EGFR Inhibition in Glioma Cells Modulates Rho Signaling to Inhibit Cell Motility and Invasion and Cooperates with Temozolomide to Reduce Cell Growth

Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G1. Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27kip1 levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion

Nanostructured Pt(NH3)4Cl2/SiO2 for nanomedicine: catalytic degradation of DNA in cancer cells

In vivo suppression of glioblastoma multiforme (GBM) in Wistar rats using silica-shelled biocatalytic Pt(NH3)4Cl2 nanoparticles is reported. These nanoparticles were synthesized by a sol-gel technique and characterized by SEM and HRTEM imaging. We confirmed morphological uniformity (30 nm) and surface acidity of the nanoparticles, respectively, by TEM imaging and FTIR spectral analysis. Interestingly, treatment of Wistar rats intraperitoneally inoculated with C6 cells using the biocatalysts resulted in considerable tumor shrinkage. Efficiency of the biocatalyst to shrink a tumor is superior to that by the commercial cytotoxic agent cisplatin. The tumor suppression property of Pt(NH3)4Cl2 nanoparticles is attributed to catalytic damage of DNA in C6 cells

Concurrent hypermethylation of DNMT1, MGMT and EGFR genes in progression of gliomas

<p>Abstract</p> <p>Background</p> <p>Gliomas are the most common neoplasm of the brain. High-grade gliomas often resist treatment even with aggressive surgical resection and adjuvant radiation and chemotherapy. Despite the combined treatment, they frequently recur with the same or higher-grade histology. Genetic instability is commonly associated with inactivation of the normal DNA repair function and tumour suppressor genes as well as activation of oncogenes resulting from alterations of promoter hypermethylation, but the molecular mechanisms of the histological and clinical progression of gliomas are still poorly understood.</p> <p>Methods</p> <p>This study involved longitudinal analysis samples of primary and recurrent gliomas to determine whether the progression of low- and high-grade gliomas is associated with the promoter methylation of the DNMT1, MGMT and EGFR genes by PCR-based restriction enzyme assay. Epigenetic inactivation of these three important glioma-associated genes was analyzed in paired biopsy samples from 18 patients with tumour recurrence.</p> <p>Results</p> <p>The methylation analysis of the CpG sites in the DNA methyltransferase (DNMT1) promoter revealed a total of 6 hypermethylations (6/18), the methylguanine-DNA methyltransferase (MGMT) promoter revealed a total of 10 hypermethylations (10/18) and the epithelial grow factor receptor (EGFR) promoter revealed a total of 12 (12/18) hypermethylations respectively in recurrent gliomas. The results demonstrated that DNMT1 promoter hypermethylation does not occur in low-grade gliomas, it was mainly observed in secondary glioblastomas. Additionally, the MGMT and EGFR promoter was hypermethylated in both low-and high-grade GLs and their corresponding histological transformed GLs.</p> <p>Conclusion</p> <p>This study has provided further evidence that the histological transformation and progression of gliomas may be associated with the inactivation of the EGFR and MGMT genes. It seems that EGFR and MGMT promoter hypermethylations are early events in the clonal evolution of gliomas and this gene inactivation has proved to be stable even in tumour recurrence. However, the DNMT hypermethylation is a late part of glioma progression.</p> <p>Virtual slides</p> <p>The virtual slide(s) for this article can be found here: <url>http://www.diagnosticpathology.diagnomx.eu/vs/1935054011612460</url></p

Specific Visualization of Glioma Cells in Living Low-Grade Tumor Tissue

BACKGROUND: The current therapy of malignant gliomas is based on surgical resection, radio-chemotherapy and chemotherapy. Recent retrospective case-series have highlighted the significance of the extent of resection as a prognostic factor predicting the course of the disease. Complete resection in low-grade gliomas that show no MRI-enhanced images are especially difficult. The aim in this study was to develop a robust, specific, new fluorescent probe for glioma cells that is easy to apply to live tumor biopsies and could identify tumor cells from normal brain cells at all levels of magnification. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation we employed brightly fluorescent, photostable quantum dots (QDs) to specifically target epidermal growth factor receptor (EGFR) that is upregulated in many gliomas. Living glioma and normal cells or tissue biopsies were incubated with QDs coupled to EGF and/or monoclonal antibodies against EGFR for 30 minutes, washed and imaged. The data include results from cell-culture, animal model and ex vivo human tumor biopsies of both low-grade and high-grade gliomas and show high probe specificity. Tumor cells could be visualized from the macroscopic to single cell level with contrast ratios as high as 1000: 1 compared to normal brain tissue. CONCLUSIONS/SIGNIFICANCE: The ability of the targeted probes to clearly distinguish tumor cells in low-grade tumor biopsies, where no enhanced MRI image was obtained, demonstrates the great potential of the method. We propose that future application of specifically targeted fluorescent particles during surgery could allow intraoperative guidance for the removal of residual tumor cells from the resection cavity and thus increase patient survival

