Chronic Exposure to Carbon Nanotubes Induces Invasion of Human Mesothelial Cells through Matrix Metalloproteinase‑2

Malignant mesothelioma is one of the most aggressive forms of cancer known. Recent studies have shown that carbon nanotubes (CNTs) are biopersistent and induce mesothelioma in animals, but the underlying mechanisms are not known. Here, we investigate the effect of long-term exposure to high aspect ratio CNTs on the aggressive behaviors of human pleural mesothelial cells, the primary cellular target of human lung mesothelioma. We show that chronic exposure (4 months) to single- and multiwalled CNTs induced proliferation, migration, and invasion of the cells similar to that observed in asbestos-exposed cells. An up-regulation of several key genes known to be important in cell invasion, notably matrix metalloproteinase-2 (MMP-2), was observed in the exposed mesothelial cells as determined by real-time PCR. Western blot and enzyme activity assays confirmed the increased expression and activity of MMP-2. Whole genome microarray analysis further indicated the importance of MMP-2 in the invasion gene signaling network of the exposed cells. Knockdown of MMP-2 in CNT and asbestos-exposed cells by shRNA-mediated gene silencing effectively inhibited the aggressive phenotypes. This study demonstrates CNT-induced cell invasion and indicates the role of MMP-2 in the process

Successive waves of CORT for 90 days greatly exacerbates LPS-induced neuroinflammation.

<p>Mice were treated with CORT (200 mg/L 0.6% EtOH) for 7 days in the drinking water followed either by 90 days without (CORT) or 90 days of bimonthly, 7 day CORT exposures (CORT+++). LPS (2 mg/kg, s.c.) was administered on day 97. TNFα, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex collected 6 hours after LPS exposure. Data represent mean ± SEM (n = 5 mice/group). Statistical significance of at least P<0.05 is denoted by * as compared to appropriate control, # as compared to LPS alone, and § as compared to CORT/LPS.</p

Chronic CORT exacerbates PIC-induced neuroinflammation.

<p>Mice were pretreated with CORT (200 mg/L 0.6% EtOH in drinking water for one week). TNFα, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex collected 6 hours after PIC exposure (12 mg/kg, i.p.). Data represent mean ± SEM (n = 5 mice/group). Statistical significance of at least P<0.05 is denoted by * as compared to respective control group.</p

Chronic CORT sensitizes the CNS to a sub-neuroinflammatory inhalation dose of LPS.

<p>Mice pretreated with CORT (200 mg/L 0.6% EtOH in drinking water for one week) were subjected to 3 hours of LPS (7.5 μg/m³) by inhalation. TNFα, IL-6, CCL2, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex collected immediately following LPS inhalation exposure. Data represent mean ± SEM (n = 5 mice/group). Statistical significance of at least P<0.05 is denoted by * as compared to respective control and # as compared to LPS alone exposed group.</p

Chronic CORT exacerbates LPS-induced neuroinflammation brain wide.

<p>The effects of chronic CORT pretreatment (200 mg/L 0.6% EtOH in drinking water for one week) on LPS (2 mg/kg, s.c.)-induced TNFα, IL-6, CCL2, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex (CTX), hippocampus (HIP), striatum (STR), hypothalamus (HYPO), olfactory bulb (OB), and cerebellum (CB) 6 hours after LPS exposure. Data represents mean ± SEM (n = 5 mice/group). Statistical significance of at least p<0.05 is denoted by * as compared to control, # as compared to LPS alone.</p

Chronic CORT pretreatment primes the neuroinflammatory response for 30 days.

<p>Mice were pretreated with CORT (200 mg/L 0.6% EtOH in drinking water for one week). Thirty days after the cessation of CORT, mice were administered LPS (2 mg/kg, s.c.). TNFα, IL-6, CCL2, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex 6 hours after LPS exposure. Data represent mean ± SEM (n = 5 mice/group). Statistical significance of at least P<0.05 is denoted by * as compared to appropriate control and # as compared to LPS alone.</p

Chronic CORT pretreatment exacerbates and prolongs LPS-induced neuroinflammation but has no effect on activation of STAT3 and does not cause astrogliosis.

<p>A) A time course of LPS (2 mg/kg, s.c.)-induced neuroinflammation with and without CORT pretreatment (200 mg/L 0.6% EtOH in drinking water for one week) is shown (saline, 2, 6 and 12 hours). TNFα, IL-6, CCL2, IL-1β, LIF, and OSM mRNA levels were measured in samples prepared from cortex. Data represents mean ± SEM (n = 5 mice/group). CTX data for LPS alone treated groups are included from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190546#pone.0190546.g001" target="_blank">Fig 1A</a> for statistical comparison. Statistical significance of at least p<0.05 is denoted by * as compared to control, # as compared to LPS alone, § as compared to 2h CORT/LPS, and ŧ as compared to 6h CORT/LPS. B & C) Effects of chronic CORT pretreatment (200 mg/L 0.6% EtOH in drinking water for one week) on LPS induced activation of the STAT3 signaling module was assessed by immunoblots of pSTAT3^Tyr705 and astrogliosis was assessed by immunoassay of GFAP. Cortex samples were analyzed for pSTAT3^Tyr705 (6 hours) and GFAP protein (72 hours) after LPS exposure of CORT pretreated animals. Data represent mean ± SEM (n = 5 mice/group). Saline and LPS alone treated groups are included from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190546#pone.0190546.g001" target="_blank">Fig 1B & 1C</a> for statistical comparison. Statistical significance of at least p<0.05 is denoted by * as compared to control, # as compared to LPS alone.</p

Induction of Stemlike Cells with Fibrogenic Properties by Carbon Nanotubes and Its Role in Fibrogenesis

We developed a three-dimensional fibroblastic nodule model for fibrogenicity testing of nanomaterials and investigated the role of fibroblast stemlike cells (FSCs) in the fibrogenic process. We showed that carbon nanotubes (CNTs) induced fibroblastic nodule formation in primary human lung fibroblast cultures resembling the fibroblastic foci in clinical fibrosis and promoted FSCs that are highly fibrogenic and a potential driving force of fibrogenesis. This study provides a predictive 3D model and mechanistic insight on CNT fibrogenesis

Effects of metallic nickel particles on p53 transcription activity and protein levels.

<p>JB6 cells, stably transfected with p53 luciferase reporter plasmid, were seeded onto a 24-well plate and incubated overnight. Cells were treated with/without metallic nickel nano- or fine particles for 24 h. The p53 transcription activity and protein levels were measured by the luciferase activity assay (A) and western-blot (B), respectively. Metallic nickel nanoparticles caused a greater decrease of p53 transcription activity and protein levels than fine particles. * <i>P</i><0.05 <i>versus</i> control. TPA (20 ng/ml) or UVB (4 kJ/m²) were set as a positive control.</p

Effects of metallic nickel particles on protein expressions of R-Ras, c-myc, c-Jun, p65, p50 and HIF-1alpha.

<p>JB6 cells were treated with 20 µg/cm² metallic nickel nano- or fine particles for 1, 3, 6 and 8 h. Western blot results show that both metallic nickel nano- and fine particles induced up-regulation of protein expressions of R-Ras, c-myc, c-Jun, p65, and p50 in a time-dependent manner. Metallic nickel nanoparticles elicited a higher stimulation on these protein expressions compared to fine particles. Nanoparticles induced a significant HIF-1alpha up-regulation after 6 and 8 h treatment.</p