Arti Shukla

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Exposure to asbestos causes cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The pathogenesis of asbestos-related diseases is complicated and still poorly understood. Studies on animal models and cell cultures have indicated that asbestos fibers generate reactive oxygen and nitrogen species (ROS/RNS) and cause oxidation and/or nitrosylation of proteins and DNA. The ionic state of iron and its ability to be mobilized determine the oxidant-inducing potential of pathogenic iron-containing asbestos types. In addition to their capacity to damage macromolecules, oxidants play important roles in the initiation of numerous signal transduction pathways that are linked to apoptosis, inflammation, and proliferation. There is strong evidence supporting the premise that oxidants contribute to asbestos-induced lung injury; thus, strategies for reducing oxidant stress to pulmonary cells may attenuate the deleterious effects of asbestos.

High levels of ambient air pollution are associated with exacerbation of asthma and respiratory morbidity, yet little is known concerning the mechanisms of inflammation and toxicity by components of inhaled particulate matter (PM). Brief inhalation of PM(2.5) (particles of an aerodynamic diameter of < 2.5 microns) (300 microg/m(3) air for 6 h followed by a period of 24 h in clean air) by either C3H/HeJ or C57/BL6 mice caused significant (P </= 0.05) increases in steady-state messenger RNA (mRNA) levels of a number of nuclear factor (NF)-kappaB-associated and/ or -regulated genes, including tumor necrosis factor-alpha and -beta, interleukin-6, interferon-gamma, and transforming growth factor-beta. Lung mRNA levels of lymphotoxin-beta and macrophage migration inhibitory factor were unchanged. In murine C10 alveolar cells and an NF-kappaB-luciferase reporter cell line, exposure to PM(2.5) at noncytotoxic concentrations resulted in increases in transcriptional activation of NF-kappaB-dependent gene expression which were inhibited in the presence of catalase. Early and persistent increases in intracellular oxidants, as measured by flow cytometry and cell imaging using the oxidant probe 2'-7'-dichlorofluoroscin diacetate, were observed in epithelial cells exposed to PM(2.5) and ultrafine carbon black particles. Studies here are the first to show NF-kappaB-related inflammatory and cytokine gene expression after inhalation of PM(2.5) and oxidant-dependent induction of NF-kappaB activity by PM(2.5) in pulmonary epithelial cells.

Asiaticoside derived from the plant Centella asiatica is known to possess good wound healing activity. Enhanced healing activity has been attributed to increased collagen formation and angiogenesis. Since antioxidants have been reported to play a significant role in the wound healing process we studied the effect of asiaticoside on the levels of certain antioxidants in the wound so as to explore the possible involvement of such a mechanism in the asiaticoside induced wound healing. Asiaticoside application (0.2%, topical) twice daily for 7 days to excision-type cutaneous wounds in rats led to increased enzymatic and non-enzymatic antioxidants, namely superoxide dismutase (35%), catalase (67%), glutathione peroxidase (49%), vitamin E (77%) and ascorbic acid (36%) in newly formed tissues. It also resulted in a several fold decrease in lipid peroxide levels (69%) as measured in terms of thiobarbituric acid reactive substance. However, continued application for 14 days showed no significant difference in these antioxidants compared with their values in vehicle treated wound tissue. It appears from the present study that asiaticosides enhanced induction of antioxidant levels at an initial stage of healing which may be an important contributory factor in the healing properties of this substance.

1. Male albino rats of 21 days age were exposed to 10 p.p.m. cadmium (CdCl2 salt) in drinking water, ad libitum, for 90 days. It increased the brain cadmium levels by 76% (P < 0.05) and 165% (P < 0.001) respectively at 30 and 90 days of exposure compared to controls. 2. Cadmium increased blood-brain barrier permeability of fluoroscein dye (24%, P < 0.02) and the levels of brain microvessel malondialdehyde (31%, P < 0.01) at 90 days of exposure. However, these parameters did not alter significantly at 30 days of exposure. 3. Increased activities of microvessel superoxide dismutase (18%, P < 0.02), glutathione peroxidase (20%, P < 0.01) and catalase (28%, P < 0.01) were observed at 30 days of exposure. 4. The continuation of the Cd treatment for 90 days decreased the levels of superoxide dismutase (30%, P < 0.001), glutathione peroxidase (23%, P < 0.005), catalase (25%, P < 0.005), glutathione reductase (18%, P < 0.02), vitamin E (20%, P < 0.01), glutathione (26%, P < 0.01), ascorbic acid (18%, P < 0.05) and ceruloplasmin (13%, P < 0.05) in the microvessal preparation compared to controls. 5. It appears that Cd-induced blood-brain barrier dysfunction may be related to the depletion of microvessel antioxidant substances along with increase in lipid peroxidation at 90 days of exposure.

The mode of cadmium-induced cell death was investigated in a rat lung epithelial cell line. Cells, grown to near confluence, were exposed to 0-30 microM CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staining showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation was validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in the cytosol. Using this technique, a maximum level of apoptosis (5-fold higher than control) was observed in cultures exposed for 48 h to 20 microM CdCl2. The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling method (TUNEL) was subsequently used to determine the percentage of cells that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 microM Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates apoptosis in these cells are presently not known, reactive oxygen species are likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by the up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of redox sensitive transcription factors (AP-1 and NF-kappaB), and changes in various forms of glutathione (reduced, oxidized, and protein-bound).

In the present investigation the involvement ot free radicals in a self-healing cutaneous wound has been demonstrated. The levels of different enzymatic and non-enzymatic antioxidants have been studied in 2, 4, 7 and 14 days old wounds and compared with normal skin. Except for glutathione reductase (GR), all other cnzymatic- and non-enzymatic antioxidants were found to decrease following wounding. The decrease was 60–70%, in superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) at 2,4 and 7 days, while in the case of catalase (CAT) the decrease was 40-60% during this period. Although a complete recovery in the activity of CAT was observed, SOD and GPx did not recover completely and GST was found to be slightly elevated on 14th day post wounding. Non-enzymatic antioxidants viz. ascorbic acid, vitamin E and glutathione were also found to decrease to about 60-70% and except glutathione none of them was foud to recover completely at 14th day postwound-ing. Interestingly thiobarbituric acid reactive substance (TBARS) expressed as malondialdehvde (MDA) equivalent, a marker of lipid peroxidation, decreased following wounding which could be because of meagre availability of lipid substrate and/or of ascorbic acid. The results indicate that wounding results in loss of different free radical scavengers both enzymatic and non-enzymatic which either partially or completely recover following healing.

Abstract Nanomaterials are commonly defined as particles or fibers of less than 1 µm in diameter. For these reasons, they may be respirable in humans and have the potential, based upon their geometry, composition, size, and transport or durability in the body, to cause adverse effects on human health, especially if they are inhaled at high concentrations. Rodent inhalation models to predict the toxicity and pathogenicity of nanomaterials are prohibitive in terms of time and expense. For these reasons, a panel of in vitro assays is described below. These include cell culture assays for cytotoxicity (altered metabolism, decreased growth, lytic or apoptotic cell death), proliferation, genotoxicity, and altered gene expression. The choice of cell type for these assays may be dictated by the procedure or endpoint selected. Most of these assays have been standardized in our laboratory using pathogenic minerals (asbestos and silica) and non‐pathogenic particles (fine titanium dioxide or glass beads) as negative controls. The results of these in vitro assays should predict whether testing of selected nanomaterials should be pursued in animal inhalation models that simulate physiologic exposure to inhaled nanomaterials. Conversely, intrathoracic or intrapleural injection of nanomaterials into rodents can be misleading because they bypass normal clearance mechanisms, and non‐pathogenic fibers and particles can test positively in these assays. WIREs Nanomed Nanobiotechnol 2010 2 219–231 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine &gt; Toxicology of Nanomaterials

This investigation studied the contribution of antioxidants in delaying healing in excision cutaneous wounds (8 mm) in diabetic, aged and immunocompromised animals. Skin levels of catalase, glutathione (GSH), ascorbic acid (AA) and vitamin E in streptozotocin-induced diabetic rat were lower as compared to nondiabetics. The 7-d wound tissue of diabetic rats showed an increased vitamin E level along with depleted GSH content. In aged rats (18 months old), higher levels of skin superoxide dismutase (SOD), glutathione peroxidase (Gpx) and thiobarbituric acid reactive substances (TBARS) and lower levels of catalase and GSH were found as compared to their values in young rats (3-4 months old). The levels of SOD, GPx, catalase, AA, GSH and vitamin E in 7-d wound tissue of aged rats were significantly lower in comparison to those in young rats. However, TBARS were elevated in these wound tissues. The non-wounded skin of immunocompromised (athymic) mice showed lower levels of SOD, catalase, and TBARS and higher GSH and GPx levels in comparison to those present in normal mouse skin. Surprisingly, the analysis of 7-d wound tissue showed higher levels of SOD, catalase, GPx, and GSH and lower TBARS level in athymic mice compared to the wound tissue of normal mice. Thus low levels of antioxidants accompanied by raised levels of markers of free radical damage play a significant role in delaying wound healing in aged rats. In diabetic rats reduced glutathione levels may have a contributory role in delaying the healing process. However, in immunocompromised mice the antioxidant status following injury showed an adapted response.

Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.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-1β, 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.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.

This review illustrates how observations on key factors in the pathogenesis of MMs leads to design of therapies based on these experimental and preclinical studies. In concert, data suggest that MMs are a complex, pleiomorphic group of tumors with their phenotypes governed by a plethora of cytokines and growth factors produced in an autocrine fashion or by components of their microenvironment. Novel therapeutic approaches have been based on exploiting mechanisms important in the pathogenesis of MMs and might include promising combined approaches using immunotherapy, sequential blocking of antiapoptotic pathways,122Du X. Xiang L. Mackall C. Pastan I. Killing of resistant cancer cells with low Bak by a combination of an antimesothelin immunotoxin and a TRAIL receptor 2 agonist antibody.Clin Cancer Res. 2011; 17: 5926-5934Crossref PubMed Scopus (6) Google Scholar, 123Lee M.J. Ye A.S. Gardino A.K. Heijink A.M. Sorger P.K. MacBeath G. Yaffe M.B. Sequential application of anticancer drugs enhances cell death by rewiring apoptotic signaling networks.Cell. 2012; 149: 780-794Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar and targeting cell-cycle promoting and susceptibility genes.124Mizuno T. Murakami H. Fujii M. Ishiguro F. Tanaka I. Kondo Y. Akatsuka S. Toyokuni S. Yokoi K. Osada H. Sekido Y. YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes.Oncogene. 2012; 31: 5117-5122Crossref PubMed Scopus (16) Google Scholar, 125Ladanyi M. Zauderer M.G. Krug L.M. Ito T. McMillan R. Bott M. Giancotti F. New strategies in pleural mesothelioma: bAP1 and NF2 as novel targets for therapeutic development and risk assessment.Clin Cancer Res. 2012; 18: 4485-4490Crossref PubMed Scopus (6) Google Scholar

Exosomes are membrane-bound, intercellular communication shuttles that are defined by their endocytic origin and size range of 30-140 nm. Secreted by nearly all mammalian cell types and present in myriad bodily fluids, exosomes confer messages between cells, proximal and distal, by transporting biofunctional cargo in the form of proteins, nucleic acids, and lipids. They play a vital role in cellular signaling in both normal physiology and disease states, particularly cancer. Exosomes are powerful progenitors in altering target cell phenotypes, particularly in tumorigenesis and cancer progression, with the ability to alter tumor microenvironments and to assist in establishing the pre-metastatic niche. Many aspects of exosomes present them as novel means to identify cancer biomarkers for early detection and therapeutic targets, and using intrinsic and engineered characteristics of exosomes as therapeutic devices to ameliorate the progression of the disease. This review outlines some of the recent and major findings with regard to exosomes in cancer, and their utilization as therapeutic tools.

Abstract Exposure to asbestos is causally associated with the development of malignant mesothelioma, a cancer of cells lining the internal body cavities. Malignant mesothelioma is an aggressive cancer resistant to all current therapies. Once inhaled or ingested, asbestos causes inflammation in and around tissues that come in contact with these carcinogenic fibers. Recent studies suggest that inflammation is a major contributing factor in the development of many types of cancer, including malignant mesothelioma. The NALP3/NLRP3 inflammasome, including the component ASC, is thought to be an important mediator of inflammation in cells that sense extracellular insults, such as asbestos, and activate a signaling cascade resulting in release of mature IL1β and recruitment of inflammatory cells. To determine if inflammasome-mediated inflammation contributes to asbestos-induced malignant mesothelioma, we chronically exposed Asc-deficient mice and wild-type littermates to asbestos and evaluated differences in tumor incidence and latency. The Asc-deficient mice showed significantly delayed tumor onset and reduced malignant mesothelioma incidence compared with wild-type animals. We also tested whether inflammation-related release of IL1β contributes to tumor development in an accelerated mouse model of asbestos-induced malignant mesothelioma. Nf2+/−;Cdkn2a+/− mice exposed to asbestos in the presence of anakinra, an IL1 receptor (IL1R) antagonist, showed a marked delay in the median time of malignant mesothelioma onset compared with similarly exposed mice given vehicle control (33.1 weeks vs. 22.6 weeks, respectively). Collectively, these studies provide evidence for a link between inflammation-related IL1β/IL1R signaling and the development of asbestos-induced malignant mesothelioma. Furthermore, these findings provide rationale for chemoprevention strategies targeting IL1β/IL1R signaling in high-risk, asbestos-exposed populations. Cancer Prev Res; 9(5); 406–14. ©2016 AACR.

Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time‐dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM‐derived IL‐6, IL‐8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free‐floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs.

Abstract There is growing interest in developing non-pharmacological treatments that could boost natural defenses against cancer and contribute to primary and secondary cancer prevention. Recent studies have shown that gentle daily stretching for 10 minutes can reduce local connective tissue inflammation and fibrosis. Because mechanical factors within the stroma can influence the tumor microenvironment, we hypothesized that stretching would reduce the growth of tumors implanted within locally stretched tissues and tested this hypothesis in a mouse orthotopic breast cancer model. Female FVB mice (N = 66) underwent bilateral injection of p53/PTEN double-null primary mouse mammary tumor cells into the third mammary fat pad. Mice were randomized to stretch vs. no stretch, and treated for 10 minutes once a day, for four weeks. Tumor volume at end-point was 52% smaller in the stretch group, compared to the no-stretch group (p &lt; 0.001) in the absence of any other treatment. Cytotoxic immune responses were activated and levels of Specialized Pro-Resolving Mediators were elevated in the stretch group. These results suggest a link between immune exhaustion, inflammation resolution and tumor growth. Stretching is a gentle, non-pharmacological intervention that could become an important component of cancer treatment and prevention.

