Flavopiridol inhibits the Growth of GL261 Gliomas In Vivo: Implications for Malignant Glioma Therapy

The mechanism of action of many chemotherapeutic agents targets the cell cycle. Recently, we demonstrated cytotoxic and other anti-tumor effects of flavopiridol, the first synthetic cyclin dependent kinase (CDK) inhibitor to enter clinical trials, on the murine GL261 glioma cell line in vitro (Newcomb et al., Cell Cycle 2003; 2:243). Given that flavopiridol has demonstrated anti-tumor activity in several human xenograft models, we wanted to evaluate it for anti-glioma activity in vivo in our established subcutaneous and intracranial GL261 experimental tumor models. In particular, the intracranial animal model recapitulates many of the histopathological and biological features of human high-grade glioma including both necrosis with pseudopalisading and invasion of the brain adjacent to tumor. Here we tested the activity of flavopiridol against tumors formed by GL261 cells, first as subcutaneous implants, and then in the intracranial model. We demonstrate efficacy of flavopiridol as a single modality treatment in delaying tumor growth in both animal models. We hypothesize that flavopiridol treatment induced tumor growth delay by two possible mechanisms involving growth arrest combined with recruitment of tumor cells to S-phase. Based on our findings, flavopiridol should be considered as a treatment approach for patients with high-grade glioma.     

Experimental immunotherapy for malignant glioma: lessons from two decades of research in the GL261 model

Nearly twenty years of experimental immunotherapy for malignant glioma yielded important insights in the mechanisms governing glioma immunology. Still considered promising, it is clear that immunotherapy does not on its own represent the magic bullet in glioma therapy. In this review, we summarize the major immunotherapeutic achievements in the mouse GL261 glioma model, which has emerged as the gold standard syngeneic model for experimental glioma therapy. Gene therapy, monoclonal antibody treatment, cytokine therapy, cell transfer strategies and dendritic cell therapy were hereby considered. Apart from the considerable progress made in understanding glioma immunology in this model, we also addressed its most pertinent issues and shortcomings. Despite these, the GL261 model will remain indispensable in glioma research since it is a fast, highly reproducible and easy-to-establish model system.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Radiation Sensitivity of GL261 Murine Glioma Model and Enhanced Radiation Response by Flavopiridol

Response of a solid tumor to radiation treatment depends, in part, on the intrinsic radiosensitivity of tumor cells, the proliferation rate of tumor cells between radiation treatments and the hypoxic state of the tumor cells. A successful radiosensitizing agent would target S-phase cells and hypoxia. Recently, we demonstrated the anti-tumor effects of flavopiridol in the GL261 murine glioma model might involve 1) recruitment of tumor cells to S-phase (Newcomb et al., Cell Cycle 2004; 3:230-234) and 2) an anti-angiogenic effect on the tumor vasculature by downregulation of hypoxia-inducible factor -1? (HIF-1?) (Newcomb et al., Neuro-Oncology 2005; 7:225-235). Given that flavopiridol has demonstrated radiosensitizing activity in several murine tumor models, we tested whether it would enhance the response of GL261 tumors to radiation. In the present study, we evaluated the intrinsic radiation sensitivity of the GL261 glioma model using the tumor control/cure dose of radiation assay (TCD50). We found that a single dose of 65 Gy (CI 57.1-73.1) was required to cure 50% of the tumors locally. Using the tumor growth delay assay, fractionated radiation (5 fractions of 5 Gy over 10 days) combined with flavopiridol (5 mg/kg) given three times weekly for 3 cycles produced a significant growth delay. Our results indicate that the GL261 murine glioma model mimics the radioresistance encountered in human gliomas, and thus should prove useful in identifying promising new investigational radiosensitizers for use in the treatment of glioma patients.     

Characterization of ATP‐induced cell death in the GL261 mouse glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010. © 2010 Wiley‐Liss, Inc.     

Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac)

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 μM tetrac (1 h at 37oC) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.     

Flavopiridol Induces Mitochondrial-Mediated Apoptosis in Murine Glioma GL261 Cells via Release of Cytochrome c and Apoptosis Inducing Factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. Over 80% of adult high-grade astrocytomas show inactivation of the Rb tumor suppressor pathway. Therefore, one possible therapeutic strategy would be to directly modulate cyclin dependent kinase (CDK) activity resulting in inhibition of Rb phosphorylation and cell cycle progression. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to:

1.inhibit cell growth;

2.inhibit cell migration;

3.decrease expression of CDK inhibitor cyclin D1, CDK4 and p21;

4.induce apoptosis in cells with high levels of p27 expression; and

5.decrease the expression of the anti-apoptotic protein Bcl-2.

The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs markedly from the mechanism of flavopiridol-induced apoptosis cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug induces apoptosis inkills different types of glioma cells by different mitochondrial-dependent pathways.     

Pigment epithelium-derived factor inhibits glioma cell growth in vitro and in vivo

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of several tumors. This study was designed to determine whether gliomas angiogenesis and tumor growth could be inhibited by PEDF. We found that PEDF down-regulated expression levels of vascular endothelial growth factor and up-regulated the expression of thrombospondin-2 and augmented apoptosis in a dose-dependent manner in both A172 and U87 glioma cells lines after 48 h of treatment. Analysis of the cell cycle showed arrest in the G₁ phase and block in S phase of the cell cycle. Meanwhile PEDF induced apoptosis was associated with increases of p53 and Bax and inhibition of Bcl-2. Conditioned medium with PEDF showed a significantly reductive effect on migration in vitro accompanied with a significant reduction of matrix metalloproteinase-9 expression. PEDF suppressed glioma cell migration in vitro and tumor burden in athymic nude mice. These results demonstrate for the first time inhibitory effects of PEDF on the growth and migration of human gliomas via induction of apoptosis and blocking of migratory-related factors. PEDF activation can be a novel approach for future therapeutic purposes against gliomas.     

