At this time, brain tumor stem cells remain a controversial hypothesis while malignant brain tumors continue to present a dire prognosis of severe morbidity and mortality. Yet, brain tumor stem cells may represent an essential cellular target for glioma therapy as they are postulated to be the tumorigenic cells responsible for recurrence. Targeting oncogenic pathways that are essential to the survival and growth of brain tumor stem cells represents a promising area for developing therapeutics. However, due to the multiple oncogenic pathways involved in glioma, it is necessary to determine which pathways are the essential targets for therapy. Furthermore, research still needs to comprehend the morphogenic processes of cell populations involved in tumor formation. Here, we review research and discuss perspectives on models of glioma in order to delineate the current issues in defining brain tumor stem cells as therapeutic targets in models of glioma.
1-Phosphatidylinositol 3-Kinase/genetics/metabolism ; Animals ; Brain Neoplasms/genetics/*metabolism/*pathology/therapy ; Glioma/genetics/*metabolism/*pathology/therapy ; Humans ; Neoplastic Stem Cells/*metabolism/*pathology ; Receptors, Notch/genetics/metabolism ; Signal Transduction/genetics/physiology

To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1alpha, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis-related genes.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; drug effects ; Genetic Therapy ; Glioma ; genetics ; pathology ; Humans ; Interleukins ; genetics ; metabolism ; Recombination, Genetic ; Tumor Cells, Cultured

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Adenoviridae/genetics ; Animals ; Blood-Brain Barrier ; Brain/drug effects/*metabolism/pathology ; Brain Neoplasms/genetics/metabolism/pathology/*therapy ; Clinical Trials, Phase I as Topic ; DNA, Viral/metabolism ; Disease Models, Animal ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; *Gene Therapy ; Glioma/genetics/metabolism/pathology/*therapy ; Humans ; Liver/drug effects/metabolism/pathology ; Protein Multimerization/genetics ; Rats ; Spleen/drug effects/metabolism/pathology ; TNF-Related Apoptosis-Inducing Ligand/genetics/*pharmacokinetics

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Adenoviridae/genetics ; Animals ; Blood-Brain Barrier ; Brain/drug effects/*metabolism/pathology ; Brain Neoplasms/genetics/metabolism/pathology/*therapy ; Clinical Trials, Phase I as Topic ; DNA, Viral/metabolism ; Disease Models, Animal ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; *Gene Therapy ; Glioma/genetics/metabolism/pathology/*therapy ; Humans ; Liver/drug effects/metabolism/pathology ; Protein Multimerization/genetics ; Rats ; Spleen/drug effects/metabolism/pathology ; TNF-Related Apoptosis-Inducing Ligand/genetics/*pharmacokinetics

OBJECTIVETo explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment.

METHODSTo tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus. The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by (125)I uptake assay, (125)I influx-course and (125)I-efflux-course. A glioma model was established with inoculation of the U251 cells in nude mice, and the inhibiting effect of (131)I on the tumor growth was tested in the mouse models.

RESULTSThe hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus. The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene. The uptake of (125)I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells. The tumor volume of the mice after (131)I treatment was significantly decreased in comparison with that before treatment.

CONCLUSIONRadioactive (131)I treatment after HNIS-based gene transfer can be enhanced and effectively inhibit the tumor growth in nude mice.

Adenoviridae ; genetics ; Animals ; Brain Neoplasms ; pathology ; therapy ; Genetic Therapy ; Glioma ; pathology ; therapy ; Humans ; In Vitro Techniques ; Iodides ; Iodine Radioisotopes ; metabolism ; therapeutic use ; Mice, Nude ; Symporters ; genetics ; Transfection

OBJECTIVETo study the anti-glioma effect of recombinant adenovirus mediated combined gene therapy of bFGF-siRNA and HIV1-Vpr in vivo.

METHODSMouse glioma model was established by injecting 5 × 10(6) LN229 cells into BALB/c-nu nude mice. 30 nude mice were randomly divided into 5 groups: the negative control group, mock group, bFGF-siRNA group, Vpr group and combined therapy group, which at regular intervals were injected with PBS, rAd5-null, rAd5-bFGF-siRNA, rAd5-Vpr, rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volume was recorded every third day to draw a growth curve. After four weeks treatment, the mice were killed and specimens were taken. HE, immunohistochemical and TUNEL staining were performed to observe the cell morphology, detect the changes of relevant target proteins and cell apoptosis, respectively. Also the ultrastructural changes were observed by electron microscopy.

RESULTSThe tumor growth inhibition rates were 36.9%, 37.2% and 58.6% in the bFGF-siRNA group, Vpr group and combined therapy group, respectively, and the combined therapy group showed the most significant effect (P < 0.05). Also the results of HE, immunohistochemical and TUNEL staining revealed that the combined therapy group had the best effects on proliferation inhibition and apoptosis induced in glioma cells (P < 0.05). The most significant ultrastructural changes were observed in the combined therapy group.