Abstract Malignant mesothelioma (MM) is an asbestos-induced cancer arising on the mesothelial surface of organ cavities. MM is essentially incurable without a means of early diagnosis and no successful standard of care. These facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome-contained microRNAs (miRNAs). We discovered that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly miR-16-5p. This observation lead us to hypothesize that MM cells preferentially secreted tumor-suppressor miRNAs via exosomes. Through separate avenues of potential therapeutic advance, we embarked on an innovative strategy to kill MM tumor cells. We employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16-5p exosome loss and replenishing cellular miR-16-5p leading to reduced tumorigenic capacity and miR-16-5p target oncoproteins CCND1 and BCL2. Additionally, we force-fed MM tumor exosomes back to MM tumor cells, which led to cell death, and a reduction in the same oncoproteins. We recapitulated these results with direct transfection of miR-16-5p, confirmed that this is a cancer-cell specific effect, and elucidated a part of the miR-16-5p mechanism of exosome loading.

Occupational exposure to crystalline silica is associated with the development of pulmonary inflammation and silicosis, yet how silica initiates pulmonary fibrosis and which cell types are involved are unclear. In studies here, we hypothesized that silica particles interact initially with pulmonary epithelial cells and alveolar macrophages (AMs) to cause transcriptional activation of nuclear factor (NF)-kappaB-regulated genes encoding inflammatory cytokines. Exposure of NF-kappaB luciferase reporter mice intratracheally to silica or lipopolysaccharide (LPS), but not the nonfibrogenic particle titanium dioxide (TiO(2)), increased immunoreactivity of luciferase protein in bronchiolar epithelial cells and AMs. Ribonuclease protection assays revealed significant (P < or = 0.05) increases in mRNA levels of inducible nitric oxide synthase, tumor necrosis factor-alpha, macrophage inflammatory protein-2, macrophage chemotactic protein-1 (MCP-1), interferon-gamma, interleukin (IL)-6, and IL-12 in lung homogenates of reporter mice after exposures to silica or LPS. Immunoreactivity of MCP-1 in these animals was localized to AMs and epithelial cells. These data are the first to show activation of NF-kappaB in situ by fibrogenic particles in pulmonary epithelial cells and AMs. Increased expression of NF-kappaB-related inflammatory cytokines by these cell types, which first encounter silica after inhalation, may be critical to the initiation of silica-associated lung diseases, thus providing a rationale for focusing on NF-kappaB in preventive and therapeutic strategies.

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.

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. U0126 significantly modulated endogenous expression of several important drug resistance (BCL2, ABCB1, ABCC3), prosurvival (BCL2), DNA repair (BRCA1, BRCA2), hormone receptor (AR, ESR2, PPARγ) and drug metabolism (CYP3A4) 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 (ABCG1, ABCA5, ABCA2, MDR/TAP, ABCA1, ABCA8, ABCC2) 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. 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.

Exposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression. Microarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 106μm2/cm2) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 106μm2/cm2) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (FOS, ATF3, IL6 and IL8) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells. Cristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses in vitro are suggested as predictive in determining the pathogenicity of potentially harmful particulates.

Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.

Abstract 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)-1β 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-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β 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-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. Cancer Prev Res; 7(3); 330–40. ©2014 AACR.

Recent studies have demonstrated growth factors and other cellular proteins as being important in the healing process. In this study, we have investigated the differential expression of proteins in wound tissues of normal and chronic animal models. Proteins were identified by specific antibodies, partial N-terminal amino acid sequence, and molecular weight homology. In normal wound tissues de novo synthesis of a heat shock protein, platelet derived growth factor (PDGF), and fibroblast growth factor (FGF) was induced within 24 h of skin injury. Proteins resembling vascular endothelial growth factor, receptors for PGDF, FGF, and epidermal growth factor, were synthesized. The elevated synthesis declined to a basal level in 7 to 14 days after skin injury which coincided with healing of wounds. These changes occurred only in wound site tissues but not in distal tissues. In contrast, the chronic wounds presented a different picture. The expressions of these proteins were either delayed or inhibited. This suggested the role of these proteins during normal and chronic wound healing. The proteins which were down regulated in chronic wounds may be used in the management of wounds and exploited as targets for therapeutic development.

Signaling pathways initiated at the external cell surface or within the cytoplasm regulate transactivation of transcription factors and gene expression that are causally related to a number of critical cellular outcomes including proliferation, apoptosis, cell survival, and production of inflammatory cytokines. Asbestos, a ubiquitous pathogenic group of mineral fibers, can stimulate gene expression in a variety of cell types in the lung via intracellular signaling pathways. These cell signaling cascades may be initiated through receptor-mediated events or integrins. Alternatively, they may be stimulated by oxidants generated both during phagocytosis of minerals and/or by redox reactions on the mineral surface. Once initiated, these pathways can lead to promotion of gene expression critical to cellular injury, proliferation and inflammation—events leading to the development of fibroproliferative diseases of the lung and pleura. The elucidation and relevance of critical signaling cascades to lung injury or repair following asbestos exposure could aid in developing strategies to prevent or treat asbestos-associated lung and pleural diseases.

Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 mm 2 /cm 2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO 2 ), or glass beads for 8 or 24 hours.RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR.Gene changes by asbestos were concentration-and time-dependent.At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 hours and of 205 genes at 24 hours, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 hours.Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos.Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 hours and no changes at 24 hours, whereas expression levels of 30 genes were elevated at 8 hours at high talc concentrations.Fine TiO 2 or glass beads caused no changes in gene expression.In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of two genes (NR4A2, MIP2) at 8 hours and 16 genes at 24 hours that were distinct from those elevated in mesothelial cells.Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches.Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1b, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.

Abstract Members of the extracellular signal‐regulated kinase (ERK) family may have distinct roles in the development of cell injury and repair, differentiation and carcinogenesis. Here, we show, using a synthetic small‐molecule MEK1/2 inhibitor (U0126) and RNA silencing of ERK1 and 2, comparatively, that ERK2 is critical to transformation and homeostasis of human epithelioid malignant mesotheliomas (MMs), asbestos‐induced tumors with a poor prognosis. Although MM cell (HMESO) lines stably transfected with shERK1 or shERK2 both exhibited significant decreases in cell proliferation in vitro , injection of shERK2 cells, and not shERK1 cells, into immunocompromised severe combined immunodeficiency (SCID) mice showed significant attenuated tumor growth in comparison to shControl (shCon) cells. Inhibition of migration, invasion and colony formation occurred in shERK2 MM cells in vitro , suggesting multiple roles of ERK2 in neoplasia. Microarray and quantitative real‐time PCR analyses revealed gene expression that was significantly increased ( CASP1 , TRAF1 and FAS ) or decreased ( SEMA3E, RPS6KA2, EGF and BCL2L1 ) in shERK2‐transfected MM cells in contrast to shCon‐transfected MM cells. Most striking decreases were observed in mRNA levels of Semaphorin 3 ( SEMA3E ), a candidate tumor suppressor gene linked to inhibition of angiogenesis. These studies demonstrate a key role of ERK2 in novel gene expression critical to the development of epithelioid MMs. After injection of sarcomatoid human MM (PPMMill) cells into SCID mice, both shERK1 and shERK2 lines showed significant decreased tumor growth, suggesting heterogeneous effects of ERKs in individual MMs.

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-1β 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.

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. 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. 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. 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.

Malignant mesothelioma is a devastating disease with a need for new treatment strategies. In the present study, we showed the importance of extracellular signal-regulated kinase 5 (ERK5) in malignant mesothelioma tumor growth and treatment.ERK5 as a target for malignant mesothelioma therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft severe combined immunodeficient (SCID) mouse models.We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in malignant mesothelioma cells. ERK5 silencing increased doxorubicin-induced cell death and doxorubicin retention in malignant mesothelioma cells. In addition, shERK5 malignant mesothelioma lines exhibited both attenuated colony formation on soft agar and invasion of malignant mesothelioma cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion, and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 malignant mesothelioma cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from intraperitoneal shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis-related (interleukin-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the intraperitoneal model of tumor growth.ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in patients with malignant mesothelioma.

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-1β and IL-18 production in human mesothelial cells. Combined with biopersistence of asbestos fibers, we hypothesize that this creates an environment of chronic IL-1β 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-1β, and IL-1 receptor antagonist confirmed the role of NLRP3 inflammasome-dependent IL-1β 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.

The effect of different bioagents such as Trichoderma harzianum, T. viride, T. virens, Pseudomonas fluorescens, and Bacillus subtilis was studied on seed mycoflora, seed germination, root/shoot length, and seedling vigour of cucumber var. Solan Srijan under in vitro conditions. Alternaria sp., Aspergillus sp., and Fusarium spp. were observed on cucumber as seed mycoflora, with T. harzianum showing the greatest inhibition for Alternaria sp. and Fusarium spp., and T. viride showing the greatest inhibition for Aspergillus sp. Cucumber var. Solan Srijan seeds were treated with various bio agents, with T. harzianum being the most effective in increasing seed germination (88.75%), root length (13.58 cm), shoot length (14.58 cm), and seedling vigour (2501.31).

Alveolar epithelial and mesothelial cells undergo apoptosis in response to asbestos, a phenomenon that may be important in injury and/or initiation of compensatory proliferation. Here, we report a functional role of protein kinase (PKC)delta in apoptosis by crocidolite asbestos. We first show that asbestos increases the kinase activity of PKC delta in alveolar type II epithelial cells (C10 line) and causes its translocation to mitochondria, events associated with caspase-9 cleavage and apoptosis as detected by the Apostain technique. Pretreatment of C10 cells with rottlerin (Rot), a PKC delta-selective inhibitor, before addition of asbestos prevented cleavage of caspase-9 and blocked the appearance of apoptotic cells. Asbestos-induced apoptosis also was inhibited in cells stably expressing a dominant-negative kinase-deficient mutant of PKC delta (dnPKC delta), but not dnPKC alpha. Activities of PKC alpha and PKC zeta increased after exposure to asbestos, but neither isoform migrated to mitochondria. A general inhibitor of PKCs, bisindolylmaleimide I, had no effect on asbestos-induced apoptosis. Hydrogen peroxide (H2O2) induced activation of PKCs delta, alpha, zeta, and theta, translocation of PKC delta to mitochondria, and caspase-9 cleavage. However, H2O2-induced apoptosis was not inhibited by cell lines stably expressing either dnPKC delta or dnPKC alpha, suggesting that activation of PKC delta has a distinct role in the development of asbestos-induced apoptosis.

The signaling pathways leading to the development of asbestos-associated diseases are poorly understood. Here we used normal and protein kinase C (PKC)-delta knockout (PKCdelta-/-) mice to demonstrate multiple roles of PKC-delta in the development of cell proliferation and inflammation after inhalation of chrysotile asbestos. At 3 days, asbestos-induced peribronchiolar cell proliferation in wild-type mice was attenuated in PKCdelta-/- mice. Cytokine profiles in bronchoalveolar lavage fluids showed increases in interleukin (IL)-1beta, IL-4, IL-6, and IL-13 that were decreased in PKCdelta-/- mice. At 9 days, microarray and quantitative reverse transcriptase-polymerase chain reaction analysis of lung tissues revealed increased mRNA levels of the profibrotic cytokine, IL-4, in asbestos-exposed wild-type mice but not PKCdelta-/- mice. PKCdelta-/- mice also exhibited decreased lung infiltration of polymorphonuclear cells, natural killer cells, and macrophages in bronchoalveolar lavage fluid and lung, as well as increased numbers of B lymphocytes and plasma cells. These changes were accompanied by elevated mRNA levels of immunoglobulin chains. These data show that modulation of PKC-delta has multiple effects on peribronchiolar cell proliferation, proinflammatory and profibrotic cytokine expression, and immune cell profiles in lung. These results also implicate targeted interruption of PKC-delta as a potential therapeutic option in asbestos-induced lung diseases.

