Human Malignant Glioma Tissue Research Articles

Bone Morphogenetic Protein 7 Effect on Human Glioblastoma Cell Transmigration and Migration.

Glioblastoma, World Health Organization—grade IV, is the most malignant glioma type and it is still an incurable tumor due to the high level of heterogeneity and uncontrolled metastatic nature. In addition to the tumorigenicity-suppressing activity, bone morphogenetic protein 7 (BMP7) has recently been found for its invasion-promoting role in glioblastoma. However, the detailed and precise mechanism in this issue should have more elucidation. Thus, in this study, we determined the BMP7 effect on glioblastoma transmigration and migration regulations and the underlying mechanisms. Human LN18/LN229 glioblastoma cells were used in this study. Our results showed a higher BMP7/pSmad5 level in human malignant glioma tissues compared to healthy brain tissues. In addition, it was demonstrated that endogenous and exogenous BMP7 stimulation could increase the transmigration and migration capabilities of human LN18/LN229 glioblastoma cells. Moreover, this event is regulated by Smad5 and p75 neurotrophin receptor (p75NTR) signaling. Furthermore, unexpected data are that the Smad1 gene knockdown could lead to the cell death of human LN18 glioblastoma cells. Overall, the present study finds that the invasion-promoting activity of BMP7 might be an autocrine stimulation of glioblastoma and this effect could be regulated by Smad5-p75NTR signaling.

TRPM7 channel inhibition mediates midazolam-induced proliferation loss in human malignant glioma.

The melastatin-like transient receptor potential 7 (TRPM7) has been implicated in proliferation or apoptosis of some cancers, indicating the potential of TRPM7 as an anti-anaplastic target. Here, we identified the characteristic TRPM7 channel currents in human malignant glioma MGR2 cells, which could be blocked by a pharmacologic inhibitor Gd3+. We mined the clinical sample data from Oncomine Database and found that human malignant glioma tissues expressed higher TRPM7 mRNA than normal brain ones. Importantly, we identified a widely used clinical anesthetic midazolam as a TRPM7 inhibitor. Midazolam treatment for seconds suppressed the TRPM7 currents and calcium influx, and treatment for 48h inhibited the TRPM7 expression. The inhibitory effect on TRPM7 accounts for the proliferation loss and G0/G1 phase cell cycle arrest induced by midazolam. Our data demonstrates that midazolam represses proliferation of human malignant glioma cells through inhibiting TRPM7 currents, which may be further potentiated by suppressing the expression of TRPM7. Our result indicates midazolam as a pharmacologic lead compound with brain-blood barrier permeability for targeting TRPM7 in the glioma.

Tryptophan-2,3-dioxygenase is regulated by prostaglandin E2 in malignant glioma via a positive signaling loop involving prostaglandin E receptor-4.

Malignant gliomas and other types of tumors generate a local immunosuppressive microenvironment, which prohibits an effective anti-tumor immune response and promotes tumor growth. Along with others, we have recently demonstrated that catabolism of the essential amino acid tryptophan via tryptophan-2,3-dioxygenase (TDO) is an important mechanism mediating tumor-associated immunosuppression particularly in gliomas. The pathways regulating TDO in tumors, however, are poorly understood. Here, we show that prostaglandins enhance TDO expression and enzymatic activity in malignant gliomas via activation of prostaglandin E receptor-4 (EP4). Stimulation with prostaglandin E2 (PGE2 ) up-regulated TDO-mediated kynurenine release in human glioma cell lines, whereas knockdown of the PGE2 receptor EP4 inhibited TDO expression and activity. In human malignant glioma tissue expression of the PGE2 -producing enzyme cyclooxygenase-2 (COX2) and its receptor EP4 were associated with TDO expression both on transcript and protein level. High expression of EP4 correlated with poor survival in malignant glioma patients WHO III-IV. Importantly, treatment of glioma cells with an EP4 inhibitor decreased TDO expression and activity. Moreover, TDO-over-expressing murine gliomas showed increased COX2 and EP4 expression suggesting a positive feedback mechanism invivo. In summary, targeting EP4 may inhibit - in addition to immunosuppressive COX2 signaling - tryptophan degradation as another important immunosuppressive pathway and thus, could provide a dual clinically relevant immunotherapeutic avenue for the treatment of malignant gliomas. We proposed that in malignant gliomas prostaglandin E2 (PGE2 ) produced by cyclooxygenases (COX) up-regulates tryptophan-2,3-dioxygenase (TDO) expression and enzyme activity through binding to its Gs-coupled receptor EP4 and therefore may mediate tumor immune escape in part through aryl hydrocarbon receptor (AHR) activation. Moreover, TDO activity itself seems to induce intratumoral PGE2 metabolism suggesting an immunosuppressive loop involving COX/EP4/TDO.