CONCLUSIONThe combined gene therapy of bFGF-siRNA with Vpr shows a prominent and synergistic anti-glioma effect compared with that of mono-gene therapy in nude mice.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Brain Neoplasms ; metabolism ; pathology ; therapy ; Cell Line, Tumor ; Cell Proliferation ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Gene Products, vpr ; genetics ; metabolism ; Genetic Therapy ; Glioma ; metabolism ; pathology ; therapy ; HIV-1 ; genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Small Interfering ; genetics ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo.

METHODSTo construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods.

RESULTSAfter transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited.

CONCLUSIONGrowth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Blotting, Western ; Brain Neoplasms ; pathology ; therapy ; Cell Line, Tumor ; Cell Proliferation ; DNA, Antisense ; genetics ; metabolism ; Flow Cytometry ; Genetic Therapy ; methods ; Glioma ; pathology ; therapy ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; PTEN Phosphohydrolase ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection ; Tumor Burden ; Xenograft Model Antitumor Assays

OBJECTIVETo construct recombinant expression vectors of small interfering RNA (siRNA) targeting survivin and investigate apoptosis of glioma cell line U251 mediated by the survivin-targeting siRNA.

METHODSAccording to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGenesil-1, and the resulted vector pGenesil-1/survivin was transfected into U251 cells using Metafectene following the standard protocols. Real-time PCR and Western blotting were performed to evaluate survivin gene silencing induced by siRNA transfection at the RNA and protein levels, respectively. Flow cytometry analysis with Annexin-V/PI double staining was used to determine the cell apoptosis.

RESULTSReal-time RT-PCR and Western blotting revealed significantly lowered survivin expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly higher apoptosis rate after transfection as shown by flow cytometry analysis.

CONCLUSIONRNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.

Apoptosis ; physiology ; Brain Neoplasms ; metabolism ; pathology ; Cell Division ; Cell Line, Tumor ; Genetic Therapy ; Glioma ; metabolism ; pathology ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Antisense ; genetics ; RNA, Small Interfering ; Transfection

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2\'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.
Animals ; Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Brain/drug effects/metabolism/pathology ; Brain Neoplasms/drug therapy/*genetics/pathology ; DNA (Cytosine-5-)-Methyltransferase/antagonists & inhibitors/*genetics/metabolism ; DNA Methylation ; Dacarbazine/*analogs & derivatives/pharmacology/therapeutic use ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation, Neoplastic ; Glioma/drug therapy/*genetics/pathology ; Humans ; Mice, Inbred C57BL ; MicroRNAs/*genetics ; Promoter Regions, Genetic

OBJECTIVETo explore if folic acid/polyamide-amine (FA/PAMAM) enhances the therapeutic effect of miR-7gene therapy for glioma in vivo.

METHODSThe miR-7 gene was transfected into U251 glioma cells by FA/PAMAM. The efficiency of gene transfection was assessed by fluorescence microscopy. The miR-7 level was detect by quantitative RT-PCR. Intracranial glioma models were established in thymectomized mice, and FA/PAMAM nanoparticles were transplanted into the tumors in situ 3 days later. The animal survival was recorded and the gross tumor volume and degree of edema were observed by MRI. Apoptosis in the glioma cells and expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) were assessed by immunohistochemistry, and EGFR and AKT-2 protein expression was detected by Western blot assay.

RESULTSCompared with the liposomes, the FA/PAMAM nanoparticles were more efficient to transfer miR-7 gene into U251 glioma cells, MRI showed that the tumor growth was much slower in the FA/PAMAM/miR-7 group, and the animal survival time was longer. The apoptosis rate was (5.3 ± 0.9)% in the control group, (11.4 ± 2.4)% in the liposome/miR-7 group, and (17.7 ± 3.7)% in the FA/PAMAM/miR-7 group. The immunohistochemical assay showed that the levels of PCNA, MMP-2 and MMP-9 protein in the FA/PAMAM/miR-7 group were (34.6 ± 5.4)%, (24.5 ± 4.1)%, (25.4 ± 5.1)%, respectively, significantly lower than those in the liposome/miR-7 group (49.3 ± 5.9)%, (31.7 ± 7.1)% and (39.4 ± 6.4)%, respectively, and those in the control group (57.3 ± 7.4)%, (45.4 ± 6.9)% and (55.1 ± 7.3)%, respectively (all P < 0.05). The expressions of EGFR and AKT-2 proteins were 1.09 ± 0.12 and 0.62 ± 0.10 in the control group, 0.63 ± 0.11 and 0.43 ± 0.07 in the liposome/miR-7 group, and significantly deceased (0.47 ± 0.09 and 0.31 ± 0.04, respectively) in the FA/PAMAM/miR-7 group (all P < 0.05).

CONCLUSIONCompared with the liposomes, FA/PAMAM can transfect miR-7 into glioma cells with a higher efficiency in vivo, makes a longer time of the drug action, and shows a certain inhibitory effect on the growth of glioma, therefore, might become a new drug targeting agent in gene therapy forglioma.

Animals ; Apoptosis ; Brain Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Dendrimers ; chemistry ; Folic Acid ; chemistry ; Genetic Therapy ; methods ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Nude ; MicroRNAs ; genetics ; metabolism ; Nanoparticles ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Thymectomy ; Transfection