Antitumor effects of a xenogeneic survivin bone marrow derived dendritic cell vaccine against murine GL261 gliomas

Survivin is a member of the inhibitor of apoptosis protein family. Gliomas and many other tumors express survivin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, survivin represents a tumor-specific target for cancer vaccine therapy. It has been shown that it is possible to produce a MHC-I-restricted cellular immunologic response to survivin vaccines. To study differences in immunogenicity between murine and human survivin proteins, we vaccinated C57BL/6 mice with bone marrow dendritic cells (BMDC) transfected with expression vectors containing the murine and human survivin genes. Mice vaccinated with BMDCs expressing a truncated human survivin protein developed cytotoxic T lymphocyte to subcutaneous GL261 glioma cells and exhibited prolonged tumor-free survival compared to mice vaccinated with BMDCs transfected with vector alone (P<0.01). While mice challenged with intracerebral GL261 cells had increased survival, no cures were observed. In contrast, vaccinated mice that fully resisted subcutaneous tumor challenge were rendered resistant to intracerebral GL261 re-challenge. BMDCs transfected with the full-length human survivin molecule were significantly more effective at prolonging survival than BMDCs expressing the full-length murine survivin gene (P=0.0175). Therefore, xenogeneic differences between human and murine sequences might be exploited to develop more immunogenic tumor vaccines.     

Pirfenidone inhibits TGF-beta expression in malignant glioma cells

Due to its immunosuppressive properties, the cytokine transforming growth factor (TGF)-beta has become a promising target in the experimental treatment of human malignant gliomas. Here, we report that the antifibrotic drug 5-methyl-1-phenyl-2-(1H)-pyridone (pirfenidone, PFD) elicits growth-inhibitory effects and reduces TGF-beta2 protein levels in human glioma cell lines. This reduction in TGF-beta2 is biologically relevant since PFD treatment reduces the growth inhibition of TGF-beta-sensitive CCL-64 cells mediated by conditioned media of glioma cells. The downregulation of TGF-beta is mediated at multiple levels. PFD leads to a reduction of TGF-beta2 mRNA levels and of the mature TGF-beta2 protein due to decreased expression and direct inhibition of the TGF-beta pro-protein convertase furin. In addition, PFD reduces the protein levels of the matrix metalloproteinase (MMP)-11, a TGF-beta target gene and furin substrate involved in carcinogenesis. These data define PFD or PFD-related agents as promising agents for human cancers associated with enhanced TGF-beta activity.     

Aurantiamide acetate suppresses the growth of malignant gliomas in vitro and in vivo by inhibiting autophagic flux

We aim to investigate the effect of aurantiamide acetate isolated from the aerial parts of Clematis terniflora DC against gliomas. Human malignant glioma U87 and U251 cells were incubated with different concentrations (0–100 μM) of aurantiamide acetate. Aurantiamide acetate greatly decreased the cell viability in a dose‐ and time‐dependent manner. It induced moderate mitochondrial fragmentation and the loss of mitochondrial membrane potential. No significant difference was found in the alternation of other intracellular organelles, although F‐actin structure was slightly disturbed. Apparent ultrastructure alternation with increased autophagosome and autolysosome accumulation was observed in aurantiamide acetate‐treated cells. The expression of LC3‐II was greatly up‐regulated in cells exposed to aurantiamide acetate (P < 0.05 compared with control). The cytoplasmic accumulation of autophagosomes and autolysosomes induced by aurantiamide acetate treatment was confirmed by fluorescent reporter protein labelling. Administration of chloroquine (CQ), which inhibits the fusion step of autophagosomes, further increased the accumulation of autophagosomes in the cytoplasm of U87 cells. Autophagy inhibition by 3‐methyladenine, Bafilomycin A1 or CQ had no influence on aurantiamide acetate‐induced cytotoxicity, whereas autophagy stimulator rapamycin significantly suppressed aurantiamide acetate‐induced cell death. The anti‐tumour effects of aurantiamide acetate were further evaluated in tumour‐bearing nude mice. Intratumoural injection of aurantiamide acetate obviously suppressed tumour growth, and increased number of autophagic vacuoles was observed in tumour tissues of animals receiving aurantiamide acetate. Our findings suggest that aurantiamide acetate may suppress the growth of malignant gliomas by blocking autophagic flux.     

Glutamate release promotes growth of malignant gliomas

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.     

HIV-1 dynamics in vivo: implications for therapy

The advent of potent combination antiretroviral therapy has been an important breakthrough in the treatment of HIV-1 infection, resulting in marked reductions in HIV-1-related morbidity and mortality. Antiretroviral therapy has also provided researchers with a powerful tool to perturb the equilibrium of viral production and viral clearance, allowing them to dissect the underlying dynamics that control the pathogenesis of AIDS. Here, we review our current understanding of the sources of HIV-1 production, the estimates for the virion and the host-cell half-lives, and the pathways of virion trafficking and clearance. We also discuss the obstacles that result from the ability of HIV-1 to remain dormant for a prolonged period of time in a subset of long-lived cells, despite an apparently effective antiretroviral treatment.     

Sodium vanadate inhibits apoptosis in malignant glioma cells: A role for Akt/PKB

The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and shown that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.     

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.     

Minocycline inhibits the growth of glioma by inducing autophagy

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.     

Ivermectin inhibits the growth of glioma cells by inducing cell cycle arrest and apoptosis in vitro and in vivo

Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma.