Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin

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

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

<p>Abstract</p> <p>Background</p> <p>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 (15×10⁶μm²/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 (15×10⁶μm²/cm²) and toxic concentrations (75×10⁶μm²/cm²) of Libby six-mix.</p> <p>Results</p> <p>Exposure to 15×10⁶μm²/cm²Libby six-mix elicited significant (p < 0.05) upregulation of one gene (<it>SOD2</it>; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in <it>SOD2</it>. 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 (75×10⁶μm²/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 75×10⁶μm²/cm²caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (<it>HO-1</it>) in LP9/TERT-1 and HKNM-2 cells.</p> <p>Conclusions</p> <p>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.</p

In silico toxicology protocols

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information

The role of matrix metalloproteinases in zebrafish (danio rerio) embryogenesis and their regulation by glucocorticoids

Matrix metalloproteinases (MMPs) play a pivotal role during development due to their ability to remodel the extracellular matrix, a function necessary for proper cellular migration and tissue morphogenesis. Studies have demonstrated that MMP gene expression can be either inhibited or induced by glucocorticoids in a variety of model systems and that exposure to glucocorticoids causes developmental abnormalities in several species. We hypothesize that glucocorticoid-induced teratogenesis is mediated through the glucocorticoid receptor (GR) and results from altering the expression and activity of the MMPs. Using the zebrafish (Danio rerio) as a model of development, the data presented here demonstrate that embryonic exposure to the glucocorticoids dexamethasone or hydrocortisone (100 mg/L) increased expression of the gelatinases MMP-2 (~1.5 fold) and MMP-9 (7.6 to 9.0-fold), and the collagenase MMP-13 (2.5 to 4.9-fold) at 72 hours post fertilization (hpf). In situ hybridization experiments confirmed these increases in MMP expression and demonstrated that the majority of transcript was localized rostrally. Enzyme activity was also increased at 72 hpf for both MMP-2 and MMP-9 (~3-fold), and MMP-13 (~13-fold) following glucocorticoid treatment, and substrate specificity was confirmed via several MMP inhibitors. Acute exposure to glucocorticoids resulted in numerous developmental abnormalities, most commonly altered craniofacial morphogenesis. Morpholino knockdown studies demonstrated that appropriate expression of MMP-2, MMP-9, and MMP-13 are necessary for proper zebrafish embryogenesis since morphants exhibited deleterious alterations in phenotype, and revealed that MMP-2 may compensate for loss of MMP-9 function. Co-treatment of zebrafish embryos with each glucocorticoid and the GR antagonist RU486 resulted in attenuation of glucocorticoid-induced increases in MMP expression (52-84% decrease) and activity (41-94% decrease), as well as a partial rescue in the abnormal craniofacial phenotypes. Taken collectively, these data show that dysregulation of MMP-2, MMP-9, and MMP-13 during embryogenesis, whether increased (as with glucocorticoids) or decreased (as with morpholinos), can lead to irregular developmental phenotypes. The teratogenic effects resulting from prolonged treatment with glucocorticoids may stem from this dysregulation of the MMPs. Finally, these results suggest that in the embryonic zebrafish, dexamethasone and hydrocortisone function through the GR, and that activation of this receptor can modulate MMP expression.Ph.D.Includes bibliographical references (p. 135-152)

A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas

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