Development, characterization, and in vitro trials of chloroaluminum phthalocyanine-magnetic nanoemulsion to hyperthermia and photodynamic therapies on glioblastoma as a biological model

A glioblastoma multiforme (GBM) is the highest grade glioma tumor (grade IV) and is the most malignant form of astrocytomas. Grade IV tumors, which are the most malignant and aggressive, affect people between the ages of 45 and 70 years. A GBM exhibits remarkable characteristics that include excessive proliferation, necrosis, genetic instability, and chemoresistance. Because of these characteristics, GBMs are difficult to treat and have a poor prognosis with a median survival of less than one year. New methods to achieve widespread distribution of therapeutic agents across infiltrative gliomas significantly improve brain tumor therapy. Photodynamic therapy (PDT) and hyperthermia (HPT) are well-established tumor therapies with minimal side effects while acting synergistically. This study introduces a new promising nanocarrier for the synergistic application of PDT and magnetic hyperthermia therapy against human glioma cell line T98 G, with cellular viability reduction down to as low as 17% compared with the control. (C) 2012 American Institute of Physics. [doi:10.1063/1.3671775]

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells

Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that significantly improve the patient survival. Altered expression of the Aurora kinases was found in different malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the effects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma. RT-PCR assays were used to determine the mRNA expression in glioblastoma tumor samples and in the cell lines. Cell proliferation was measured by XTT assay, and apoptosis was determined by flow cytometry. Drug combination analyses were made based in Chou-Talalay method. Gamma radiation for clonogenic survival used the doses of 2, 4 and 6 Gy. Changes in Aurora B level were assessed by Western blot analysis. Aurora A and B were expressed in glioblastoma samples as well as in the glioblastoma cell lines (n = 6). Moreover, ZM447439, a selective Aurora kinase inhibitor, decreased the proliferation separately and synergistically with temozolomide in primary cultures and cell lines of glioblastoma. ZM also enhanced the effects of radiation on the two cell lines studied (U343 and U251), mainly when associated with TMZ in U343 cells. Treatment with ZM induced apoptotic cell death and diminished Aurora B protein level. These data suggest that Aurora kinase inhibition may be a target for glioblastoma treatment and could be used as adjuvant to chemo- and radiotherapy.

Monitoramento por imagem de ressonância magnética do crescimento tumoral no modelo C6 de glioblastoma com perspectivas de avaliação da terapia de magnetohipertemia

Objetivo: Estabelecer um padrão de crescimento tumoral (volume) em ratos Wistar submetidos ao modelo C6 de glioblastoma multiforme por meio de imagens de ressonância magnética para posterior verificação de redução de volume tumoral com a terapia de magnetohipertermia. Métodos: Para o modelo C6, utilizamos ratos Wistar, machos, jovens, pesando entre 250 e 300 g. Após anestesiados (cetamina 55 mg/kg e xilazina 11 mg/kg) foram injetadas estereotaxicamente células tumorigênicas linhagem C6 suspensas em meio de cultura (105 células em 10 µL) no córtex frontal direito (coordenadas a partir do bregma: anteroposterior = 2,0 mm; látero-lateral = 3,0 mm; profundidade = 2,5 mm) com uma seringa Hamilton. No Grupo Controle, houve a injeção do meio de cultura sem as células. Posteriormente, foram feitas imagens mediante a técnica de imagem por ressonância magnética em 14, 21 e 28 dias após a injeção em um escâner de imagem por ressonância magnética 2.0 T (Bruker BioSpec, Germany). Para o exame, os animais foram anestesiados com cetamina 55 mg/kg e xilazina 11 mg/kg. Multifatias coronais foram adquiridas utilizando uma sequência spin-echo padrão com os seguintes parâmetros: TR/TE = 4,000 ms/67,1 ms, FOV = 3,50, Matrix 192, slice thickness = 0,4 mm e slice separation = 0 mm. Resultados: A análise das imagens de ressonância magnética do tumor possibilitou a clara visualização da massa tumoral, sendo possível ainda estabelecer parâmetros de volume tumoral nos diferentes dias analisados. O volume de 14 dias após a indução do foi de 13,7 ± 2,5 mm3 . Aos 21 dias, o volume alcançado foi de 31,7 ± 6,5 mm3 e, aos 28 dias, a massa tumoral atingiu 122,1 ± 11,8 mm3 . Conclusão: Estes resultados mostraram a possiblidade de avaliação do volume tumoral no modelo C6 em ratos, o que possibilitará, no futuro, a aplicação da terapia de magnetohipertermia bem como verificação de seus resultados.