To investigate the role of bronchiolar epithelial NF-kappaB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IkappaBalpha mutant (IkappaBalphasr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IkappaBalphasr Tg(+) and Tg(-) mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1beta were increased (p < or = 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p < or = 0.05) in Tg(+) vs Tg(-) mice. Asbestos also caused increases in IL-4, MIP-1beta, and MCP-1 in BALF that were more elevated (p < or = 0.05) in Tg(+) mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p < or = 0.05) in Tg(+) mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p < or = 0.05) in Tg(+) vs Tg(-) mice. Our findings demonstrate that airway epithelial NF-kappaB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy. Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy. Malignant mesotheliomas (MMs) are derived from the mesothelial cells of the pleural, peritoneal, or pericardial cavities. Exposure to asbestos is a major risk factor for MM as ∼80% of MM patients have known exposure to asbestos.1Mossman BT Bignon J Corn M Seaton A Gee JB Asbestos: scientific developments and implications for public policy.Science. 1990; 247: 294-301Crossref PubMed Scopus (590) Google Scholar, 2Mossman BT Gee JB Asbestos-related diseases.N Engl J Med. 1989; 320: 1721-1730Crossref PubMed Scopus (337) Google Scholar, 3Robinson BW Lake RA Advances in malignant mesothelioma.N Engl J Med. 2005; 353: 1591-1603Crossref PubMed Scopus (918) Google Scholar MMs are increasing worldwide, and most patients survive <12 months after initial diagnosis.1Mossman BT Bignon J Corn M Seaton A Gee JB Asbestos: scientific developments and implications for public policy.Science. 1990; 247: 294-301Crossref PubMed Scopus (590) Google Scholar, 2Mossman BT Gee JB Asbestos-related diseases.N Engl J Med. 1989; 320: 1721-1730Crossref PubMed Scopus (337) Google Scholar, 3Robinson BW Lake RA Advances in malignant mesothelioma.N Engl J Med. 2005; 353: 1591-1603Crossref PubMed Scopus (918) Google Scholar, 4Carbone M Albelda SM Broaddus VC Flores RM Hillerdal G Jaurand MC Kjaerheim K Pass HI Robinson B Tsao A Eighth International Mesothelioma Interest Group.Oncogene. 2007; 26: 6959-6967Crossref PubMed Scopus (27) Google Scholar Thus, effective therapeutic strategies for MM are desperately needed. cAMP response element binding protein (CREB1 or CREB) is a 43-kDa basic/leucine zipper transcription factor that regulates gene expression through activation of cAMP-dependent or -independent signal transduction pathways. CREB1 binds to an octanucleotide cAMP CRE consensus sequence in promoters of target genes as a homodimer or heterodimer with other members of the CREB/ATF superfamily. Phosphorylation of CREB1 at Ser-133 is essential for CREB-mediated transcription.5Mayr B Montminy M Transcriptional regulation by the phosphorylation-dependent factor CREB.Nat Rev Mol Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2084) Google Scholar Ser-133 phosphorylation promotes target gene activation in part through recruitment of the coactivator paralogs, CREB-binding protein and p300.6Goodman RH Smolik S CBP/p300 in cell growth, transformation, and development.Genes Dev. 2000; 14: 1553-1577Crossref PubMed Google Scholar Recruitment of CREB-binding protein by phospho-CREB1 (pCREB1) appears sufficient for CREB-mediated gene activation.7Cardinaux JR Notis JC Zhang Q Vo N Craig JC Fass DM Brennan RG Goodman RH Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation.Mol Cell Biol. 2000; 20: 1546-1552Crossref PubMed Scopus (155) Google Scholar, 8Du K Asahara H Jhala US Wagner BL Montminy M Characterization of a CREB gain-of-function mutant with constitutive transcriptional activity in vivo.Mol Cell Biol. 2000; 20: 4320-4327Crossref PubMed Scopus (90) Google Scholar The transcriptional coactivator pCREB-binding protein/p300 is also a histone acetyltransferase that regulates gene expression by acetylating histones and other transcription factors. CREB has been classically studied in the physiology of nerve or contractile cells and most recently in some cancers.9Shankar DB Cheng JC Kinjo K Federman N Moore TB Gill A Rao NP Landaw EM Sakamoto KM The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia.Cancer Cell. 2005; 7: 351-362Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10Pigazzi M Ricotti E Germano G Faggian D Arico M Basso G cAMP response element binding protein (CREB) overexpression CREB has been described as critical for leukemia progression.Haematologica. 2007; 92: 1435-1437Crossref PubMed Scopus (42) Google Scholar, 11Chhabra A Fernando H Watkins G Mansel RE Jiang WG Expression of transcription factor CREB1 in human breast cancer and its correlation with prognosis.Oncol Rep. 2007; 18: 953-958PubMed Google Scholar, 12Aggarwal S Kim SW Ryu SH Chung WC Koo JS Growth suppression of lung cancer cells by targeting cyclic AMP response element-binding protein.Cancer Res. 2008; 68: 981-988Crossref PubMed Scopus (83) Google Scholar, 13Arany I Herbert J Herbert Z Safirstein RL Restoration of CREB function ameliorates cisplatin cytotoxicity in renal tubular cells.Am J Physiol. 2008; 294: F577-F581Google Scholar Signaling cascades responsible for CREB activation by extracellular stimuli include protein kinase A (PKA), protein kinase C (PKC), Ca2+/calmodulin-dependent kinase (CaM kinases), p90 ribosomal S6 kinase, and extracellular signal-regulated kinases (ERK1/2).14Roux PP Richards SA Blenis J Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity.Mol Cell Biol. 2003; 23: 4796-4804Crossref PubMed Scopus (155) Google Scholar, 15Shaywitz AJ Greenberg ME CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.Annu Rev Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1797) Google Scholar Since both PKC and ERK1/2 have been linked to cell proliferation, fibrogenesis, and mesothelial cell transformation by asbestos,16Lounsbury KM Stern M Taatjes D Jaken S Mossman BT Increased localization and substrate activation of protein kinase Cδ in lung epithelial cells following exposure to asbestos.Am J Pathol. 2002; 160: 1991-2000Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 17Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-δ knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Ramos-Nino ME Timblin CR Mossman BT Mesothelial cell transformation requires increased AP-1 binding activity and ERK-dependent Fra-1 expression.Cancer Res. 2002; 62: 6065-6069PubMed Google Scholar, 19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar we hypothesized that activated CREB was critical to the development and chemoresistance of MMs. Here, we first explored signaling pathways leading to phosphorylation of CREB1 and functional ramifications of silencing CREB in human mesothelial cells exposed to asbestos. We then studied activation and function of CREB in human MM cells in vitro in response to Dox/Adriamycin, a drug used in single-agent trials20Pass H Carbone M Vogelzang N Malignant Mesothelioma: Advances in Pathogenesis, Diagnosis, and Translational Therapies. Springer Science+Business Media, Inc., New York2005: 854Google Scholar and in a recent phase III study with Onconase.4Carbone M Albelda SM Broaddus VC Flores RM Hillerdal G Jaurand MC Kjaerheim K Pass HI Robinson B Tsao A Eighth International Mesothelioma Interest Group.Oncogene. 2007; 26: 6959-6967Crossref PubMed Scopus (27) Google Scholar We demonstrate that crocidolite asbestos, the most potent asbestos type in the causation of MM,1Mossman BT Bignon J Corn M Seaton A Gee JB Asbestos: scientific developments and implications for public policy.Science. 1990; 247: 294-301Crossref PubMed Scopus (590) Google Scholar, 2Mossman BT Gee JB Asbestos-related diseases.N Engl J Med. 1989; 320: 1721-1730Crossref PubMed Scopus (337) Google Scholar, 3Robinson BW Lake RA Advances in malignant mesothelioma.N Engl J Med. 2005; 353: 1591-1603Crossref PubMed Scopus (918) Google Scholar causes CREB activation in human mesothelial cells via EGF receptor (EGFR) and PKA-dependent pathways. Moreover, we show that human MM cell lines and human MM tissue arrays show high endogenous activation of CREB1 that is further increased by Dox. Silencing of CREB in asbestos-exposed human mesothelial cells or Dox-treated MMs by transfection of small interfering CREB renders them more sensitive to asbestos- or Dox-induced apoptosis. Data show roles of CREB in the development, migration, and chemoresistance of MMs. Human peritoneal mesothelial LP9/TERT-1 (LP9) cells, an hTERT-immortalized cell line phenotypically and functionally resembling normal human mesothelial cells,21Dickson MA Hahn WC Ino Y Ronfard V Wu JY Weinberg RA Louis DN Li FP Rheinwald JG Human keratinocytes that express hTERT and also bypass a p16INK4a-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics.Mol Cell Biol. 2000; 20: 1436-1447Crossref PubMed Scopus (803) Google Scholar were obtained from Dr. J. Rheinwald (Brigham and Women’s Hospital, Harvard University, Boston, MA). This cell line was used to examine effects of asbestos on CREB activation, CREB-related gene expression, and apoptosis by asbestos. Sarcomatous (Mont) and epithelioid (Me26) human pleural MM cell lines were obtained from Drs. L. Mutti, (Maugeri Foundation, Pavia, Italy) and M. Bocchetta (Loyola University, Mayfield, IL), respectively. NYU474 pleural mesothelial cells, Gard and Hmeso MM lines were contributed by Drs. H. I. Pass (New York University, New York, NY) and J. Testa (Fox Chase Cancer Center, Philadelphia, PA), respectively. Hmeso cells, originally designated H-MESO-1, were isolated by Reale et al.22Reale FR Griffin TW Compton JM Graham S Townes PL Bogden A Characterization of a human malignant mesothelioma cell line (H-MESO-1): a biphasic solid and ascitic tumor model.Cancer Res. 1987; 47: 3199-3205PubMed Google Scholar All cells were incubated at 37°C and 5% CO2 and grown to ∼80 to 90% confluency in complete medium consisting of Dulbecco’s modified Eagle’s medium/F12 50/50 and 10% fetal bovine serum (Mediatech, Herndon, VA), 0.1 μg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 2.5 μg/ml insulin, 2.5 μg/ml transferrin, 2.5 ng/ml sodium selenite (Sigma-Aldrich), and penicillin-streptomycin (50 U/ml penicillin G and 50 μg/ml streptomycin sulfate) (Invitrogen, Carlsbad, CA). The physical and chemical characterization of the National Institute on Environmental Health Sciences reference sample of crocidolite asbestos has been reported previously.23Campbell WJ Huggins CW Wylie AG Chemical and physical characterization of amosite, chrysotile, crocidolite, and nonfibrous tremolite for oral ingestion studies. National Institute of Environmental Health Sciences, 1980: 1Google Scholar After sterilization under UV light overnight, particulates were suspended in HBSS at 1 mg/ml, sonicated for 15 minutes in a water bath sonicator, and triturated five times through a 22-gauge needle. A volume of this suspension was added to cells in medium to achieve the desired final concentration of 5 μg/cm2 area dish, a concentration causing apoptosis and compensatory proliferation of surrounding pleural mesothelial cells.24Goldberg JL Zanella CL Janssen YM Timblin CR Jimenez LA Vacek P Taatjes DJ Mossman BT Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos.Am J Respir Cell Mol Biol. 1997; 17: 265-271Crossref PubMed Scopus (77) Google Scholar The EGFR inhibitor, AG1478 (10 and 20 μmol/L), the ERK1/2 inhibitor, U0126 (10 and 20 μmol/L), the general PKC inhibitor (Bisindolymaleimide I; 5 μmol/L), a PKCδ-specific inhibitor Rottlerin (5 μmol/L), and the CaM kinase II inhibitor, KN93 (30 μmol/L), or its inactive isomer, KN92 (30 μmol/L), were obtained from Calbiochem (La Jolla, CA). The PKA inhibitor, H89 (10 μmol/L), was obtained from BIOMOL (Plymouth Meeting, PA). Forskolin (10 μmol/L for 15 minutes), an activator of CREB, and Dox were purchased from Sigma-Aldrich. All inhibitors were added at effective concentrations reported previously in the literature19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar, 25Yuan Z Taatjes DJ Mossman BT Heintz NH The duration of nuclear extracellular signal-regulated kinase 1 and 2 signaling during cell cycle reentry distinguishes proliferation from apoptosis in response to asbestos.Cancer Res. 2004; 64: 6530-6536Crossref PubMed Scopus (32) Google Scholar, 26Barlow CA Shukla A Mossman BT Lounsbury KM Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells.Am J Respir Cell Mol Biol. 2006; 34: 7-14Crossref PubMed Scopus (24) Google Scholar for 1 hour before asbestos exposure. Control cultures received medium without inhibitors but with vehicle (≤1% DMSO) instead and were treated identically. All experiments were performed in triplicate or more. On-Target plus Non-Targeting small-interfering RNA (siRNA) number 1 (scrambled control) and On-Target plus SMARTpool human CREB siRNA (100 nmol/L, Dharmacon, Lafayette, CO) were transfected into 95% confluent cells using Lipofectamine 2000 (Invitrogen), following the manufacturer’s protocol. The efficiency of CREB protein knockdown was determined by quantitative RT-PCR and Western blot analysis after 24, 48, and 72 hours. Cells were exposed to agents as described above, the medium aspirated, and cells washed twice with ice-cold PBS before collection in 4× sample buffer (200 μmol/L Tris (pH 6.8), 4% SDS, 4 mg/ml β-mercaptoethanol, 40% glycerol, and 2 μmol/L pyronin-Y). The amount of protein was determined using the RC DC protein assay (Bio-Rad, Hercules, CA). A total of 60 μg protein were separated by 10% SDS-PAGE and transferred to nitrocellulose. Western blots were performed as previously described,19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar using antibodies specific to total and phosphorylated CREB (rabbit polyclonal anti-CREB, 1/1000, and rabbit polyclonal anti-phospho-Ser 133-CREB1/500; Cell Signaling Technology, Danvers, MA) and total β-actin 1/2000 (Abcam, Cambridge, MA). QuantityOne was used to quantify band density, and phosphorylated protein levels were normalized to respective total protein levels, ie, pCREB/CREB). Blots are representative of at least three different experiments. Total RNA was prepared using an RNeasy plus mini kit according to the manufacturer’s protocol (Qiagen, Valencia, CA) as described previously.19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar Total RNA (1 μg) was reverse-transcribed with random primers using the Promega AMV Reverse Transcriptase kit (Promega, Madison, WI) according to the recommendations of the manufacturer. To quantify gene expression, the cDNA was amplified by TaqMan Real Time Q-PCR using the 7900HT Sequence Prism Detector (Applied Biosystems, Foster City, CA). Triplicate assays were performed with RNA samples isolated from at least three independent experiments. Fold changes in gene expression were calculated using the δ-delta Ct method. The values obtained from cDNAs and hypoxanthine phosphoribosyl transferase (HPRT) controls provided relative gene expression levels for the gene locus investigated. The Assay on Demand primers and probes used were purchased from Applied Biosystems. To determine whether modulation of CREB levels altered cell death in LP9 human mesothelial and MM cells, detection of apoptosis was performed using the Apostain technique as described previously.27Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C δ-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (55) Google Scholar Briefly, cells grown on glass coverslips were transfected with siCREB or scrambled control. After exposure to either asbestos or Dox (25 μmol/L) for 24 hours, coverslips were processed to determine the numbers of apoptotic cells and total cell numbers per field. Five random fields were evaluated at ×400 magnification on each coverslip. Migration was assessed using 6-well Transwell polycarbonate filters (Corning Costar, Corning, NY) with an 8-μm pore size. Approximately 2.5 × 104 cells transfected with either siCREB or control siRNA (siC) were seeded in the upper chamber of the Transwell inserts and incubated for 72 hours at 37°C in serum-free medium. Dulbecco’s modified Eagle’s medium/F12 containing 10% FBS was used as a chemoattractant in the bottom chamber. Cells that did not migrate through the pores of the Transwell inserts were manually removed with a cotton swab. Cells that migrated to the bottom of the membrane were fixed in cold methanol for 10 minutes and then stained with 0.01% crystal violet in 20% ethanol. After incubating for 10 minutes, filters were washed thoroughly in water and suspended in 200 μl of 5% acetic acid and 5% methanol. Colorimetric readings were taken at OD595. Three MM tissue arrays (obtained from Dr. H. I. Pass) were examined. Each array contained 10 to 15 MM sections from different patients with pleural mesothelioma (N = 40 total), 1 section of lung carcinoma (epithelioid clear cell carcinoma) and 1 section of normal lung, kidney, and liver. In addition, we evaluated three reactive mesothelial hyperplasias and four additional sections from normal lungs. In brief, slides containing 5-μm-thick sections were deparaffinized in xylene and ethanol. After antigen retrieval at 95°C in DakoCytomation (Carpinteria, CA) target retrieval solution, sections were blocked with “peroxidase block” and then with “protein block” sequentially for 30 minutes in a humidified chamber. After washing in PBS, sections were incubated with 1/100 dilution of polyclonal anti-rabbit pCREB (Cell Signaling Technology) overnight at 4°C in a humidified chamber. After washing in PBS, biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) was applied to sections for 1 hour at room temperature. Each section was then treated with streptavidin-horseradish peroxidase for 30 minutes in a humidified chamber, and color was developed using DAB chromogen for 5 minutes. Each slide was counterstained with Gill’s hematoxylin. After brief washes in 100% ethanol and xylene, slides were air dried, and coverslips were mounted with Permaslip. For the negative control, one slide was stained as described above but without primary antibody. Slides were examined by a board certified pathologist (Dr. M. W. Bosenberg) using a blind coding system for the localization and intensity of pCREB expression (0 = no staining; 1 = weak; 2 = moderate; and 3 = strong). For all in vitro experiments, at least three independent experiments were performed (N = 2 to 4 samples/group/experiment). Statistical significance was evaluated by analysis of variance using the Student Neuman-Keul’s procedure for adjustment of multiple pairwise comparisons between treatment groups or the nonparametric Kruskal-Wallis, Mann-Whitney, or Tukey honestly significant difference tests. Values of P < 0.05 were considered statistically significant. Since asbestos activates a number of protein kinase cascades in lung epithelial cells and mesothelial cells after phosphorylation of the EGFR,19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar, 27Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C δ-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (55) Google Scholar, 28Zanella CL Posada J Tritton TR Mossman BT Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.Cancer Res. 1996; 56: 5334-5338PubMed Google Scholar, 29Zanella CL Timblin CR Cummins A Jung M Goldberg J Raabe R Tritton TR Mossman BT Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis.Am J Physiol. 1999; 277: L684-L693PubMed Google Scholar, 30Pache JC Janssen YM Walsh ES Quinlan TR Zanella CL Low RB Taatjes DJ Mossman BT Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.Am J Pathol. 1998; 152: 333-340PubMed Google Scholar, 31Barlow CA Barrett TF Shukla A Mossman BT Lounsbury KM Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2.Am J Physiol. 2007; 292: L1361-L1369Google Scholar we hypothesized that one or more of these pathways might be linked to CREB1 phosphorylation by asbestos in human mesothelial cells. In both LP9 cells (Figure 1A) and freshly isolated human mesothelial cells (NYU474) (Figure 1B), asbestos (5 μg/cm2 dish) caused increases (P < 0.05) in CREB1 phosphorylation that persisted for 24 hours. The positive control, Forskolin (10 μmol/L; 15 minutes), also phosphorylated CREB1 and ATF1 in human mesothelial cells (Figure 1, A and B). After addition of asbestos, more protracted phosphorylation of proteins over time may be related to the fact that asbestos fibers are insoluble and slowly precipitate on or remain within mesothelial cells. To assess the mechanisms of activation of CREB1 by asbestos in LP9 cells, we used small-molecule inhibitors known to regulate CREB activation in other cell types.12Aggarwal S Kim SW Ryu SH Chung WC Koo JS Growth suppression of lung cancer cells by targeting cyclic AMP response element-binding protein.Cancer Res. 2008; 68: 981-988Crossref PubMed Scopus (83) Google Scholar, 19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar, 26Barlow CA Shukla A Mossman BT Lounsbury KM Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells.Am J Respir Cell Mol Biol. 2006; 34: 7-14Crossref PubMed Scopus (24) Google Scholar, 27Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C δ-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (55) Google Scholar, 28Zanella CL Posada J Tritton TR Mossman BT Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.Cancer Res. 1996; 56: 5334-5338PubMed Google Scholar, 29Zanella CL Timblin CR Cummins A Jung M Goldberg J Raabe R Tritton TR Mossman BT Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis.Am J Physiol. 1999; 277: L684-L693PubMed Google Scholar, 30Pache JC Janssen YM Walsh ES Quinlan TR Zanella CL Low RB Taatjes DJ Mossman BT Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.Am J Pathol. 1998; 152: 333-340PubMed Google Scholar, 31Barlow CA Barrett TF Shukla A Mossman BT Lounsbury KM Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2.Am J Physiol. 2007; 292: L1361-L1369Google Scholar These included specific small-molecule inhibitors of ERK1/2 (U0126; 10 and 20 μmol/L), PKA (H89, 10 μmol/L), CaM kinase II (KN93 and its inactive analog, KN92; 30 μmol/L), general PKCs (Bisindolymaleimide I; 5 μmol/L), PKCδ (Rottlerin; 5 μmol/L), and EGFR phosphorylation (AG1478; 10 and 20 μmol/L) at nontoxic concentrations. As shown in Figure 1C, the PKA inhibitor, H89, completely abolished the increased CREB1 phosphorylation by asbestos, whereas the MEK1/2 inhibitor U0126 had no effect. In contrast, the EGFR inhibitor, AG1478, blocked asbestos-induced CREB activation significantly at both concentrations (10 and 20 μmol/L) (Figure 1D). Inhibitors of CaM kinase II, general PKCs, and PKCδ had no effects on asbestos-induced CREB1 activation (data not shown). These results show that asbestos-induced CREB activation involves signaling via the EGFR and PKA. It should be noted that the pCREB antibody used here also reacted with pATF1, another CREB family member (shown just below the pCREB band in Western blots). To study whether expression of CREB-regulated genes (BCL2, cFOS, MKP1, EGR1, and MMP2, MMP9, and MMP13) was increased in LP9 mesothelial cells exposed to asbestos, RNA was prepared and reverse-transcribed as described in Materials and Methods. We chose to examine gene expression related to regulation of “early response” cell signaling (cFOS, EGR1, and MKP1), apoptosis (BCL2), and extracellular matrix (MMP2, MMP9, and MMP13), responses linked to asbestos after in vitro exposures and inhalation.19Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar, 25Yuan Z Taatjes DJ Mossman BT Heintz NH The duration of nuclear extracellular signal-regulated kinase 1 and 2 signaling during cell cycle reentry distinguishes proliferation from apoptosis in response to asbestos.Cancer Res. 2004; 64: 6530-6536Crossref PubMed Scopus (32) Google Scholar, 26Barlow CA Shukla A Mossman BT Lounsbury KM Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells.Am J Respir Cell Mol Biol. 2006; 34: 7-14Crossref PubMed Scopus (24) Google Scholar, 27Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C δ-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (55) Google Scholar, 28Zanella CL Posada J Tritton TR Mossman BT Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.Cancer Res. 1996; 56: 5334-5338PubMed Google Scholar, 29Zanella CL Timblin CR Cummins A Jung M Goldberg J Raabe R Tritton TR Mossman BT Asbestos-induced phospho