Over-expression of tetraspanin 8 in malignant glioma regulates tumor cell progression

Tumor cell invasion and proliferation remain the overwhelming causes of death for malignant glioma patients. To establish effective therapeutic methods, new targets implied in these processes have to be identified. Tetraspanin 8 (Tspn8) forms complexes with a large variety of trans-membrane and/or cytosolic proteins to regulate several important cellular functions. In the current study, we found that Tspn8 was over-expressed in multiple clinical malignant glioma tissues, and its expression level correlated with the grade of tumors. Tspn8 expression in malignant glioma cells (U251MG and U87MG lines) is important for cell proliferation and migration. siRNA-mediated knockdown of Tspn8 markedly reduced in vitro proliferation and migration of U251MG and U87MG cells. Meanwhile, Tspn8 silencing also increased the sensitivity of temozolomide (TMZ), and significantly increased U251MG or U87MG cell death and apoptosis by TMZ were achieved with Tspn8 knockdown. We observed that Tspn8 formed a complex with activated focal adhesion kinase (FAK) in both human malignant glioma tissues and in above glioma cells. This complexation appeared required for FAK activation, since Tspn8 knockdown inhibited FAK activation in U251MG and U87MG cells. These results provide evidence that Tspn8 contributes to the pathogenesis of glioblastoma probably by promoting proliferation, migration and TMZ-resistance of glioma cells. Therefore, targeting Tspn8 may provide a potential therapeutic intervention for malignant glioma.

Neurotrophin Signaling via TrkB and TrkC Receptors Promotes the Growth of Brain Tumor-initiating Cells

Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma.

Elevated cell invasion in a tumor sphere culture of RSV-M mouse glioma cells.

Cancer stem cells (CSCs) are the sole population possessing high self-renewal activity in tumors, with their existence affecting tumor recurrence. However, the invasive activity of CSCs has yet to be fully understood. In this article, we established a tumor sphere culture of RSV-M mouse glioma cells (RSV-M-TS) and evaluated their migration and invasion activities. Histological analysis of a tumor formed by cranial injection of the RSV-M-TS cells showed highly invasive properties and similarities with human malignant glioma tissues. When the migration activity of both RSV-M and RSV-M-TS cells were compared by intracranial injection, rapid migration of RSV-M-TS cells was observed. To confirm the invasive capabilities of RSV-M-TS cells, a three-dimensional collagen invasion assay was performed in vitro using RSV-M, RSV-M-TS, and RSV-M-TS cells cultured with medium containing serum. RSV-M and RSV-M-TS cultured with medium containing serum for 8 days indicated low migration activity, while moderate invasion activity was observed in RSV-M-TS cells. This activity was further enhanced by incubation with medium containing serum overnight. To identify the genes involved in this invasion activity, we performed quantitative polymerase chain reaction (PCR) array analysis of RSV-M and RSV-M-TS cells. Of 84 cancer metastasis-related genes, up-regulation was observed in 24 genes, while 4 genes appeared to be down-regulated in RSV-M-TS cells. These results suggest that the enhanced invasive activity of glioma sphere cells correlates with a number of tumor metastasis-related genes and plays a role in the dissemination and invasion of glioma cells.

