Jedd M. Hillegass

Assessing nanotoxicity in cells in vitro

Nanomaterials are commonly defined as particles or fibers of less than 1 microm 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.

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

BackgroundPleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.ResultsWe 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.ConclusionsThese 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.

Inflammation precedes the development of human malignant mesotheliomas in a SCID mouse xenograft model

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.

Alterations in Gene Expression in Human Mesothelial Cells Correlate with Mineral Pathogenicity

Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 microm(2)/cm(2) 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-1 beta, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.

Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin

BackgroundMalignant 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.ResultsU0126 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.ConclusionsThese 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.

ERK2 is essential for the growth of human epithelioid malignant mesotheliomas

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.

Utilization of Gene Profiling and Proteomics to Determine Mineral Pathogenicity in a Human Mesothelial Cell Line (LP9/TERT-1)

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.

Gd-labeled microparticles in MRI: in vivo imaging of microparticles after intraperitoneal injection.

Particulate materials have recently been studied for in vivo applications such as imaging, drug delivery, and even combined therapy/imaging agents for the simultaneous detection and treatment of diseases.[1–4] Nanoparticles have received much attention in the drug delivery field;[5, 6] however, because they can enter organelles and distribute widely throughout the body,[7–10] there is some uncertainty about the acute and/or chronic toxicities of nanoparticles. Microparticles, with their larger size, may be a less toxic alternative that still can be modified with the same array of functionalities as nanoparticles. We recently showed that acid-prepared mesoporous spheres (APMS),[11, 12] composed of silica and with an average diameter of 1.5 µm, are readily taken up by cells when modified with tetraethylene glycol (TEG) on their external surfaces. These microparticles also readily deliver chemotherapeutic agents in vitro and in vivo,[13, 14] and can be bifunctionally modified with both TEG and antibodies for targeted uptake by cancer cells.[15] A unique feature of our microparticles is that they are not present within endosomes after uptake but instead are directly surrounded by cytosol, in contrast to nanoparticles. Thus, molecular cargo including DNA and RNA is released directly to the cytosol and is not subject to the enzymatic and pH-based degradation mechanisms within endosomes. Our current interests include studies on the molecular mechanisms and treatment of malignant mesothelioma (MM)[16] and tumors of the intraperitoneal (IP) cavity, such as pancreatic and ovarian cancers.[17] To visualize our particles in vivo, we used a known ligand to immobilize gadolinium(III) to the pores of APMS and imaged animals via magnetic resonance imaging (MRI).[18] Nanoparticle-based MRI contrast agents have been reported recently;[19, 20] however, we report here for the first time that porous silica microparticles containing a Gd chelate can be injected IP and tracked in real-time by MRI. Surprisingly, a fraction of the particles were excreted via the urine, a finding we confirmed by analyzing the urine and tissues of the treated animals.

Increased efficacy of doxorubicin delivered in multifunctional microparticles for mesothelioma therapy

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 < 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.

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

BackgroundExposures 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.ResultsExposure 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.ConclusionsLibby 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.