Malignant mesothelioma (MM) remains a highly deadly malignancy with poor treatment option. The MM cells further promote a highly inflammatory microenvironment, which contributes to tumor initiation, development, severity and propagation. We reasoned that the anti-inflammatory actions of mesenchymal stromal cells (MSCs) and further antitumor effects of MSCs engineered to overexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein (MSC-TRAIL) would effectively inhibit mesothelioma growth. Using a mouse xenograft model of intraperitoneal human mesothelioma, native mouse (mMSCs) or human (hMSC) MSCs were administered either systemically (intravenously or intraperitoneally) at various times following tumor inoculation. Both mMSCs and hMSCs localized at the sites of MM tumor growth in vivo and decreased local inflammation. Further, a trend towards decrease in tumor burden was observed. Parallel studies of in vitro exposure of nine primary human mesothelioma cell lines to mMSCs or hMSCs demonstrated reduced tumor cell migration. MSC-TRAIL exposure induced apoptosis of TRAIL-sensitive MM cells in vitro, and both mouse and human MSC-TRAIL significantly reduced the inflammatory tumor environment in vivo. Moreover, human MSC-TRAIL administration significantly reduced peritoneal tumor burden in vivo and increased tumor cell apoptosis. These proof-of-concept studies suggest that TRAIL-expressing MSCs may be useful against malignant mesothelioma.

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.

Journal of Cellular BiochemistryVolume 116, Issue 8 p. 1540-1552 Article Differential Susceptibility of Human Pleural and Peritoneal Mesothelial Cells to Asbestos Exposure Julie Dragon, Julie Dragon Department of Microbiology and Molecular Genetics, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorJoyce Thompson, Joyce Thompson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorMaximilian MacPherson, Maximilian MacPherson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorArti Shukla, Corresponding Author Arti Shukla Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405 Correspondence to: Arti Shukla, Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail: Arti.Shukla@med.uvm.eduSearch for more papers by this author Julie Dragon, Julie Dragon Department of Microbiology and Molecular Genetics, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorJoyce Thompson, Joyce Thompson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorMaximilian MacPherson, Maximilian MacPherson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorArti Shukla, Corresponding Author Arti Shukla Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405 Correspondence to: Arti Shukla, Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail: Arti.Shukla@med.uvm.eduSearch for more papers by this author First published: 10 March 2015 https://doi.org/10.1002/jcb.25095Citations: 18 Conflict of interest: The authors declare no competing interests. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat ABSTRACT 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-1β, 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. J. Cell. Biochem. 116: 1540–1552, 2015. © 2015 Wiley Periodicals, Inc. Citing Literature Supporting Information Additional supporting information may be found in the online version of this article at the publisher's web-site Filename Description jcb25095-sup-0001-SuppData-S1.xls38.5 KB Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume116, Issue8August 2015Pages 1540-1552 RelatedInformation

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.

Significance: The lung is a unique organ, as it is constantly exposed to air, and thus it requires a robust antioxidant defense system to prevent the potential damage from exposure to an array of environmental insults, including oxidants. The peroxiredoxin (PRDX) family plays an important role in scavenging peroxides and is critical to the cellular antioxidant defense system. Recent Advances: Exciting discoveries have been made to highlight the key features of PRDXs that regulate the redox tone. PRDXs do not act in isolation as they require the thioredoxin/thioredoxin reductase/NADPH, sulfiredoxin (SRXN1) redox system, and in some cases glutaredoxin/glutathione, for their reduction. Furthermore, the chaperone function of PRDXs, controlled by the oxidation state, demonstrates the versatility in redox regulation and control of cellular biology exerted by this class of proteins. Critical Issues: Despite the long-known observations that redox perturbations accompany a number of pulmonary diseases, surprisingly little is known about the role of PRDXs in the etiology of these diseases. In this perspective, we review the studies that have been conducted thus far to address the roles of PRDXs in lung disease, or experimental models used to study these diseases. Intriguing findings, such as the secretion of PRDXs and the formation of autoantibodies, raise a number of questions about the pathways that regulate secretion, redox status, and immune response to PRDXs. Future Directions: Further understanding of the mechanisms by which individual PRDXs control lung inflammation, injury, repair, chronic remodeling, and cancer, and the importance of PRDX oxidation state, configuration, and client proteins that govern these processes is needed.

Adverse lung outcomes from exposure to per-and polyfluoroalkyl substances (PFAS) are known; however, the mechanism of action is poorly understood. To explore this, human bronchial epithelial cells were grown and exposed to varied concentrations of short-chain (perfluorobutanoic acid, perflurobutane sulfonic acid and GenX) or long-chain (PFOA and perfluorooctane sulfonic acid (PFOS)) PFAS, alone or in a mixture to identify cytotoxic concentrations. Non-cytotoxic concentrations of PFAS from this experiment were selected to assess NLRP3 inflammasome activation and priming. We found that PFOA and PFOS alone or in a mixture primed and activated the inflammasome compared with vehicle control. Atomic force microscopy showed that PFOA but not PFOS significantly altered the membrane properties of cells. RNA sequencing was performed on the lungs of mice that had consumed PFOA in drinking water for 14 weeks. Wild type (WT), PPARα knock-out (KO) and humanized PPARα (KI) were exposed to PFOA. We found that multiple inflammation- and immune-related genes were affected. Taken together, our study demonstrated that PFAS exposure could alter lung biology in a significant manner and may contribute to asthma/airway hyper-responsiveness.

The role of nitric oxide (NO), a well known vasodilator, in the regulation of blood-brain barrier (BBB) permeability is not clear. Therefore, the present study was planned to assess the role of NO-releasing compounds like sodium nitroprusside (SNP) and the active metabolite of molsidomine, SIN-1, as well as a precursor of NO, L-arginine, on this physiological barrier. The permeability was assessed by using several tracers. All three agents increased the permeability of BBB to the tracer. The increase in permeability caused by L-arginine was not blocked by N-nitro-L-arginine methyl ester (L-NAME). L-Arginine-treated brains did not show an elevation of nitrite content, thus ruling out the possibility of NO generation and its involvement in BBB permeability alteration. It is concluded that NO itself causes an increase in the permeability of BBB. However arginine-induced opening is not NO mediated.

Eight weeks of latent iron deficiency in weaned rats maintained on an experimental low iron content diet (18-20 mg/kg) did not significantly alter the packed cell volume and hemoglobin concentration; however, the hepatic and brain nonheme iron contents decreased by 66% and 21% (p less than 0.001), respectively. The tryptophan concentration decreased by 31% and 34% in liver and brain, respectively, in rats on experimental diet (p less than 0.01). The brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid contents were reduced by 21% and 23% (p less than 0.01 and p less than 0.02), respectively. However, in the brain, weight, protein, DNA, and the activities of monoamine oxidase, aldehyde dehydrogenase, and liver tryptophan oxygenase were found to remain unaltered. When rehabilitated with a diet containing 390 mg/kg iron, rats previously maintained on the experimental diet for 2 weeks showed partial recovery in tryptophan levels both in liver and brain. However, brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels remained unaltered. The hepatic iron content improved without any change in brain iron content. The latent iron deficiency produced significant alterations in the metabolism of 5-hydroxytryptamine and brain iron content that could not be recovered 2 weeks after the iron rehabilitation.

Asbestos is a known inflammatory, carcinogenic, and fibrotic agent, but the mechanisms leading to asbestos-induced lung diseases are unclear. Using a murine inhalation model of fibrogenesis, we show that asbestos causes significant increases in mRNA levels of lung matrix metalloproteinases (MMPs 12 and 13) and tissue inhibitor of metalloproteinases (TIMP1), as well as increased activities of MMP 2, 9, and 12 in bronchoalveolar lavage fluids (BALF). Asbestos-exposed PKCdelta knockout (PKCdelta-/-) mice exhibited decreased expression of lung MMP12 and MMP13 compared with asbestos-exposed wild-type mice. Studies using small molecule inhibitors in murine alveolar epithelial type II cells (C10) and primary lung fibroblasts confirmed that asbestos transcriptionally up-regulates MMPs via an EGFR (or other growth factor receptors)/PI3K/PKCdelta/ERK1/2 pathway. Moreover, use of a broad-spectrum MMP inhibitor showed that MMPs play an important role in further enhancing asbestos-induced signaling events by activating EGFR. These data reveal a potentially important link between asbestos signaling and integrity of the extracellular matrix (ECM) that likely contributes to asbestos-induced lung remodeling and diseases.

