ABSTRACT
Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p < 0.0001). IMP3 is an RNA binding protein known to bind to the 5'-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression- and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.
Subject(s)
Biomarkers, Tumor/metabolism , Glioblastoma/metabolism , Glioblastoma/pathology , Insulin-Like Growth Factor II/metabolism , MAP Kinase Signaling System , RNA-Binding Proteins/metabolism , Adolescent , Adult , Aged , Animals , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Glioblastoma/genetics , Glioblastoma/physiopathology , Humans , Insulin-Like Growth Factor II/biosynthesis , Insulin-Like Growth Factor II/genetics , Mice , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Neoplasm Invasiveness , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Prognosis , Protein Biosynthesis , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Survival Analysis , Up-Regulation , Young AdultABSTRACT
Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn(GTSC)]3, complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, [Zn(GTSC)]3 had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that [Zn(GTSC)]3 had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that [Zn(GTSC)]3 appeared to have a nuclear localization within 30 min of addition to MCF7 cells. Moreover, [Zn(GTSC)]3 showed minimal cytotoxicity compared with Zn(ATSM), suggesting that [Zn(GTSC)]3 may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel [Zn(GTSC)]3 complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.
Subject(s)
Fluorescent Dyes/chemistry , Molecular Imaging , Organometallic Compounds/chemistry , Thiosemicarbazones/chemistry , Zinc/chemistry , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival/drug effects , Crystallography, X-Ray , Flow Cytometry , Fluorescence , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Fluorescent Dyes/toxicity , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Models, Molecular , Molecular Structure , Organometallic Compounds/chemical synthesis , Organometallic Compounds/metabolism , Organometallic Compounds/toxicity , Stereoisomerism , Structure-Activity Relationship , Thiosemicarbazones/toxicity , Tumor Cells, CulturedABSTRACT
Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin α2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = <0.0001 and <0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.
Subject(s)
Glioblastoma/diagnosis , Haptoglobins/analysis , Haptoglobins/physiology , Proteomics/methods , Animals , Astrocytes/chemistry , Astrocytes/pathology , Biomarkers, Tumor/blood , Case-Control Studies , Cell Line, Tumor , Cell Movement , Cell Proliferation , Haptoglobins/genetics , Humans , Melanoma/chemistry , Melanoma/pathology , Mice , Up-RegulationABSTRACT
Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo IIα inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.