plays a significant role in asbestos-induced inflammasome activation in mesothelial cells in vitro.

Asbestos exposure leads to malignant mesothelioma (MM), a deadly neoplasm of mesothelial cells of various locations. Although there is no doubt about the role of asbestos in MM tumorigenesis, mechanisms are still not well explored. Recently, our group demonstrated that asbestos causes inflammasome priming and activation in mesothelial cells, which in part is dependent on oxidative stress. Our current study sheds light on yet another mechanism of inflammasome activation by asbestos. Here we show the role of actin polymerization in asbestos-induced activation of the nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Using human mesothelial cells, we first demonstrate that asbestos and carbon nanotubes induced caspase-1 activation and high-mobility group box 1, interleukin 1 beta and interleukin 18 secretion was blocked by Cytochalasin D (Cyto D) an actin polymerization inhibitor. Next, to understand the mechanism, we assessed whether phagocytosis of fibers by mesothelial cells is affected by actin polymerization inhibition. Transmission electron microscopy showed the inhibition of fiber uptake by mesothelial cells in the presence of Cyto D. Furthermore, localization of components of the inflammasome, apoptotic speck-like protein containing a CARD domain (ASC) and NLRP3, to the perinuclear space in mitochondria or endoplasmic reticulum in response to fiber exposure was also interrupted in the presence of Cyto D. Taken together, our studies suggest that actin polymerization plays important roles in inflammasome activation by fibers via regulation of phagocytosis and/or spatial localization of inflammasome components.

Inflammasome Modulation by Chemotherapeutics in Malignant Mesothelioma.

Malignant mesothelioma (MM) is a fatal disease in dire need of therapy. The role of inflammasomes in cancer is not very well studied, however, literature supports both pro-and anti-tumorigenic effects of inflammasomes on cancer depending upon the type of cancer. Asbestos is a causative agent for MM and we have shown before that it causes inflammasome priming and activation in mesothelial cells. MM tumor cells/tissues showed decreased levels of inflammasome components like NLRP3 and caspase-1 as compared to human mesothelial cells or normal tissue counterpart of tumor. Based on our preliminary findings we hypothesized that treatment of MMs with chemotherapeutic drugs may elevate the levels of NLRP3 and caspase-1 resulting in increased cell death by pyroptosis while increasing the levels of IL-1ß and other pro-inflammatory molecules. Therefore, a combined strategy of chemotherapeutic drug and IL-1R antagonist may play a beneficial role in MM therapy. To test our hypothesis we used two human MM tumor cell lines (Hmeso, H2373) and two chemotherapeutic drugs (doxorubicin, cisplatin). Through a series of experiments we showed that both chemotherapeutic drugs caused increases in NLRP3 levels, caspase-1 activation, pyroptosis and pro-inflammatory molecules released from MM cells. In vivo studies using SCID mice and Hmeso cells showed that tumors were smaller in combined treatment group of cisplatin and IL-1R antagonist (Anakinra) as compared to cisplatin alone or untreated control groups. Taken together our study suggests that chemotherapeutic drugs in combination with IL-1R antagonist may have a beneficial role in MM treatment.

Differential Susceptibility of Human Pleural and Peritoneal Mesothelial Cells to Asbestos Exposure.

Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1ß, IL-1α, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer.

Exploratory use of docetaxel loaded acid-prepared mesoporous spheres for the treatment of malignant melanoma.

INTRODUCTION: Five year survival for metastatic melanoma (MM) is very low at <10%. Therapeutic options have been limited secondary to systemic toxicity. As a result there has been a growing movement towards developing targeted drug delivery models. Prior research of this group has demonstrated the effectiveness of acid-prepared mesoporous spheres (APMS-TEG) in delivering chemotherapeutic agents at a lower effective dose than systemic administration. This study aims to assess the ability of the previously developed APMS-TEG particles to deliver therapeutic doses of docetaxel for the treatment of melanoma. METHODS: In vitro experiments were performed to assess docetaxel loading onto APMS-TEG particles and release kinetics. Toxicity experiments were performed using docetaxel and docetaxel loaded APMS-TEG. The effect on cell growth was assessed using the MelJuSo, UACC903, and WM1205 melanoma cell lines. RESULTS: Docetaxel demonstrated statistically significant dose dependent reduction in growth of melanoma cells. In all three cell lines, doses of 1 nM were sufficient to produce statistically significant reduction in cell growth. Scanning electron micrographs demonstrate increased uptake of APMS-TEG particles by melanoma cells in the first 24 hours, with the majority within the first 4 hours. Unloaded APMS particles had no effect on the melanoma cells, demonstrating that the particles themselves are not toxic. APMS-TEG particles had a peak release of drug within the first hour, with equilibration thereafter. The 5, 10, and 20 nM loaded particles all had statistically significant reduction in cell growth than the control groups. DISCUSSION: The high potency against melanoma cells makes docetaxel a suitable choice for loading into APMS-TEG particles. Docetaxel loaded APMS-TEG particles demonstrate significant activity against malignant melanoma and thus offer an innovative approach to the treatment of metastatic melanoma.

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.

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.

Extracellular signal-regulated kinase 5 and cyclic AMP response element binding protein are novel pathways inhibited by vandetanib (ZD6474) and doxorubicin in mesotheliomas.

Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos. All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent. Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer. However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM. Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined. Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells. Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox. After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed. Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM. Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.

Curcumin: a double hit on malignant mesothelioma.

Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1ß and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using in vitro models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1ß and IL-18. Absence of IL-1ß processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1ß priming through inhibition of the NF-κB pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-κB, toll-like receptors (TLR), and IL-1ß. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation.

Microspheres targeted with a mesothelin antibody and loaded with doxorubicin reduce tumor volume of human mesotheliomas in xenografts.

BACKGROUND: Malignant mesotheliomas (MMs) are chemoresistant tumors related to exposure to asbestos fibers. The long latency period of MM (30-40 yrs) and heterogeneity of tumor presentation make MM difficult to diagnose and treat at early stages. Currently approved second-line treatments following surgical resection of MMs include a combination of cisplatin or carboplatin (delivered systemically) and pemetrexed, a folate inhibitor, with or without subsequent radiation. The systemic toxicities of these treatments emphasize the need for more effective, localized treatment regimens. METHODS: Acid-prepared mesoporous silica (APMS) microparticles were loaded with doxorubicin (DOX) and modified externally with a mesothelin (MB) specific antibody before repeated intraperitoneal (IP) injections into a mouse xenograft model of human peritoneal MM. The health/weight of mice, tumor volume/weight, tumor necrosis and cell proliferation were evaluated in tumor-bearing mice receiving saline, DOX high (0.2 mg/kg), DOX low (0.05 mg/kg), APMS-MB, or APMS-MB-DOX (0.05 mg/kg) in saline. RESULTS: Targeted therapy (APMS-MB-DOX at 0.05 mg/kg) was more effective than DOX low (0.05 mg/kg) and less toxic than treatment with DOX high (0.2 mg/kg). It also resulted in the reduction of tumor volume without loss of animal health and weight, and significantly decreased tumor cell proliferation. High pressure liquid chromatography (HPLC) of tumor tissue confirmed that APMS-MB-DOX particles delivered DOX to target tissue. CONCLUSIONS: Data suggest that targeted therapy results in greater chemotherapeutic efficacy with fewer adverse side effects than administration of DOX alone. Targeted microparticles are an attractive option for localized drug delivery.

Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells.

BACKGROUND: Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear. RESULTS: We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1ß, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model. CONCLUSIONS: These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.