Abstract New and effective treatment strategies are desperately needed for malignant mesothelioma (MM), an aggressive cancer with a poor prognosis. We have shown previously that acid‐prepared mesoporous microspheres (APMS) are nontoxic after intrapleural or intraperitoneal (IP) administration to rodents. The purpose here was to evaluate the utility of APMS in delivering chemotherapeutic drugs to human MM cells in vitro and in two mouse xenograft models of MM. Uptake and release of doxorubicin (DOX) alone or loaded in APMS (APMS‐DOX) were evaluated in MM cells. MM cell death and gene expression linked to DNA damage/repair were also measured in vitro . In two severe combined immunodeficient mouse xenograft models, mice received saline, APMS, DOX or APMS‐DOX injected directly into subcutaneous (SC) MM tumors or injected IP after development of human MMs peritoneally. Other mice received DOX intravenously (IV) via tail vein injections. In comparison to DOX alone, APMS‐DOX enhanced intracellular uptake of DOX, MM death and expression of GADD34 and TP73 . In the SC MM model, 3× weekly SC injections of APMS‐DOX or DOX alone significantly inhibited tumor volumes, and systemic DOX administration was lethal. In mice developing IP MMs, significant ( p &lt; 0.05) inhibition of mesenteric tumor numbers, weight and volume was achieved using IP administration of APMS‐DOX at one‐third the DOX concentration required after IP injections of DOX alone. These results suggest APMS are efficacious for the localized delivery of lower effective DOX concentrations in MM and represent a novel means of treating intracavitary tumors.

Asbestos-related diseases continue to result in approximately 120,000 deaths every year in the United States and worldwide. Although extensive research has been conducted on health effects of occupational exposures to asbestos, many issues related to environmental asbestos exposures remain unresolved. For example, environmental asbestos exposures associated with a former mine in Libby, Montana, have resulted in high rates of nonoccupational asbestos-related disease. Additionally, other areas with naturally occurring asbestos deposits near communities in the United States and overseas are undergoing investigations to assess exposures and potential health risks. Some of the latest public health, epidemiological, and basic research findings were presented at a workshop on asbestos at the 2014 annual meeting of the Society of Toxicology in Phoenix, Arizona. The following focus areas were discussed: a) mechanisms resulting in fibrosis and/or tumor development; b) relative toxicity of different forms of asbestos and other hazardous elongated mineral particles (EMPs); c) proper dose metrics (e.g., mass, fiber number, or surface area of fibers) when interpreting asbestos toxicity; d) asbestos exposure to susceptible populations; and e) using toxicological findings for risk assessment and remediation efforts. The workshop also featured asbestos research supported by the National Institute of Environmental Health Sciences, the Agency for Toxic Substances and Disease Registry, and the U.S. Environmental Protection Agency. Better protection of individuals from asbestos-related health effects will require stimulation of new multidisciplinary research to further our understanding of what constitutes hazardous exposures and risk factors associated with toxicity of asbestos and other hazardous EMPs (e.g., nanomaterials).

Abstract Diet has a profound impact on health and susceptibility to cancer. Adaptation to a Western diet is strongly associated with increased colorectal cancer (CRC) and obesity prevalence. This study demonstrates the epigenetic changes in intestinal stem cells (ISCs) induced by a pro-obesity high-fat Western diet (HFD) and the heightened risk of oncogenic transformation. A HFD induces a phenotype characterized by increased ISC frequency, proliferation, regenerative capacity, and ability to initiate adenomas. We hypothesized that a HFD generates lasting epigenetic alterations in ISCs thereby enhancing oncogenic susceptibility. We evaluated the chromatin landscape of intestinal stem cells (ISCs) and the reversibility of the epigenetic changes. Mice carrying a fluorescent reporter of the ISC marker Lgr5, were subjected to a control diet, HFD, or HFD with a return to control. We isolated ISCs by flow cytometry and used an assay for transposase-accessible chromatin sequencing (ATAC-seq) to identify differentially accessible genomic regions correlated with the ISC phenotype. We found that a HFD induces distinct changes in the chromatin landscape that persist after removal from the HFD. These accessible regions are associated with genes involved in lipid metabolism and are enriched with PPAR (Peroxisome Proliferator-Activated Receptor) binding motifs. We demonstrate that these lipid metabolism regulators, PPAR-delta and PPAR-alpha, are necessary for the majority of significant accessible regions. We further show that HFD-induced accessibility significantly correlates with early chromatin alterations seen by loss of tumor suppressor APC. Our data supports a mechanistic pathway for diet-induced epigenetic reprogramming due to PPAR activity and suggests that the HFD imposes a preneoplastic stem cell chromatin state that stokes the risk of oncogenic change. Citation Format: Dominic R. Saiz, Thomas Hartley-McDerrmott, Yesenia Barrera-Millan, Karla Fabiola Castro-Ochoa, Abhigyan Shukla, Matthew Torel, Miyeko D. Mana. PPARsing epigenetic memory in intestinal stem cells: High-fat diet and oncogenic susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1247.

Fibrosis is a consequence of injury which is characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and function. Transforming growth factor‐β, a potent wound healing agent, has also been shown to be an agent that can produce fibrosis because it is a potent stimulator of collagen synthesis. Both glucocorticoids and bleomycin have recently been shown to affect collagen synthesis in opposite directions, by utilizing a common pathway of involving transforming growth factor‐β activator protein binding to the transforming growth factor‐β element. This article presents a mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor‐β pathway.

Exposure to ambient particulate matter (PM) is linked to increases in respiratory morbidity and exacerbation of cardiopulmonary diseases. However, the important components of PM and their mechanisms of action in lung disease are unclear. We demonstrate the development of dose-related proliferation and apoptosis after exposure of an alveolar epithelial cell line (C10) to PM or to ultrafine carbon black (ufCB), a component of PM. Ribonuclease protection assays demonstrated that increases in mRNA levels of the early response protooncogenes c-jun, junB, fra-1, and fra-2 accompanied cell proliferation at low concentrations of PM whereas apoptotic concentrations of PM caused transient increases in expression of fos and jun family members and dose responsive increases in mRNA levels of receptor-interacting protein, Fas-associated death domain, and caspase-8. Significant increases in steady-state mRNA levels of protooncogenes and apoptosis-associated genes, TNFR-associated death domain, and Fas were also observed after exposure of epithelial cells to ufCB, but not fine carbon black or glass beads, respectively, suggesting that the ultrafine particulate component of PM is critical to its biological activity.

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Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development of malignant and fibrotic lung diseases. Asbestos exposure leads to apoptosis, followed by compensatory proliferation, yet many of the signaling cascades coupled to these outcomes are unclear. Because CREs (Ca(2+)/cAMP-response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB (CRE binding protein) is likely to play an important role in the development of asbestos-mediated lung injury. To explore this possibility, we tested the hypotheses that asbestos exposure leads to CREB phosphorylation in lung epithelial cells and that protein kinase A (PKA) and extracellular signal-regulated kinases 1/2 (ERK1/2) are central regulators of the CREB pathway. Persistent CREB phosphorylation was observed in lung sections from mice following inhalation of crocidolite asbestos. Exposure of C10 lung epithelial cells to crocidolite asbestos led to rapid CREB phosphorylation and apoptosis that was decreased by the inhibition of PKA or ERK1/2 using the specific inhibitors H89 and U0126, respectively. Furthermore, crocidolite asbestos selectively induced a sustained increase in MAP kinase phosphatase-1 mRNA and protein. Silencing CREB protein dramatically reduced asbestos-mediated ERK1/2 phosphorylation, yet significantly increased the number of cells undergoing asbestos-induced apoptosis. These data reveal a novel and selective role for CREB in asbestos-mediated signaling through pathways regulated by PKA and ERK1/2, further providing evidence that CREB is an important regulator of apoptosis in asbestos-induced responses of lung epithelial cells.

Identifying and understanding the early molecular events that underscore mineral pathogenicity using in vitro screening tests is imperative, especially given the large number of synthetic and natural fibers and particles being introduced into the environment. The purpose of the work described here was to examine the ability of gene profiling (Affymetrix microarrays) to predict the pathogenicity of various materials in a human mesothelial cell line (LP9/TERT-1) exposed to equal surface area concentrations (15 × 106 or 75 × 106 μm2/cm2) of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 h. Since crocidolite asbestos caused the greatest number of alterations in gene expression, multiplex analysis (Bio-Plex) of proteins released from LP9/TERT-1 cells exposed to crocidolite asbestos was also assessed to reveal if this approach might also be explored in future assays comparing various mineral types. To verify that LP9/TERT-1 cells were more sensitive than other cell types to asbestos, human ovarian epithelial cells (IOSE) were also utilized in microarray studies. Upon assessing changes in gene expression via microarrays, principal component analysis (PCA) of these data was used to identify patterns of differential gene expression. PCA of microarray data confirmed that LP9/TERT-1 cells were more responsive than IOSE cells to crocidolite asbestos or nonfibrous talc, and that crocidolite asbestos elicited greater responses in both cell types when compared to nonfibrous talc, TiO2, or glass beads. Bio-Plex analysis demonstrated that asbestos caused an increase in interleukin-13 (IL‐13), basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF). These responses were generally dose-dependent (bFGF and G-CSF only) and tumor necrosis factor (TNF)-α independent (except for G‐CSF). Thus, microarray and Bio-Plex analyses are valuable in determining early molecular responses to fibers/particles and may directly contribute to understanding the etiology of diseases caused by them. The number and magnitude of changes in gene expression or “profiles” of secreted proteins may serve as valuable metrics for determining the potential pathogenicity of various mineral types. Hence, alterations in gene expression and cytokine/chemokine changes induced by crocidolite asbestos in LP9/TERT-1 cells may be indicative of its increased potential to cause mesothelioma in comparison to the other nonfibrous materials examined.

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 (15×10(6) μm2/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 (15×10(6) μm2/cm2) and toxic concentrations (75×10(6) μm2/cm2) of Libby six-mix.Exposure to 15×10(6) μm2/cm2 Libby six-mix elicited significant (p < 0.05) upregulation of one gene (SOD2; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in SOD2. 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 (75×10(6) μm2/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 75×10(6) μm2/cm2 caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (HO-1) in LP9/TERT-1 and HKNM-2 cells.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.

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.

The objective of the present investigation was to study the involvement of NO in regulating the permeability of the blood-brain barrier (BBB) during infections, since NOS is known to be induced following infections. The administration of inactivated Escherichia coli (a source of lipopolysaccharide) or poly (I:C), an interferon inducer, to rats increased the permeability of BBB significantly. This increase was found to be potentiated in the presence of L-arginine, a substrate for NOS, while D-arginine had no such effect. N-nitro L-arginine methyl ester, an inhibitor of NOS, and dexamethasone, an inhibitor of NOS induction, blocked the E. coli-induced effects. These results suggest that during infections, NOS inductions causes the release of large quantities of NO, resulting in increased BBB permeability.

