Five year survival for metastatic melanoma (MM) is very low at <10%. Therapeutic options have been limited secondary to systemic toxicity. As a result there has been a growing movement towards developing targeted drug delivery models. Prior research of this group has demonstrated the effectiveness of acid-prepared mesoporous spheres (APMS-TEG) in delivering chemotherapeutic agents at a lower effective dose than systemic administration. This study aims to assess the ability of the previously developed APMS-TEG particles to deliver therapeutic doses of docetaxel for the treatment of melanoma.
In vitro experiments were performed to assess docetaxel loading onto APMS-TEG particles and release kinetics. Toxicity experiments were performed using docetaxel and docetaxel loaded APMS-TEG. The effect on cell growth was assessed using the MelJuSo, UACC903, and WM1205 melanoma cell lines.
Docetaxel demonstrated statistically significant dose dependent reduction in growth of melanoma cells. In all three cell lines, doses of 1 nM were sufficient to produce statistically significant reduction in cell growth. Scanning electron micrographs demonstrate increased uptake of APMS-TEG particles by melanoma cells in the first 24 hours, with the majority within the first 4 hours. Unloaded APMS particles had no effect on the melanoma cells, demonstrating that the particles themselves are not toxic. APMS-TEG particles had a peak release of drug within the first hour, with equilibration thereafter. The 5, 10, and 20 nM loaded particles all had statistically significant reduction in cell growth than the control groups.
The high potency against melanoma cells makes docetaxel a suitable choice for loading into APMS-TEG particles. Docetaxel loaded APMS-TEG particles demonstrate significant activity against malignant melanoma and thus offer an innovative approach to the treatment of metastatic melanoma.
Melanoma; Mesoporous; Silica; Microsphere; Nanoparticle; Docetaxel
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.
malignant mesothelioma; asbestos; mesothelium; inflammation
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.
Asbestos; Malignant mesothelioma; Thioredoxin; Thioredoxin interacting protein; Inflammasomes
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.
Malignant mesothelioma (MM) is a devastating disease with a need for new treatment strategies. In the present study we demonstrated the importance of ERK5 in MM tumor growth and treatment.
ERK5 as a target for MM therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft 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 MM cells. ERK5 silencing increased DOX-induced cell death and DOX retention in MM cells. In addition, shERK5 MM lines exhibited both attenuated colony formation on soft agar and invasion of MM 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 MM 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 IP shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis related (IL-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the IP model of tumor growth.
; ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in MM patients.
Malignant mesothelioma; asbestos; Mitogen activated protein kinases; Extracellular signal regulated kinase 5; Gene expression
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.
Targeted therapy; Mesoporous silica; Peritoneum; Chemotherapy; Microparticles
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.
Asbestos; Mesothelioma; Mesothelium; Inflammasomes; NLRP3
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 mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced 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 dose-responsive 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, tumor-specific 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.
mesothelioma; asbestos; toxicity; epidermal growth factor receptor; protein kinase B/AKT
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 SCID 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, 3X 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<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.
Microparticles; Mesoporous silica; Mesothelioma; Doxorubicin; Intracavitary tumors
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.
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. Whereas 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 SCID mice showed significant attenuated tumor growth in comparison to shControl cells. Inhibition of migration, invasion, and colony formation occurred in shERK2 MM cells in vitro, suggesting multiple roles of ERK2 in neoplasia. Microarray and qRT-PCR analyses revealed gene expression that was significantly increased (CASP1, TRAF1, FAS) or decreased (SEMA3E, RPS6KA2, EGF, BCL2L1) in shERK2-transfected MM cells in contrast to shControl-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.
Asbestos; mesothelioma; extracellular signal regulated kinase (ERK1/2); Mitogen activated protein kinases; gene expression
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.
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.
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 (15×106 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 (15×106 μm2/cm2) and toxic concentrations (75×106 μm2/cm2) of Libby six-mix.
Exposure to 15×106 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 (75×106 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 75×106 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 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.
cytokines; chemokines; asbestos; inflammation; mesothelioma
Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 μm2/cm2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), 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 TiO2 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-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.
mesothelioma; crocidolite asbestos; talc; titanium dioxide; gene profiling
Nanomaterials are commonly defined as particles or fibers of less than 1 micron 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, silica) and nonpathogenic 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 as they bypass normal clearance mechanisms, and nonpathogenic fibers and particles can test positively in these assays.
Nanoparticles; Nanofibers; Nanotubes; Nanospheres; Asbestos
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 TNF-α-independent (except for G-CSF).
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.
Oxidant stress–mediated regulation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic outcomes in lung epithelium, yet a role for Ca2+ and Ca2+/cAMP-response element binding protein (CREB) in ERK1/2 signaling has not been defined. In this study, we tested the hypotheses that oxidants induce Ca2+-mediated phosphorylation of ERK and CREB, and that CREB is required for oxidant-induced proliferation and apoptosis. H2O2 initiated an influx of extracellular Ca2+Ca2+Ca2+Ca2+ that was required for phosphorylation of both ERK and CREB in C10 lung epithelial cells. H2O2-mediated CREB phosphorylation was sensitive to MEK inhibition, suggesting that crosstalk between Ca2+, ERK, and CREB signaling pathways contributes to the oxidant-induced response. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos steady-state mRNA levels, but unexpectedly enhanced bcl-2 steady-state mRNA levels after H2O2 exposure. Whereas inhibition of CREB activity had no detectable effect on H2O2 stimulation of cell cycle, loss of CREB activity significantly reduced the number of cells undergoing apoptosis. These data support a novel communication between Ca2+-ERK1/2 and CREB elicited by H2O2, and further provide evidence that CREB is an important regulator of apoptosis in oxidant-mediated responses of lung epithelial cells.
calcium signaling; lung pathology; mitogen-activated protein kinase; reactive oxygen species
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.
asbestos; mesothelioma; lung cancer; threshold; proliferation; protooncogenes