The growth of glioblastoma orthotopic xenografts in nude mice is directly correlated with impaired object recognition memory

Cognitive dysfunction is found in patients with brain tumors and there is a need to determine whether it can be replicated in an experimental model. In the present study, the object recognition (OR) paradigm was used to investigate cognitive performance in nude mice, which represent one of the most important animal models available to study human tumors in vivo. Mice with orthotopic xenografts of the human U87MG glioblastoma cell line were trained at 9, 14, and 18days (D9, D14, and D18, respectively) after implantation of 5×10(5) cells. At D9, the mice showed normal behavior when tested 90min or 24h after training and compared to control nude mice. Animals at D14 were still able to discriminate between familiar and novel objects, but exhibited a lower performance than animals at D9. Total impairment in the OR memory was observed when animals were evaluated on D18. These alterations were detected earlier than any other clinical symptoms, which were observed only 22-24days after tumor implantation. There was a significant correlation between the discrimination index (d2) and time after tumor implantation as well as between d2 and tumor volume. These data indicate that the OR task is a robust test to identify early behavior alterations caused by glioblastoma in nude mice. In addition, these results suggest that OR task can be a reliable tool to test the efficacy of new therapies against these tumors.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

Background: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. Results: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. Conclusions: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas

The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the Sao Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.

Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas

Mutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas (<10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.

N-4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N-4-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene) hydrazinecarbothioamide) and its N-4-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N-4-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N-4-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N-4-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC50: MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5) M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization. (C) 2012 Elsevier Ltd. All rights reserved.

Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

Abstract Background The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated β-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. Results The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated β-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. Conclusion Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.

Antiproliferative activity of Eremanthus crotonoides extracts and centratherin demonstrated in brain tumor cell lines

The genus Eremanthus is recognized by the predominance of sesquiterpene lactones from the furanoheliangolide type, a class of substances extensively tested against cancer cell lines. Thus, the species E. crotonoides (DC.) Sch. Bip., Asteraceae, obtained on "restinga" vegetation was evaluated against U251 and U87-MG glioma cell lines using the MTT colorimetric assay. Dichloromethane fraction was cytotoxic to both glioblastoma multiforme cell lines. We then conducted UPLC-PDA-ESI-MS/MS analysis of the dichloromethane fraction, which allowed the identification of the sesquiterpene lactones centratherin and goyazensolide. The isolation of centratherin was performed using chromatographic techniques and the identification of this substance was confirmed according to NMR data. Cytotoxic activity of centratherin alone was also evaluated against both U251 and U87-MG cells, which showed IC50 values comparable with those obtained for the commercial anticancer drug doxorubicin. All the tested samples showed cytotoxic activity against glioblastoma multiforme cells which suggests that E. crotonoides extracts may be important sources of antiproliferative substances and that the centratherin may serve as prototype for developing new antiglioblastoma drugs.

Monitoramento in vivo por imagem por ressonância magnética de células C6 de glioma marcadas com nanopartículas superparamagnéticas de óxido de ferro

OBJECTIVE: The aim of the current study was to monitor the migration of superparamagnetic iron oxide nanoparticle (SPION)-labeled C6 cells, which were used to induce glioblastoma tumor growth in an animal model, over time using magnetic resonance imaging (MRI), with the goal of aiding in tumor prognosis and therapy. METHODS: Two groups of male Wistar rats were used for the tumor induction model. In the first group (n=3), the tumors were induced via the injection of SPION-labeled C6 cells. In the second group (n=3), the tumors were induced via the injection of unlabeled C6 cells. Prussian Blue staining was performed to analyze the SPION distribution within the C6 cells in vitro. Tumor-inducing C6 cells were injected into the right frontal cortex, and subsequent tumor monitoring and SPION detection were performed using T2- and T2*-weighted MRI at a 2T field strength. In addition, cancerous tissue was histologically analyzed after performing the MRI studies. RESULTS: The in vitro qualitative evaluation demonstrated adequate distribution and satisfactory cell labeling of the SPIONs. At 14 or 21 days after C6 injection, a SPION-induced T2- and T2*-weighted MRI signal reduction was observed within the lesion located in the left frontal lobe on parasagittal topography. Moreover, histological staining of the tumor tissue with Prussian Blue revealed a broad distribution of SPIONs within the C6 cells. CONCLUSION: MRI analyses exhibit potential for monitoring the tumor growth of C6 cells efficiently labeled with SPIONs.