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos. Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos. Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.1Mossman BT Bignon J Corn M Seaton A Gee JB Asbestos: scientific developments and implications for public policy.Science. 1990; 247: 294-301Crossref PubMed Scopus (578) Google Scholar, 2Mossman BT Churg A Mechanisms in the pathogenesis of asbestosis and silicosis.Am J Respir Crit Care Med. 1998; 157: 1666-1680Crossref PubMed Scopus (745) Google Scholar The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.3Mossman BT Lounsbury KM Reddy SP Oxidants and signaling by mitogen-activated protein kinases in lung epithelium.Am J Respir Cell Mol Biol. 2006; 34: 666-669Crossref PubMed Scopus (138) Google Scholar, 4Shukla A Gulumian M Hei TK Kamp D Rahman Q Mossman BT Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases.Free Radic Biol Med. 2003; 34: 1117-1129Crossref PubMed Scopus (232) Google Scholar, 5Jung M Davis WP Taatjes DJ Churg A Mossman BT Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo.Free Radic Biol Med. 2000; 28: 1295-1299Crossref PubMed Scopus (38) Google Scholar, 6Panduri V Surapureddi S Soberanes S Weitzman SA Chandel N Kamp DW p53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am J Respir Cell Mol Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.2Mossman BT Churg A Mechanisms in the pathogenesis of asbestosis and silicosis.Am J Respir Crit Care Med. 1998; 157: 1666-1680Crossref PubMed Scopus (745) Google Scholar Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,5Jung M Davis WP Taatjes DJ Churg A Mossman BT Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo.Free Radic Biol Med. 2000; 28: 1295-1299Crossref PubMed Scopus (38) Google Scholar, 6Panduri V Surapureddi S Soberanes S Weitzman SA Chandel N Kamp DW p53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am J Respir Cell Mol Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar which may lead to compensatory cell proliferation7Buder-Hoffmann S Palmer C Vacek P Taatjes D Mossman B Different accumulation of activated extracellular signal-regulated kinases (ERK 1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells.Am J Respir Cell Mol Biol. 2001; 24: 405-413Crossref PubMed Scopus (85) Google Scholar, 8Sabo-Attwood T Ramos-Nino M Bond J Butnor K Heintz N Gruber A Steele C Taatjes D Vacek P Mossman B Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis.Am J Pathol. 2005; 167: 1243-1256Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and the production of inflammatory and fibrogenic cytokines.8Sabo-Attwood T Ramos-Nino M Bond J Butnor K Heintz N Gruber A Steele C Taatjes D Vacek P Mossman B Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis.Am J Pathol. 2005; 167: 1243-1256Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Haegens A Barrett TF Gell J Shukla A Macpherson M Vacek P Poynter ME Butnor KJ Janssen-Heininger YM Steele C Mossman BT Airway epithelial NF-{kappa}B activation modulates asbestos-induced inflammation and mucin production in vivo.J Immunol. 2007; 178: 1800-1808PubMed Google Scholar, 10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),3Mossman BT Lounsbury KM Reddy SP Oxidants and signaling by mitogen-activated protein kinases in lung epithelium.Am J Respir Cell Mol Biol. 2006; 34: 666-669Crossref PubMed Scopus (138) Google Scholar, 7Buder-Hoffmann S Palmer C Vacek P Taatjes D Mossman B Different accumulation of activated extracellular signal-regulated kinases (ERK 1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells.Am J Respir Cell Mol Biol. 2001; 24: 405-413Crossref PubMed Scopus (85) Google Scholar, 11Cummins AB Palmer C Mossman BT Taatjes DJ Persistent localization of activated extracellular signal-regulated kinases (ERK1/2) is epithelial cell-specific in an inhalation model of asbestosis.Am J Pathol. 2003; 162: 713-720Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 12Robledo RF Buder-Hoffmann SA Cummins AB Walsh ES Taatjes DJ Mossman BT Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.Am J Pathol. 2000; 156: 1307-1316Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar nuclear factor-κB (NF-κB),9Haegens A Barrett TF Gell J Shukla A Macpherson M Vacek P Poynter ME Butnor KJ Janssen-Heininger YM Steele C Mossman BT Airway epithelial NF-{kappa}B activation modulates asbestos-induced inflammation and mucin production in vivo.J Immunol. 2007; 178: 1800-1808PubMed Google Scholar, 13Janssen YM Barchowsky A Treadwell M Driscoll KE Mossman BT Asbestos induces nuclear factor kappa B (NF-kappa B) DNA-binding activity and NF-kappa B-dependent gene expression in tracheal epithelial cells.Proc Natl Acad Sci USA. 1995; 92: 8458-8462Crossref PubMed Scopus (152) Google Scholar, 14Yang H Bocchetta M Kroczynska B Elmishad AG Chen Y Liu Z Bubici C Mossman BT Pass HI Testa JR Franzoso G Carbone M TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis.Proc Natl Acad Sci USA. 2006; 103: 10397-10402Crossref PubMed Scopus (230) Google Scholar and the protein kinase (PK)C10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Robledo RF Buder-Hoffmann SA Cummins AB Walsh ES Taatjes DJ Mossman BT Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.Am J Pathol. 2000; 156: 1307-1316Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar, 16Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C delta-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (54) Google Scholar and A families.17Barlow CA Barrett TF Shukla A Mossman BT Lounsbury KM Asbestos-mediated Creb phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2.Am J Physiol Lung Cell Mol Physiol. 2007; 292: L1361-L1369Crossref PubMed Scopus (28) Google Scholar A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Robledo RF Buder-Hoffmann SA Cummins AB Walsh ES Taatjes DJ Mossman BT Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.Am J Pathol. 2000; 156: 1307-1316Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C delta-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (54) Google Scholar via increased formation of diacylglycerol.18Sesko A Cabot M Mossman B Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.Proc Natl Acad Sci USA. 1990; 87: 7385-7389Crossref PubMed Scopus (31) Google Scholar We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.16Shukla A Stern M Lounsbury KM Flanders T Mossman BT Asbestos-induced apoptosis is protein kinase C delta-dependent.Am J Respir Cell Mol Biol. 2003; 29: 198-205Crossref PubMed Scopus (54) Google Scholar Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Shukla A Barrett TF Nakayama KI Nakayama K Mossman BT Lounsbury KM Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung.FASEB J. 2006; 20: 997-999Crossref PubMed Scopus (34) Google Scholar A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.19Lucas M Sanchez-Margalet V Protein kinase C involvement in apoptosis.Gen Pharmacol. 1995; 26: 881-887Crossref PubMed Scopus (100) Google Scholar, 20Whelan RD Parker PJ Loss of protein kinase C function induces an apoptotic response.Oncogene. 1998; 16: 1939-1944Crossref PubMed Scopus (136) Google Scholar In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca2+/calmodulin-dependent kinase superfamily.21Rozengurt E Rey O Waldron RT Protein kinase D signaling.J Biol Chem. 2005; 280: 13205-13208Crossref PubMed Scopus (360) Google Scholar PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.22Valverde AM Sinnett-Smith J Van Lint J Rozengurt E Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain.Proc Natl Acad Sci USA. 1994; 91: 8572-8576Crossref PubMed Scopus (356) Google Scholar The other two members of this family include PKD223Sturany S Van Lint J Muller F Wilda M Hameister H Hocker M Brey A Gern U Vandenheede J Gress T Adler G Seufferlein T Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases.J Biol Chem. 2001; 276: 3310-3318Crossref PubMed Scopus (156) Google Scholar and PKD3, (originally PKCν).24Hayashi A Seki N Hattori A Kozuma S Saito T PKCnu, a new member of the protein kinase C family, composes a fourth subfamily with PKCmu.Biochim Biophys Acta. 1999; 1450: 99-106Crossref PubMed Scopus (171) Google Scholar Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.25Waldron RT Rozengurt E Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C.J Biol Chem. 2000; 275: 17114-17121Crossref PubMed Scopus (111) Google Scholar In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,26Storz P Doppler H Toker A Protein kinase Cdelta selectively regulates protein kinase D-dependent activation of NF-kappaB in oxidative stress signaling.Mol Cell Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (200) Google Scholar as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.28Lemonnier J Ghayor C Guicheux J Caverzasio J Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation.J Biol Chem. 2004; 279: 259-264Crossref PubMed Scopus (112) Google Scholar Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,21Rozengurt E Rey O Waldron RT Protein kinase D signaling.J Biol Chem. 2005; 280: 13205-13208Crossref PubMed Scopus (360) Google Scholar, 26Storz P Doppler H Toker A Protein kinase Cdelta selectively regulates protein kinase D-dependent activation of NF-kappaB in oxidative stress signaling.Mol Cell Biol. 2004; 24: 2614-2626Crossref PubMed Scopus (200) Google Scholar, 29Storz P Doppler H Toker A Activation loop phosphorylation controls protein kinase D-dependent activation of nuclear factor kappaB.Mol Pharmacol. 2004; 66: 870-879Crossref PubMed Scopus (99) Google Scholar, 30Wang QJ PKD at the crossroads of DAG and PKC signaling.Trends Pharmacol Sci. 2006; 27: 317-323Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar but how these affect functional ramifications of carcinogens, such as asbestos, are unclear. The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.31Cory S Adams JM The Bcl2 family: regulators of the cellular life-or-death switch.Nat Rev Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3258) Google Scholar, 32Puthalakath H Strasser A Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins.Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (620) Google Scholar There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).33O'Connor L Strasser A O'Reilly LA Hausmann G Adams JM Cory S Huang DC Bim: a novel member of the Bcl-2 family that promotes apoptosis.EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (943) Google Scholar BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,34Weber A Paschen SA Heger K Wilfling F Frankenberg T Bauerschmitt H Seiffert BM Kirschnek S Wagner H Hacker G BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins.J Cell Biol. 2007; 177: 625-636Crossref PubMed Scopus (74) Google Scholar whereas BimL is bound to microtubules and is less cytotoxic.35Puthalakath H Huang DC O'Reilly LA King SM Strasser A The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.Mol Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.36Lei K Davis RJ JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis.Proc Natl Acad Sci USA. 2003; 100: 2432-2437Crossref PubMed Scopus (880) Google Scholar BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)37Ewings KE Hadfield-Moorhouse K Wiggins CM Wickenden JA Balmanno K Gilley R Degenhardt K White E Cook SJ ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.EMBO J. 2007; 26: 2856-2867Crossref PubMed Scopus (137) Google Scholar and proteasomal degradation.38Ley R Balmanno K Hadfield K Weston C Cook SJ Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein Bim.J Biol Chem. 2003; 278: 18811-18816Crossref PubMed Scopus (497) Google Scholar We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases. A contact inhibited, non-transformed murine alveolar type II epithelial cell line (C10),39Malkinson AM Dwyer-Nield LD Rice PL Dinsdale D Mouse lung epithelial cell lines–tools for the study of differentiation and the neoplastic phenotype.Toxicology. 1997; 123: 53-100Crossref PubMed Scopus (115) Google Scholar was grown in CMRL 1066 medium supplemented with l-glutamine (2 mmol/L), penicillin/streptomycin (100 μg/ml), and 10% fetal bovine serum (GIBCO BRL, Rockville, MD). Cells were grown to near confluence; then complete medium was replaced with serum-free (maintenance) medium for 16 hours before exposure to asbestos or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml; Calbiochem, La Jolla, CA). Crocidolite asbestos fibers [Na2(Fe3+)2(Fe2+)3(OH)2(Si8O22)], National Institute of Environmental Health Sciences reference sample) were suspended in Hanks' balanced salt solution (GIBCO BRL, Rockville, MD) at a concentration of 1 mg/ml, sonicated, triturated 10X through a 22-gauge needle to obtain a homogenous suspension, and added directly to medium at a final concentration of 5 μg/cm2 area culture dish for the times indicated. This concentration was selected because it causes apoptosis in C10 cells at 24 hours followed by compensatory proliferation at 72 hours.7Buder-Hoffmann S Palmer C Vacek P Taatjes D Mossman B Different accumulation of activated extracellular signal-regulated kinases (ERK 1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells.Am J Respir Cell Mol Biol. 2001; 24: 405-413Crossref PubMed Scopus (85) Google Scholar In some experiments, the NF-κB inhibitor, Bay 11-7082 (5 and 10 μmol/L; Sigma, St. Louis, MO), the JNK inhibitor SP600125 (10 and 20 μmol/L; Calbiochem, La Jolla, CA), or the ERK1/2 inhibitor U0126 (10 μmol/L; Sigma, St. Louis, MO) were added to C10 cells 1 hour before asbestos at effective concentrations of inhibitors reported in the literature.40Pierce JW Schoenleber R Jesmok G Best J Moore SA Collins T Gerritsen ME Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.J Biol Chem. 1997; 272: 21096-21103Crossref PubMed Scopus (906) Google Scholar, 41Bennett BL Sasaki DT Murray BW O'Leary EC Sakata ST Xu W Leisten JC Motiwala A Pierce S Satoh Y Bhagwat SS Manning AM Anderson DW SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.Proc Natl Acad Sci USA. 2001; 98: 13681-13686Crossref PubMed Scopus (2187) Google Scholar, 42Yuan Z Taatjes DJ Mossman BT Heintz NH The duration of nuclear extracellular signal-regulated kinase 1 and 2 signaling during cell cycle reentry distinguishes proliferation from apoptosis in response to asbestos.Cancer Res. 2004; 64: 6530-6536Crossref PubMed Scopus (32) Google Scholar Control cultures received medium without agents and were treated identically. All experiments were performed in triplicate or more. Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals following protocols approved by the University of Vermont Institutional Animal Care and Use Committee. PKCδ−/− mice bred into the C57Bl/6 background were characterized as PKCδ+/+ or PKCδ−/− as described previously.10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 43Miyamoto A Nakayama K Imaki H Hirose S Jiang Y Abe M Tsukiyama T Nagahama H Ohno S Hatakeyama S Nakayama KI Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta.Nature. 2002; 416: 865-869Crossref PubMed Scopus (360) Google Scholar In brief, mice (8 to 12 weeks of age) were exposed to ambient air or the National Institute of Environmental Health Sciences reference sample of chrysotile asbestos (7 mg/m3 air, 6 hours/day, 5 days/week) for 9 or 40 days as described previously.9Haegens A Barrett TF Gell J Shukla A Macpherson M Vacek P Poynter ME Butnor KJ Janssen-Heininger YM Steele C Mossman BT Airway epithelial NF-{kappa}B activation modulates asbestos-induced inflammation and mucin production in vivo.J Immunol. 2007; 178: 1800-1808PubMed Google Scholar, 10Shukla A Lounsbury KM Barrett TF Gell J Rincon M Butnor KJ Taatjes DJ Davis GS Vacek P Nakayama KI Nakayama K Steele C Mossman BT Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-{delta} knockout mice.Am J Pathol. 2007; 170: 140-151Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12Robledo RF Buder-Hoffmann SA Cummins AB Walsh ES Taatjes DJ Mossman BT Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.Am J Pathol. 2000; 156: 1307-1316Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The mice were euthanized using an intraperitoneal injection of pentobarbital (Abbott Laboratories, Abbott Park, IL), the lungs instilled with PBS, and lobes tied off using sutures. The left lung lobe was immersed in optimal cutting temperature embedding compound (Tissue-Tek, Torrance, CA) before snap-freezing in liquid-cooled isopentane and storage at −80°C until sectioning. Other lobes were used for Western blot analyses. The siControl Non-Targeting small interfering (si)RNA #2 (scrambled control), Smartpool mouse PKD, or PKCδ siRNA (50 nmol/L, Dharmacon, Lafayette, CO) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer's protocol. The efficiency of PKD and PKCδ knockdown was determined by Western blot analysis after 48 and 72 hours. To determine whether phosphorylated PKD was expressed in lung after inhalation of asbestos, 10 μm thick sections of lung tissue were fixed in 4% paraformaldehyde for 7 minutes, washed in PBS, and permeabilized with methanol for 10 minutes at −20°C. After washing in PBS, sections were treated for antigen retrieval in 1% SDS for 5 minutes, washed with PBS, and then incubated with a blocking solution containing 10% normal goat serum in 2% bovine serum albumin (BSA)/0.1% Triton X-100/PBS solution for 1 hour at room temperature. After aspiration of blocking solution, primary antibody (rabbit polyclonal phospho-Ser744/Ser748 PKD, Cell Signaling Technology, Danvers, MA; 1:1000) diluted in 2% BSA plus 0.1% Triton X-100 in PBS (BSA/PBS-T) was added, and sections were incubated overnight at 4°C. Sections were then washed with PBS, and incubated with secondary antibody (AlexaFluor 568 goat-anti-rabbit IgG, Molecular Probes, Carlsbad, CA; 1:400 in PBS) for 1 hour at room temperature. Controls consisted of sections incubated with secondary antibody alone. Sections then were washed in PBS followed by incubation with the nuclear counterstain, YOYO-1 iodide (Molecular Probes; 1:10000), 1 unit/ml RNase, and 0.1% sodium azide in BSA/PBS-T for 30 minutes at room temperature. After washing in PBS, coverslips were mounted onto slides using AquaPolyMount (Polysciences, Inc. Warrington, PA). For each section, confocal images were collected in fluorescence modes using a Bio-Rad MRC1024ES confocal scanning laser microscope (Bio-Rad, Hercules, CA). To determine whether PKD-driven signaling pathways occurred in the same cell, confluent C10 cells were exposed to H2O2, a mediator of asbestos-induced lung injury4Shukla A Gulumian M Hei TK Kamp D Rahman Q Mossman BT Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases.Free Radic Biol Med. 2003; 34: 1117-1129Crossref PubMed Scopus (232) Google Scholar, 44Mossman BT Marsh JP Sesko A Hill S Shatos MA Doherty J Petruska J Adler KB Hemenway D Mickey R Vacek P Kagan E Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis.Am Rev Respir Dis. 1990; 141: 1266-1271Crossref PubMed Google Scholar at concentrations (0.5 and 5.0 mmol/L for 30 minutes) sufficient to activate JNK1/2 and induce cell death.45Pantano C Shrivastava P McElhinney B Janssen-Heininger Y Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase.J Biol Chem. 2003; 278: 44091-44096Crossref PubMed Scopus (73) Google Scholar C10 cells were grown to confluency in 4 well-chambered slides (BD Biosciences, San Jose, CA), and untreated

ABSTRACT Malignant mesothelioma (MM) is an aggressive cancer of the mesothelium caused by asbestos. Asbestos use has been reduced but not completely stopped. In addition, natural or man‐made disasters will continue to dislodge asbestos from old buildings into the atmosphere and as long as respirable asbestos is available, MM will continue to be a threat. Due to the long latency period of MM development, it would still take decades to eradicate this disease if asbestos was completely removed from our lives today. Therefore, there is a need for researchers and clinicians to work together to understand this deadly disease and find a solution for early diagnosis and treatment. This article focuses on developmental mechanisms as well as current therapies available for MM. J. Cell. Biochem. 115: 1–7, 2014. © 2013 Wiley Periodicals, Inc.