Overexpression of c-erbB2 is a negative prognostic factor in anaplastic astrocytomas

The epidermal growth factor receptor (EGFR) family, consisting of four tyrosine kinase receptors, c-erbB1-4, seems to be influential in gliomagenesis. The aim of this study was to investigate EGFR gene amplification and expression of c-erbB1-4 receptor proteins in human anaplastic astrocytomas. Formalin-fixed and paraffin-embedded sections from 31 cases were investigated by standard immunohistochemical procedures for expression of c-erbB1-4 receptor proteins using commercial antibodies. EGFR gene amplification was studied by fluorescence in situ hybridization using paraffin-embedded tissues. Two monoclonal antibodies, NCL-EGFR-384 and NCL-EGFR, were used for EGFR detection and they displayed positive immunoreactivity in 97% and 71%, respectively. For c-erbB2 detection three monoclonal antibodies, CB11, 3B5, and 5A2, were applied and they displayed positive immunoreactivity in 45%, 100%, and 52%, respectively. Positive immunostaining for c-erbB3 and c-erbB4 was encountered in 97% and 74%, respectively. The EGFR gene was amplified in 9 out of 31 tumors (29%). After adjusting for age, Karnofsky performance status, and extent of surgical resection, Cox multiple regression analysis with overall survival as the dependent variable revealed that c-erbB2 overexpression detected by the monoclonal antibody clone CB11 was a statistically significant poor prognostic factor (P = 0.004). This study shows the convenience and feasibility of immunohistochemistry when determining the expression of receptor proteins in tissue sections of human astrocytomas. The synchronous overexpression of c-erbB1-4 proteins in anaplastic astrocytomas supports their role in the pathogenesis of these tumors. Further, c-erbB2 overexpression seems to predict aggressive behaviour

Cell cycle and aging, morphogenesis, and response to stimuli genes are individualized biomarkers of glioblastoma progression and survival

<p>Abstract</p> <p>Background</p> <p>Glioblastoma is a complex multifactorial disorder that has swift and devastating consequences. Few genes have been consistently identified as prognostic biomarkers of glioblastoma survival. The goal of this study was to identify general and clinical-dependent biomarker genes and biological processes of three complementary events: lifetime, overall and progression-free glioblastoma survival.</p> <p>Methods</p> <p>A novel analytical strategy was developed to identify general associations between the biomarkers and glioblastoma, and associations that depend on cohort groups, such as race, gender, and therapy. Gene network inference, cross-validation and functional analyses further supported the identified biomarkers.</p> <p>Results</p> <p>A total of 61, 47 and 60 gene expression profiles were significantly associated with lifetime, overall, and progression-free survival, respectively. The vast majority of these genes have been previously reported to be associated with glioblastoma (35, 24, and 35 genes, respectively) or with other cancers (10, 19, and 15 genes, respectively) and the rest (16, 4, and 10 genes, respectively) are novel associations. <it>Pik3r1</it>, <it>E2f3, Akr1c3</it>, <it>Csf1</it>, <it>Jag2</it>, <it>Plcg1</it>, <it>Rpl37a</it>, <it>Sod2</it>, <it>Topors</it>, <it>Hras</it>, <it>Mdm2, Camk2g</it>, <it>Fstl1</it>, <it>Il13ra1</it>, <it>Mtap </it>and <it>Tp53 </it>were associated with multiple survival events.</p> <p>Most genes (from 90 to 96%) were associated with survival in a general or cohort-independent manner and thus the same trend is observed across all clinical levels studied. The most extreme associations between profiles and survival were observed for <it>Syne1</it>, <it>Pdcd4</it>, <it>Ighg1</it>, <it>Tgfa</it>, <it>Pla2g7</it>, and <it>Paics</it>. Several genes were found to have a cohort-dependent association with survival and these associations are the basis for individualized prognostic and gene-based therapies. <it>C2</it>, <it>Egfr</it>, <it>Prkcb</it>, <it>Igf2bp3</it>, and <it>Gdf10 </it>had gender-dependent associations; <it>Sox10</it>, <it>Rps20</it>, <it>Rab31</it>, and <it>Vav3 </it>had race-dependent associations; <it>Chi3l1</it>, <it>Prkcb</it>, <it>Polr2d</it>, and <it>Apool </it>had therapy-dependent associations. Biological processes associated glioblastoma survival included morphogenesis, cell cycle, aging, response to stimuli, and programmed cell death.</p> <p>Conclusions</p> <p>Known biomarkers of glioblastoma survival were confirmed, and new general and clinical-dependent gene profiles were uncovered. The comparison of biomarkers across glioblastoma phases and functional analyses offered insights into the role of genes. These findings support the development of more accurate and personalized prognostic tools and gene-based therapies that improve the survival and quality of life of individuals afflicted by glioblastoma multiforme.</p