The prognosis of patients with Glioblastoma Multiforme (GBM), the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy. Genetic heterogeneity of GBM warrants extensive studies to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. In the present study, we report a novel proteomic approach by using two-dimensional difference gel electrophoresis (2D-DIGE) followed by spot picking and analysis of proteins/peptides by Mass Spectrometry. We report Glucose Regulated Protein 78 (GRP78) as a differentially expressed protein in the GBM cell line compared to human normal Astrocyte cells.In addition to proteomic studies, we performed microarray analysis which further confirmed up regulation of GRP78 in GBM cells compared to human normal Astrocyte cells.GRP78 has long been recognized as a molecular chaperone in the endoplasmic reticulum (ER) and can be induced by the ER stress response. Besides its location in the ER, GRP78 has been found in cell plasma membrane, cytoplasm, mitochondria, nucleus and other cellular secretions. GRP78 is implicated in tumor cell proliferation, apoptosis resistance, immune escape, metastasis and angiogenesis, and its elevated expression usually correlates with a variety of tumor micro environmental stresses, including hypoxia, glucose deprivation, lactic acidosis and inflammatory response. GRP78 protein acts as a centrally located sensor of stress, which senses and facilitates the adaptation to the tumor microenvironment.Our findings showed differential expression of this gene in brain cancer GBM and thus confirm similarities in findings in existing transcriptional and translational studies. Thus, these findings could be of further importance for diagnostic, therapeutic and prognostic approaches for dealing with this highly malignant cancer.

Cisplatin, carboplatin, oxaliplatin, and related metallodrugs are extensively being used in the treatment of a variety of cancers. Unfortunately these drugs are highly toxic and tumor becomes drug-resistance. These circumstances have led researchers to look for new cytotoxic agents that may exhibit less toxicity and devoid of drug resistance. It is believed that cisplatin and related drugs directly bind to genomic DNA through purine bases. Synthesis of new metallodrugs which does not follow the above mechanism of action might yield better drugs with less toxicity and devoid of drug resistance. Recently we have demonstrated that several anticancer rhenium compounds do not directly bind to DNA. We have synthesized numerous rhenium pentylcarbonato and acetylsalicylato complexes which include (CO)3(2,2’-Bipyridyl)ReOC(O)OC5H11 (PC-1), (CO)3(1,10-Phenanthroline)ReOC(O)OC5H11 (PC-2), (CO)3(5-Methyl-1,10-Phenanthroline)Re ReOC(O)OC5H11 (PC-3), (CO)3(2,9-Dimethyl-1,10-Phenanthroline)ReOC(O)OC5H11 (PC-4), (CO)3(5,6-Dimethyl-1,10-Phenanthroline)ReOC(O)OC5H11 (PC-5), (CO)3(4,7-Diphenyl-1,10-Phenanthroline)ReOC(O)OC5H11 (PC-6), (CO)3(2,9-Dimethyl-4,7-Diphenyl-1,10-Phenanthroline)Re ReOC(O)OC5H11 (PC-7), (CO)3(2,2’-Bipyridyl)ReOC(O)C6H4·C(O)OCH3 (ASP-1), (CO)3(1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3, (ASP-2), (CO)3(5-Methyl-1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3 (ASP-3), (CO)3(2,9-Dimethyl-1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3 (ASP-4), (CO)3(5,6-Dimethyl-1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3 (ASP-5), (CO)3(4,7-Diphenyl-1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3 (ASP-6), (CO)3(2,9-Dimethyl-4,7-Diphenyl-1,10-Phenanthroline)ReOC(O)C6H4·C(O)OCH3 (ASP-7). The anticancer properties of the compounds were evaluated using human prostate, alveolar lung, brain, colon, and leukemia cancer cell lines and normal bone marrow cell lines. The results of this study demonstrate that these complexes have significant anticancer properties. Therefore, these complexes can potentially find applications in the treatment of these cancers. Acknowledgment. The work at MSU was partially supported by grants from the National Institutes of Health (Grant No. G11HD038439) and Nuclear Regulatory Commission (Grant No. NRC-HQ-12-G-27-0086). The work at ECSU-UNC was partially supported by grant from the Department of Energy (TMCF/DOE grant). Citation Format: Hirendra N. Banerjee, Deidre Vaughan, Jewe Medley, Gwyn Hyman, Christopher Krauss, Carl Parson, Santosh Mandal, Pola Olczak, Michael Mbagu, Divine Kebulu, Saroj Pramanik, Fazlul Sarkar. Anticancer properties of novel rhenium compounds against human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4485. doi:10.1158/1538-7445.AM2013-4485

Epigenetic modifications to the genome affect gene expression, causing increased risk for cancers and other diseases. DNA methylation and modification of histone proteins are ways in which this occurs. The specific aim of this study was to identify DNA methylation percentages in Glioblastoma multiforme (GBM) samples compared against normal astrocyte genomic DNA(gDNA). Within the context of DNA methylation, a global genome methylation analysis was performed to determine gDNA methylation differences between GBM and a normal astrocyte samples using an Imprint® Methylated DNA Quantification Kit (Catalog # MDQ1, Sigma®, Saint Louis USA). . Methyl-sensitive cut counting analysis (MSCC) technique was used further estimate and compare methylation of cancer and normal gDNA. DNA samples were digested using the restriction endonuclease HpaII, which is methyl-sensitive and will cut DNA at unmethylated CCGG sites. It is estimated that generally 90% of CpG islands in genomic DNA have at least one HpaII site . HpaII sites must be greater than 40 bases from another HpaII site to be designated an individual HpaII site. In this case, the identified gene sequence is considered to be a single site. DNA (2 μg) was digested using 20 units of HpaII.An adaptor with the recognition site for the MmeI restriction enzyme was added using T4 DNA ligase. The DNA was then ethanol precipitated and nicks were repaired using an 8 unit Bst DNA polymerase. MmeI (2 units) was added to the DNA, which served to cleave at 18 bases adjacent to the HpaII sites. These segments were then ligated to another adapter for PCR amplification and sequencing. Tag sizes were purified using a 10% PAGE gel and amplified with quantitative PCR and Bio-Rad iProof high-fidelity polymerase. An Illumina HiSeq 2000 Genome Analyzer at the University of Nebraska Medical Center Sequencing Core Facility was used for tag sequencing. Tag sequences were paired against the human genome (h19) library using Bowtie, a short sequence aligner . Sequence tags, representing an unmethylated HpaII site, were counted in GBM and normal astrocyte gDNA. The change in tag counts between the two samples represented a change in the methylation status of the HpaII site. The results showed GBM genomic DNA(gDNA) possessed lower gDNA methylation percentages compared to normal samples. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM gDNA methylation sites for genes PBK, KIF23, COL6A3 and LOX.Results could be used as prognostic indicators for glioblastoma therapeutic strategy. ACKNOWLEDGEMENT:Supported by a NIH-RISE GRANT to ELIZABETH CITY STATE UNIVERSITY and a TMCF-DOE award to Dr.Hirendra Banerjee. Citation Format: Hirendra N. Banerjee, GWYN HYMAN, Jeffrey Rousch, VINOD MANGALIK, Deidre Vann, Christopher Krauss, sabrina sharpe, DAVID KLINKEBIEL, SANTOSH MANDAL, MUKESH VERMA. An epigenetic study of DNA methylation and histone modification of brain cancer Glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2014-2299