Occupational and environmental exposures to airborne asbestos and silica are associated with the development of lung fibrosis in the forms of asbestosis and silicosis, respectively. However, both diseases display distinct pathologic presentations, likely associated with differences in gene expression induced by different mineral structures, composition and bio-persistent properties. We hypothesized that effects of mineral exposure in the airway epithelium may dictate deviating molecular events that may explain the different pathologies of asbestosis versus silicosis. Using robust gene expression-profiling in conjunction with in-depth pathway analysis, we assessed early (24 h) alterations in gene expression associated with crocidolite asbestos or cristobalite silica exposures in primary human bronchial epithelial cells (NHBEs). Observations were confirmed in an immortalized line (BEAS-2B) by QRT-PCR and protein assays. Utilization of overall gene expression, unsupervised hierarchical cluster analysis and integrated pathway analysis revealed gene alterations that were common to both minerals or unique to either mineral. Our findings reveal that both minerals had potent effects on genes governing cell adhesion/migration, inflammation, and cellular stress, key features of fibrosis. Asbestos exposure was most specifically associated with aberrant cell proliferation and carcinogenesis, whereas silica exposure was highly associated with additional inflammatory responses, as well as pattern recognition, and fibrogenesis. These findings illustrate the use of gene-profiling as a means to determine early molecular events that may dictate pathological processes induced by exogenous cellular insults. In addition, it is a useful approach for predicting the pathogenicity of potentially harmful materials.

The ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibits the glucosylation of ceramide and thus, by virtue of the normal catabolism of the higher glucosphingolipids, leads to a general depletion of cellular glucolipids. In a previous study with chronic administration of this inhibitor in mice, it was found that the kidneys and liver, particularly the former, grew more poorly than the organs of control mice. This study shows that the inhibitor produces rapid decreases in glucolipid concentration in kidney which are maintained for at least 5 days without noticeable harm. The changes were enhanced by inclusion of L-cycloserine in the injection scheme. Cycloserine blocks ketosphinganine synthase and thus slows the synthesis of all sphingolipids. However, sphingomyelin levels did not drop significantly in this study. The glucosyltransferase inhibitor also produced a small decrease in kidney beta-D-glucuronidase and distinct increases in the levels of glucocerebrosidase, galactocerebrosidase and sphingomyelinase. It also produced a small but distinct decrease in the level of glucosyltransferase, after a delay of a few hours, possibly because the inhibitor was metabolized to a covalently inactivating product. Comparison with kidney, liver and brain showed that the kidney was more sensitive to the action of the morpholino inhibitor.

Feeding a marginally low iron content diet (18–20 mg iron/kg diet) to weaned (21-day-old) rats for 8 weeks produced a significant decrease in liver non-heme iron (66%, p<0.001) but no change in blood hemoglobin. Total iron contents of liver (56%, p<0.01), spleen (20%, p<0.05), and kidney (19%, p<0.05) were also found to decrease along with increased zinc, copper, calcium, manganese lead and cadmium in various organs. The magnitude of alteration of a metal was different in different organs. However, liver was found to be the most affected organ. Two weeks of rehabilitation with iron-sufficient diet (390 mg iron/kg diet) normalized these altered levels.

Abstract Asbestos fibers cause persistent increases in activator protein-1 (AP-1) family member proto-oncogenes in lung epithelial and mesothelial cells that are linked to proliferation and cell transformation. Using lung epithelial cells, the progenitor cells of lung cancers, we report that crocidolite asbestos initially depletes intracellular glutathione followed by up-regulation of both catalytic and modifier subunits of γ-glutamylcysteine synthetase. In vivo asbestos inhalation experiments confirm increased protein levels of γ-glutamylcysteine synthetase in mouse lungs. We also show that asbestos-induced mRNA levels of fos/jun proto-oncogenes, fra-1 transactivation, and AP-1 to DNA binding activity are glutathione-dependent. Epidermal growth factor receptor activity by asbestos is blocked by N-acetyl-l-cysteine, suggesting that it is an initial redox-activated event leading to downstream AP-1 proto-oncogene up-regulation. The overexpression of subunits of γ-glutamylcysteine synthetase in combination completely blocked asbestos-induced up-regulation of AP-1 proto-oncogene expression. However, when overexpressed individually, the modifier subunit had more dramatic effects than the catalytic subunit. Our work shows that the glutathione-controlled redox status of the epithelial cell plays a pivotal role in asbestos-induced epidermal growth factor receptor and proto-oncogene activation as well as AP-1 activity.

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.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is an effective inhibitor of the glucosyltransferase that makes glucosylceramide. Virtually all of the hundreds of naturally occurring glycolipids are formed from this primary glycolipid, so the inhibitor acts to lower their concentrations by the process of attrition (hydrolytic catabolism). Trials with mice carrying ascites carcinoma cells showed that PDMP could produce a permanent cure in some of the animals and marked prolongation of life in the others (Inokuchi, J., I. Mason, and N.S. Radin. 1987. Cancer Lett. 38: 23-30). In order to maximize the effect, we studied the metabolism of PDMP by labeling it with [3H] on carbon one, using a labeling method that discriminated against the unwanted erythro-isomer. The active enantiomer of the inhibitor (D-) was isolated by chromatography of the camphanate esters, followed by methanolytic cleavage. Examination of the fate of the labeled drug after a single injection showed that it was very rapidly converted to several polar products that were rapidly excreted. The drug penetrated all of the organs readily and a small portion was oxidized at the C-1 position to yield 3H2O. From these findings it appeared likely that the amine is attacked by a mixed function oxidase based on cytochrome P450. This conclusion was confirmed by showing that the tissue levels of PDMP could be greatly elevated, for a much longer time, when the mice were pretreated with piperonyl butoxide or cimetidine. The amount of conversion to polar metabolites was substantially reduced and tissue levels of PDMP were maintained much longer. Analysis of mice injected with one or both drugs showed that piperonyl butoxide augmented the effects of PDMP on ceramide, glucosylceramide, and dihexosylceramide levels, as well as on the activity of glucosylceramide synthase. It is suggested that piperonyl butoxide be used as an adjuvant for the many useful drugs that are inactivated by the P450 system.

Aortic vascular smooth muscle cells (VSMC) were used to study the effect of age on responses to high glucose concentrations or the cytokine, tumor necrosis factor-alpha (TNF-alpha). Activator protein-1 (AP-1) binding to DNA increased more in VSMC from old versus young rats (P < 0.02) and was related to increased expression of its components, c-Fos, Fra-1, and JunD. The relationship to upstream signals, i.e., activities of mitogen-activated protein kinases (MAPK), was studied using antibodies to total and phosphorylated forms of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. High glucose and TNF-alpha increased ERK phosphorylation more in old (P < 0.05); whereas only TNF-alpha induced JNK activation in young (P < 0.04). PD98059, a MEK inhibitor, attenuated AP-1 activation, lowered c-Fos and Fra-1 protein levels and reduced cell number and cells positive for proliferating cell nuclear antigen in old. We concluded that age differentially influenced activation of signaling pathways in VSMC exposed to high glucose or TNF-alpha. This may contribute to the increased risk for vascular disease associated with aging and diabetes mellitus (DM).

Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.

It has been reported earlier that rat microvessels which constitute the blood-brain barrier (BBB) are rich in free radical scavenging enzymes. In the present investigation, BBB of rat was disrupted by intravenous infusion of the hypertonic saline and changes in enzymes--namely, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)--were evaluated in the brain microvessels at 30 min after the intravenous administration of hypertonic saline, being the time of peak effect. There was a significant increase in the activities of CAT (40%), GPx (26%), and SOD (16%) over the control values. In addition, within 90 min BBB was found to be reestablished and the levels of enzymes reverted to normal. Malondialdehyde (MDA) levels and activity of lactate dehydrogenase (LDH) remained unaltered during and following disruption, suggesting that there was no change in the membrane lipid environment. Similarly, there was no cell lysis. The results suggest that the disruption of BBB following hypertonic saline administration might be due to an increase in the generation of free radicals in the brain microvessels.

A diet containing 18–20 mg iron/kg to young weaned rats for 8 weeks altered the metabolism of gamma-aminobutyric acid and glutamate in the central nervous system without affecting blood hemoglobin. Subsequent rehabilitation with 390 mg iron/kg diet for 2 weeks normalized these changes.

Gangliosides stimulate the hydrolysis of glucosylceramide (GlcCer), their precursor, and therefore may lower the level of cellular GlcCer and exert a feedback control effect to slow the formation of gangliosides. Tests were made to see if a similar effect on GlcCer levels can be exerted by the action of gangliosides on GlcCer synthesis. Using a new assay procedure, we showed that gangliosides do inhibit the synthase in brain membranes quite effectively, the most active being those lipids with more sugar and sialic acid moieties. Mice injected with a mixture of brain gangliosides for 5 days were found to have a lower level of ceramide:UDP-Glc glucosyltransferase activity in brain, liver, and kidney. The inhibition seems to be exerted by competition for the active site and binding to effector site(s) on the enzyme. It is possible that the reported therapeutic actions of gangliosides on the nervous system are, in part, the result of lowered levels of GlcCer. Malignant tumors shed gangliosides into the extracellular fluid, which are believed to block the generation of antibodies by the host's immunodefense system; this effect also may be due, in part, to reduction in the GlcCer level of immunogenic cells. A new finding is that a ceramide containing phytosphingosine is a markedly better substrate for GlcCer synthase than one containing the more common base.

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)-linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal-regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis.Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill).Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels.Pretreatment with an EGFR phosphorylation or mitogenactivated protein kinase kinase 1/2 inhibitor abrogated asbestosinduced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells.Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines.Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show doseresponsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos.However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumorspecific changes in endogenous cell death-related gene expression.Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag-immortalized human mesothelial cells.They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human 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.

Abstract 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.

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.

Purpose The diversity literature has yet to investigate relationships between diversity and leader–member exchanges (LMX) at multiple levels of analysis. The purpose of this paper is to test a multilevel model of nationality diversity and LMX. In doing so, the authors investigate the role of surface- and deep-level diversity as related to leader–member exchange differentiation (LMXD) and relative LMX (RLMX), and hence to subordinate job performance. Design/methodology/approach The authors test a multilevel model of diversity and LMX using multisource survey data from subordinates nesting within supervisors. The authors do so in a context where diversity in nationality is pervasive and plays a key role in LMXs, i.e., a multinational organization in Dubai. The authors tested the cross-level moderated model using MPlus. Findings The results suggest surface-level similarity is more important to RLMX than deep-level similarity. The relationship between surface-level similarity and RLMX is moderated by workgroup nationality diversity. When workgroups are more diverse, there is a positive relationship between dyadic nationality similarity and RLMX; when workgroups are less diverse, similarity in nationality matters less. Moreover, LMXD at the workgroup level moderates the relationship between RLMX and performance at the individual level. Originality/value This study is one of very few to examine both diversity and LMX at multiple levels of analysis. This is the first study to test the workgroup diversity as a cross-level moderator of the relationship between deep-level similarity and LMX. The results challenge the prevailing notion that that deep-level similarity is more strongly related to LMX than surface-level diversity.

Asbestos is a group of naturally occurring mineral fibers which are associated in occupational settings with increased risks of malignant mesothelioma (MM), lung cancers, and pulmonary fibrosis (asbestosis). The six recognized types of asbestos fibers (chrysotile, crocidolite, amosite, tremolite, anthophyllite, and actinolite) are different chemically and physically and may have different dose-response relationships in the development of various asbestos-associated diseases. For example, epidemiologic and lung fiber content studies suggest that the pathogenic potential and durability of crocidolite is much greater than chrysotile asbestos in the causation of human MM. We have used isolated mesothelial cells, the target cells of MM, as well as epithelial cells of the lung, the target cells of lung cancers, in vitro to elucidate the dose-response relationships in expression of early response protooncogenes and other genes critical to cell proliferation and malignant transformation in cells exposed to crocidolite and chrysotile asbestos, as well as a number of nonpathogenic fibers and particles. These studies reveal distinct dose-response patterns with different types of asbestos, suggesting a threshold for effects of chrysotile both in in vitro studies and inhalation experiments. The different patterns of gene expression have been confirmed in lungs of rats exposed by inhalation to these types of asbestos. Experiments also suggest no observed adverse effect levels after evaluation of lung injury, inflammation, and fibrosis at lower concentrations of both types of asbestos.

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.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.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.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.

Oxidative stress was assesed by estimating lipid peroxidation product (LPO) in the form of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants in the form of superoxide dismutase (SOD), catalase and nonenzymatic antioxidant vitamins e.g. vitamin C, β carotene and vitamin E in either serum or plasma or erythrocytes in 190 cases of age related cataract in the age group of 50-80 years. 190 cases were grouped into three morphological types namely, 73 cases of cortical, 77 cases of posterior subcapsular and 40 cases of nuclear cataract and values of LPO and antioxidants were compared with 78 cases of age matched healthy control groups. Plasma TBARS levels were cataract cases when compared with control groups. There were no significant differences in the erythrocyte levels of catalase and plasma levels of Vit E between cataract cases and control groups. No significant changes of parameters were seen among three different morphological types of age related cataract. The present study shows that the oxidative stress may play an important role in the age related cataract.

Levels of certain antioxidants namely reduced glutathione (GSH), ascorbic acid (Vit C), alpha-tocopherol (Vit E) and antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were compared in monkey and rat brain microvessels which constitute the blood-brain barrier (BBB). The BBB of both the species contains appreciable amounts of the antioxidants to protect against oxidative damage. The level of protection in rat seems to be more efficient than monkey since rat microvessels contain higher concentrations of some of the bio-antioxidants. The comparative status of enzymatic and non-enzymatic protective system against oxidation in the brain microvessels has been discussed.

Pleural and peritoneal mesotheliomas (MMs) are chemoresistant tumors with no effective therapeutic strategies. The authors first injected multifunctional, acid-prepared mesoporous spheres (APMS), microparticles functionalized with tetraethylene glycol oligomers, intraperitoneally into rodents. Biodistribution of APMS was observed in major organs, peritoneal lavage fluid (PLF), and urine of normal mice and rats. After verification of increased mesothelin in human mesotheliomas injected into severe combined immunodeficient (SCID) mice, APMS were then functionalized with an antibody to mesothelin (APMS-MB) or bovine serum albumin (BSA), a nonspecific protein control, and tumor targeting was evaluated by inductively coupled plasma mass spectrometry and multifluorescence confocal microscopy. Some APMS were initially cleared via the urine over a 24 hr period, and small amounts were observed in liver, spleen, and kidneys at 24 hr and 6 days. Targeting with APMS-MB increased APMS uptake in mesenteric tumors at 6 days. Approximately 10% to 12% of the initially injected amount was observed in both spheroid and mesenteric MM at this time point. The data suggest that localized delivery of APMS-MB into the peritoneal cavity after encapsulation of drugs, DNA, or macromolecules is a novel therapeutic approach for MM and other tumors (ovarian and pancreatic) that overexpress mesothelin.

Because androgenic hormones produce an increase in kidney size and the production of kidney glycosphingolipids and because the level of tissue glucosylceramide (GlcCer) has been implicated as a modulator of organ size, we measured the activities of two enzymes in mouse kidney that (in part) determine the level of this glycolipid. GlcCer synthase was found to rise in specific activity with age and increased kidney size, whereas GlcCer glucosidase was found to decrease with age; both enzyme changes act to elevate the level of kidney GlcCer and point to the level of this lipid as a determinant of kidney size. The increase in synthase was especially rapid in male kidney, consistent with the more rapid growth of male kidney. Testosterone injection into both males and females produced elevated levels of GlcCer synthase, decreased levels of the hydrolase, and more rapid growth. Injection of 17 beta-estradiol, which produced slow or negative kidney growth, caused the synthase activity to decrease in female mouse kidney and the glucosidase activity to rise in both sexes. Thus the changes in kidney size produced by the two hormones paralleled the changes in GlcCer level predicted by the hormone effects on the two enzymes. In liver, estradiol produced increases in both enzymes but only the male liver increased in size. Testosterone did not affect the liver. The testes grew with age but the activities of both enzymes decreased.

Malignant mesothelioma (MM) is a deadly cancer caused by asbestos exposure that is increasing worldwide.Early diagnosis for this cancer is very difficult and MM is mostly resistant to conventional therapies.A host of factors may be responsible for development of MM and imparting drug resistance to this cancer.Understanding these processes will be important in designing therapeutic approaches for MM.Some of the conventional as well as current approaches for MM therapy are discussed in this review.

Cell signaling by pathogenic minerals may initiate the transactivation of genes that are critical to carcinogenesis and fibroproliferative diseases of the lung and pleura. We have shown previously that stimulation of the mitogen-activated protein kinase (MAPK) cascade by asbestos fibers leads to phosphorylation events involved in transactivation of lun and Fos proteins that comprise the activator protein-1 (AP-1) transcription factor. Recently, we have also used AP-1 luciferase reporter transgenic mice and immunocytochemistry to show that transactivation of AP-1 occurs in bronchiolar and alveolar epithelial cells after inhalation of asbestos fibers. After inhalation of asbestos, epithelial cells of the lung also show increased immunoreactivity of pliosphorylated extracellular signal regulated kinases (ERKs 1/2) at sites of fibrogenesis. The availability of lung epithelial cell-specific promoters has allowed the creation of transgenic mice with mutations in the transactivation domains of key receptors and protein intermediates that comprise the MAPK signaling cascade. These rodent models may reveal whether cell signaling events initiated by mineral dusts in epithelial cells are critical to the development of cell proliferation, apoptosis, and lung disease.

Agents inducing oxidative stress activate one or more pathways of the Mitogen-Activated Protein Kinases (MAPK), a family of signaling proteins leading to activation of the Fos/Jun protooncogene family that comprise the Activator Protein-1 (AP-1) transcription factor. Redox sensitive molecular targets usually contain highly conserved cysteine residues, and their oxidation, nitration and formation of disulfide links are crucial events in oxidant/redox signaling. Oxidation of sulfide groups in signaling proteins causes structural modifications, resulting in the exposure of active sites and protein activation. Thiol proteins such as intracellular glutathione (GSH) and thioredoxin (Trx) are of central importance in the regulated control of redox signaling pathways by reducing disulfide bridges or oxidized cysteine residues. We review here how oxidants interact with membrane receptors, other proteins, and Mitogen-Activated Protein Kinase Phosphatases (MKPs) to activate MAPK and AP-1 regulated gene expression critical to cell differentiation, proliferation, death/apoptosis, and disease. These pathways have been studied extensively in cells in culture after exposure to H2O2, generating systems of reactive oxygen or nitrogen species (ROS/NOS), and a number of naturally occurring oxidant stresses such as chemotherapeutic drugs or airborne pollutants. The epithelial cell of the respiratory tract and inhalation models of lung injury, inflammation, carcinogenesis, and fibrogenesis have been employed by us and others to study MAPK and AP-1 activation by soluble oxidants and insoluble particulates (asbestos, silica, particulate matter or PM) inducing oxidative stress. Responses of cells to these oxidant stresses are critical to injury or repair, inflammation, and critical outcomes of disease.

Resurrection plants are nature’s wonder with a unique water stress tolerant capacity. In recent years some of these species has drawn attention of scientific community to study their physiological mechanism and genetic makeup during water stress and hydrated conditions with a possibility to utilize them as a viable genetic tool to develop drought tolerance species to combat global climate change. This study is an overview of resurrection plant Selaginella bryopteris, a pteridophyte with lithophyte habitat, its distribution, characteristics, constituents and physiological mechanism during dehydrated and rehydrated conditions. Most of the characters except few find this species close to the mythological ‘Sanjeevani booti’. Further, its scope in the field of genetic engineering, pharmacology and medicines are also analysed. More research is required to identify various other similar plant species with a potentiality to prepare drug formulations to fight chronic diseases and to develop engineered plant with drought tolerance capacity.

Asiaticoside derived from the plant Centella asiatica is known to possess good wound healing activity. Enhanced healing activity has been attributed to increased collagen formation and angiogenesis. Since antioxidants have been reported to play a significant role in the wound healing process we studied the effect of asiaticoside on the levels of certain antioxidants in the wound so as to explore the possible involvement of such a mechanism in the asiaticoside induced wound healing. Asiaticoside application (0.2%, topical) twice daily for 7 days to excision-type cutaneous wounds in rats led to increased enzymatic and non-enzymatic antioxidants, namely superoxide dismutase (35%), catalase (67%), glutathione peroxidase (49%), vitamin E (77%) and ascorbic acid (36%) in newly formed tissues. It also resulted in a several fold decrease in lipid peroxide levels (69%) as measured in terms of thiobarbituric acid reactive substance. However, continued application for 14 days showed no significant difference in these antioxidants compared with their values in vehicle treated wound tissue. It appears from the present study that asiaticosides enhanced induction of antioxidant levels at an initial stage of healing which may be an important contributory factor in the healing properties of this substance. Copyright © 1999 John Wiley & Sons, Ltd.

As a result of impaired fatty acid oxidation, a characteristic urinary dicarboxylic aciduria occurs in the riboflavin deficient animal. We compared the occurrence of riboflavin deficiency induced by phototherapy with changes in urinary organic acid profiles in 8 full-term, breast-fed neonates who received phototherapy for hyperbilirubinemia, and in 10 full-term, breastfed controls. Riboflavin status was assessed by measuring flavin adenine dinucleotide saturation of erythrocyte glutathione reductase. All 8 neonates exposed to phototherapy developed riboflavin deficiency (p less than 0.001). Riboflavin deficiency was progressive with the duration of phototherapy. None of the controls was riboflavin deficient. Urine organic acid profiles indicative of mitochondrial acyl-CoA dehydrogenase activity (fatty acid beta-oxidation, quantitated by gas chromatography mass spectrometry) showed no changes between the study and control groups in mono-, di-, or tricarboxylic acids or other organic acids. The riboflavin deficiency induced by phototherapy in full-term neonates was not of sufficient severity to limit riboflavin-dependent fatty acid oxidation.

Abstract Inflammation plays an important role in development of various cancers including malignant mesothelioma (MM). We have shown that asbestos activates NOD-like receptor protein 3 (NLRP3), a component of the inflammasome in human macrophages. As chronic asbestos exposure is a key risk factor for the development of MM, we hypothesized that inflammasome-mediated inflammation might underlie the pathogenesis of this cancer. To show the involvement of NLRP3 in asbestos-induced mesothelioma, we demonstrated that exposure of asbestos to immortalized human mesothelial cells (LP9/hTERT), a cell type responsible for origin of MM, caused mRNA increase and activation of NLRP3 as measured by caspase-1 activation and IL-1β release. Inhibition of NLRP3 by siRNA caused significant decreases in NLRP3 mRNA levels as well as asbestos-induced IL-1β release in medium. On the other hand, human MM lines and tumor tissues showed significantly decreased levels of NLRP3 and caspase-1 as compared to LP9 or matching normal tissues respectively. Our findings suggest that initial exposure to asbestos causes increased mRNA levels and activity of NLRP3, which may help in MM development by promoting mesothelial cell transformation. However, tumor development culminates in MM with decreased NLRP3 protein and increased drug resistance which may be due to caspase-1 inactivation. Thus NLRP3 may be an appropriate target for therapy of MM, especially in combination with cytotoxic chemotherapeutic drugs and IL-1 receptor antagonists. This study is supported by a Meothelioma Applied Research Foundation (MARF) grant (AS) and by NIEHS grants T32 ES07122 (BM). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5461. doi:1538-7445.AM2012-5461

Water is a vital resource for human survival.The availability of good quality water is an indispensable feature for prevents diseases and improving quality of life.It is necessary to know details about different physical parameters like color, temperature, Total hardness, pH, sulphate, chloride, DO, BOD, COD and alkalinity used to test water quality.This paper aims to analyze water quality using the Physico-chemical parameters of water samples collected from the Bansagar dam in Vindhyan region of M.P. India.

&lt;div&gt;Abstract&lt;p&gt;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)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using &lt;i&gt;in vitro&lt;/i&gt; 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-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β 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-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. &lt;i&gt;Cancer Prev Res; 7(3); 330–40. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 62K, Supplementary Figure 1. Curcumin in combination with asbestos resulted in a synergistic effect on NLRP3 and pro-IL-1beta priming in human mesothelial cells. LP9 cells pretreated with curcumin (Cur 10 microM for 24-48 h) and exposed to asbestos (5 microg/cm2 for 24-48 h) showed increased NLRP3 (A) and pro-IL-1beta (B) mRNA levels with no significant change in caspase-1 activation (C) compared to control and asbestos exposed LP9 cells.&lt;/p&gt;

&lt;p&gt;PDF file - 61K, Supplementary Figure 2. Curcumin treatment did not reduce MM tumor burden. (A) Allograft model comparing gavage administered curcumin to control group. (B) Allograft model comparing IP administered curcumin to control, cisplatin only and cisplatin plus curcumin groups. (C) Xenograft model comparing gavage and IP administered curcumin to control groups.&lt;/p&gt;

The current study has been conducted in Solan district of Himachal Pradesh to evaluate soil physico-chemical parameters and soil health. The soil samples have been collected from farmer's field of five villages of Solan district. From each village 10 farmers were randomly selected and making the sample size 50. The findings showed thatSoil pH ranged from 6.23 to 7.34 and electrical conductivity (EC) of the entire study area remained below 0.29 dSm-1. It was discovered that there was a marked variation in chemical parameters of various samples viz. Soil Organic Carbon (SOC), available nutrients (NPK) and available micro nutrients. SOC ranged between 0.53 to 2.03 per cent, available nitrogen content varied from medium to high, phosphorus content remained at high range, but varied from 54.78 to 65.71 kg ha-1 and available potassium content varied from medium to high range. The findings also demonstrated a strong and positive relationship between organic carbon and available nitrogen. Farmers were given soil health cards after testing soil samples from various areas.

&lt;div&gt;Abstract&lt;p&gt;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)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using &lt;i&gt;in vitro&lt;/i&gt; 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-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β 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-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. &lt;i&gt;Cancer Prev Res; 7(3); 330–40. ©2014 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

The advent of both microarray and massively parallel sequencing have revolutionized high-throughput analysis of the human transcriptome. Due to limitations in microarray technology, detecting and quantifying coding transcript isoforms, in addition to non-coding transcripts, has been challenging. As a result, RNA-Seq has been the preferred method for characterizing the full human transcriptome, until now. A new high-resolution array from Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0), has been designed to interrogate all transcript isoforms in the human transcriptome with >6 million probes targeting coding transcripts, exon-exon splice junctions, and non-coding transcripts. Here we compare expression results from GeneChip HTA 2.0 and RNA-Seq data using identical RNA extractions from three samples each of healthy human mesothelial cells in culture, LP9-C1, and healthy mesothelial cells treated with asbestos, LP9-A1. For GeneChip HTA 2.0 sample preparation, we chose to compare two target preparation methods, NuGEN Ovation Pico WTA V2 with the Encore Biotin Module versus Affymetrix's GeneChip WT PLUS with the WT Terminal Labeling Kit, on identical RNA extractions from both untreated and treated samples. These same RNA extractions were used for the RNA-Seq library preparation. All analyses were performed in Partek Genomics Suite 6.6. Expression profiles for control and asbestos-treated mesothelial cells prepared with NuGEN versus Affymetrix target preparation methods (GeneChip HTA 2.0) are compared to each other as well as to RNA-Seq results.