CS-14 * NEUROTROPHIN RECEPTORS TrkB AND TrkC ARE REQUIRED FOR SURVIVAL OF BRAIN TUMOR INITIATING CELLS

Neurotrophins (NGF, BDNF and NT3) and their receptors (TrkA, TrkB, TrkC and p75NTR) are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75NTR is required for glioma invasion and proliferation. However, the role of other Trk receptors has not been examined. In this study, we investigated the importance of neurotrophin receptors TrkB and TrkC in survival of brain tumor initiating cells (BTICs). We used human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas and examined the expression of neurotrophin receptors and studied their role in survival/growth these cells in vitro, we have also examined the mechanism underlie. Here, we show that human malignant glioma tissues and tumor-initiating cells express the neurotrophin receptors TrkB and TrkC, not TrkA and they also express neurotrophins NGF, BDNF and NT3. Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 stimulates Trk phosphorylation, activates Erk and Akt and enhances tumor-initiating cell viability and growth. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition using inhibitors of Trk, Erk and Akt, decreases neurotrophin-dependent Trk phosphorylation, Erk and Akt activation and BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling and further neurotrophin signaling is required for maintains long-term BTIC growth in the absence of EGF and FGF, and this effect is attenuated with Trk, Erk and Akt inhibitors. In vivo experiments are under progress. Our results highlight a novel role for neurotrophin signaling in malignant glioma and suggest that Trks could be a target for combinatorial treatment.

CXCL12-induced upregulation of FOXM1 expression promotes human glioblastoma cell invasion

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor; it is highly aggressive and is associated with a poor prognosis. Binding of the chemokine CXCL12 to its receptors (CXCR4 and CXCR7) contributes to the activation of many downstream signaling pathways and promotes the invasion of various malignant tumor cells, including GBM cells. FOXM1, a transcription factor involved in cell cycle regulation, is overexpressed in GBM and is involved in GBM progression. However, the molecular mechanisms by which CXCL12 promotes the invasion of human GBM cells remain unclear. In this study, we demonstrate that CXCL12 increases the production of FOXM1 by binding to CXCR4 in GBM cell lines. Furthermore, pretreatment with an inhibitor of the PI3K/AKT pathway abrogated the CXCL12-induced expression of FOXM1. In addition, there was a positive correlation between CXCL12/CXCR4 expression and FOXM1 expression in human malignant glioma tissues. Finally, a functional assay revealed that CXCL12 does not stimulate GBM cell invasion when FOXM1 expression is silenced using a small interfering RNA (siRNA). Collectively, these findings suggest that CXCL12 promotes GBM cell invasion in part by increasing the expression of FOXM1, which is mediated in part by a PI3K/AKT-dependent mechanism in vitro.

Dissecting Pin1 and phospho‐pRb regulation

The activity of the Retinoblastoma protein, the master regulator of the cell cycle, is finely regulated by phosphorylation. CDKs and cyclins are major players in phosphorylation and it has been recently discovered that the prolyl isomerase Pin1 is an essential protein that orchestrates this process. In this article, we report new findings regarding the role of Pin1 in the pRb pathway. Our data suggest that PI3K, CDKs, and the Pin1 axis have a critical role in sustaining the complete phosphorylation of pRb. Furthermore, we analyze the correlation between Pin1 and pRb phosphorylation in vivo. We show that, in human malignant glioma tissue microarrays (TMA) and in Pin1 knockout (KO) mice, there is a positive correlation between Pin1 and pRb phosphorylation. Prospectively, our findings suggest that the synergism between CDKs, Pin1, and PI3K inhibitors hold great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.

Platinum distribution in malignant glioma following intraoperative intravenous infusion of carboplatin

The objective of this paper was to determine the time course and extent of platinum uptake into human malignant glioma tissue. An intraoperative, intravenous infusion of carboplatin was given to nine patients (seven glioblastoma and two anaplastic glioma) undergoing tumour excision. Carboplatin dosage was calculated individually to achieve a target systemic free carboplatin exposure.Tumour and peritumoural tissue was harvested at timed intervals following carboplatin administration. Plasma and tumour platinum concentrations were measured by graphite furnace flameless atomic absorption spectrophotometry. Histological examination was also performed on a piece of each tissue sample. The mean carboplatin dose administered was 783, SEM. 56 mg (range 485-903). Plasma pharmacokinetics showed a typical elimination curve. The mean peak plasma platinum concentration was 44, SEM 5 mug/ml (range 27-74).The mean total elemental plasma platinum area under the curve (AUC) was 9.0, SEM 1.4 mg/ml/min. Platinum was detected in 61 tumour samples, the mean peak concentration being 13 SEM 2 mug/g (range 5-21). Platinum was also detected in peritumoural brain and necrotic tumour. No correlation was apparent between the degree of necrosis in each tumour specimen and tumour platinum concentration. Platinum concentrations achieved in tumour were similar to levels that would be cytotoxic for glioma cells in vitro. The results of this study have implications for future studies using capillary permeability modifying agents as adjuncts to brain tumour chemotherapy.