Differentiation Of Glioma Cells Research Articles

HIF1α regulates single differentiated glioma cell dedifferentiation to stem-like cell phenotypes with high tumorigenic potential under hypoxia.

The standard treatment for Glioblastoma multiforme (GBM) is surgical resection and subsequent radiotherapy and chemotherapy. Surgical resection of GBM is typically restricted because of its invasive growth, which results in residual tumor cells including glioma stem cells (GSCs) and differentiated cells. Recurrence has been previously thought to occur as a result of these GSCs, and hypoxic microenvironment maintains the GSCs stemness also plays an important role. Summarizing traditional studies and we find many researchers ignored the influence of hypoxia on differentiated cells. We hypothesized that the residual differentiated cells may be dedifferentiated to GSC-like cells under hypoxia and play a crucial role in the rapid, high-frequency recurrence of GBM. Therefore, isolated CD133–CD15–NESTIN– cells were prepared as single-cell culture and treated with hypoxia. More than 95% of the surviving single differentiated CD133–CD15–NESTIN– cell dedifferentiated into tumorigenic CD133+CD15+NESTIN+ GSCs, and this process was regulated by hypoxia inducible factor-1α. Moreover, the serum also played an important role in this dedifferentiation. These findings challenge the traditional glioma cell heterogeneity model, cell division model and glioma malignancy development model. Our study also highlights the mechanism of GBM recurrence and the importance of anti-hypoxia therapy. In addition to GSCs, residual differentiated tumor cells also substantially contribute to treatment resistance and the rapid, high recurrence of GBM.

Pivotal role of high-mobility group box 1 (HMGB1) signaling pathways in glioma development and progression

Human gliomas represent the most common type of intracranial tumors, with highest morbidity and mortality. They are characterized by excessive invasiveness and cell proliferation while their unclear boundaries predispose to tumor recurrence soon after conventional treatment. Elucidation of the molecular mechanisms implicated in their development and/or treatment resistance is highly demanded. The high-mobility group box 1 (HMGB1) protein, a highly conserved nuclear protein that functions as a chromatin-binding factor, facilitating nucleosome stabilization and regulating gene transcription, has been implicated in glioma formation and progression. Extracellular released HMGB1 binds to high-affinity receptors, including the receptor for advanced glycation end-products (RAGE) and toll-like receptor (TLR)-2, TLR-4, and TLR-9. Upon receptor binding, HMGB1 triggers the activation of key signaling pathways and immune responses, involved in the regulation of cell growth, differentiation, motility, and apoptosis. Based on the type of receptor and/or cell, HMGB1 is capable to promote oncogenesis or suppress tumor growth, thus affecting treatment efficacy. Herein, we discuss recent evidence implicating HMGB1 in glioma cell differentiation, proliferation, and metastasis with both clinical and prognostic significance. In addition, potential therapeutic approaches to target this protein in order to reduce chemoresistance of glioma cells are also addressed.

Silence of bFGF enhances chemosensitivity of glioma cells to temozolomide through the MAPK signal pathway.

Basic fibroblast growth factor (bFGF) is a multifunctional growth factor in glioma cells and has been proved to be associated with the grade malignancy of glioma and prognosis of patients. Although there is evidence showing that bFGF plays an important role in proliferation, differentiation, angiogenesis, and survival of glioma cells, the effect of bFGF on chemosensitivity of glioma has not been verified. In this study, we analyzed the relationship between bFGF and chemotherapy resistance, with the objective of offering new strategy for chemotherapy of glioma patients. Here, siRNA was used to silence the expression of bFGF in glioma cell lines including U87 and U251 followed by chemotherapy of temozolomide (TMZ). Then, the characters of glioma including proliferation, apoptosis, migration, and cell cycle were studied in U87 and U251 cell lines. Our results demonstrated that silencing bFGF enhanced the effect of TMZ by inhibiting proliferation and migration, blocking cell cycle in G0/G1, and promoting apoptosis. In addition, the phosphorylation level of MAPK was measured to explore the mechanism of chemosensitization. The results showed that bFGF could promote the activation of the MAPK signal pathway. Our data indicated that bFGF might be a potential target for chemotherapy through the MAPK signal pathway.

Loss of miR-124 and miR-137 expression in glioblastoma multiforme and their roles in glioma cell proliferation and differentiation

Background: Glioblastoma Multiforme (GBM) is a common malignant brain tumor. It is characterized by rapid growth and high tumor heterogeneity. The molecular mechanisms driving GBM tumorigenesis and progression are not yet fully elucidated.Methods: miR-124, and miR-137, expression in 43 high-grade GBM tumor tissues was measured by real-time PCR and compared with expression in 24 normal brain tissues. The effects of miR-124, and miR-137, overexpression on glioma U251 cell proliferation and differentiation were analyzed. Tuj1 and GFAP expression was detected using immunofluorescence staining. Cell proliferation was detected with flow cytometry.Results: miR-124, and miR-137, expression was significantly decreased in GBM tumor tissues compared to normal brain tissues (p < 0.01). Immunofluorescence showed, after miR-124 and miR-137 transfection, normal synapse growth structure in GBM, and that neuronal differentiation factor expression significantly increased, including Tuj1 and GFAP. Flow cytometry analysis showed that GBM cell cycle extension and differentiation was repressed.Conclusion: miR-124, and miR-137, function as tumor suppressors to inhibit cell proliferation and differentiation and in GBM, their expression was significantly decreased.

Differentiation Induction Effect of Mir-429 Over-Expression in U251 Glioma Cell Line

The purpose of this research was to investigate the effect of mir-429 ectopic over-expression on U251 glioblastoma cell line. It has been shown that mir-429 could target BMK1 and reduce glioma cells invasion. We cultured U251-mir-429 cells and observed changes in their morphology for 14 days. They seemed to become wider with expanded cytoplasm and go on differentiation. Neuronal (NEFM, NSE and neurogenin) and oligodendrocyte (MBP, OLIG2 and NKX2-2) gene markers expression level was examined with real-time PCR. NSE, neurogenin, MBP, OLIG2 and NKX2-2 were significantly over-expressed, giving an evidence of cellular differentiation in glioma cells.

Abstract B16: Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy

Abstract Glioma is currently an incurable disease. Identifying the cell of origin for glioma could provide critical insights for developing effective therapies. We previously used a mouse genetic system termed Mosaic Analysis using Double Markers (MADM) to introduce p53 and NF1 mutations into neural stem cells to determine which cell type is responsible for gliomagenesis. The unequivocal GFP-labeling of sparse mutant cells generated by MADM enabled us to study the entire course of tumor development, from pre-transforming stages to full malignancy. Based on thorough histological, transcriptomic, and genetic analyses we identified oligodendrocyte precursor cells (OPCs) as the cell of origin in this glioma model. Here, we sought to understand the distinct role of p53 and NF1 in the transformation of OPCs since understanding these roles can give us a more thorough insight into how future therapies may be designed. The MADM system generates sparse GFP+ null and RFP+ sibling WT OPCs in an otherwise colorless, heterozygous mouse, allowing us to assess the impact of TSG loss on the capacity of cells to proliferate, survive, and differentiate. The loss of NF1 alone significantly increased mutant OPC proliferation with a proportional decrease in differentiation. However, massively over-expanded NF1-null OPCs did not transform into malignant gliomas, implicating the critical role of p53 in tumor suppression. Surprisingly, the loss of p53 alone had no effect on mutant OPC number, proliferation, or differentiation, suggesting that p53 function as a braking system that won't manifest its activity in the absence of driver mutation, such as NF1 loss. Next, we set out to test whether restoring either TSG function in tumor OPC cell lines would have any therapeutic efficacy. The restoration of wild type p53 led to a significant increase in both cell-cycle arrest and cell death. Since p53 function is commonly altered from mutations in patients, rather than complete loss of the protein, we tested whether restoring mutant p53 function with PRIMA-1 would have the same biological response as WT p53. We found that the restoration of mutant p53 function resulted in similar levels of cell cycle arrest and cell death as WT p53. This finding suggests that rescuing p53 function using small molecules may be of high value for future therapies. It also suggests that the loss of p53 activity is not a permanent fixture of tumor cells but that p53 can still exert it's tumor suppressor activity after transformation. Next we tested the therapeutic effects of the restoration of the GAP domain of NF1, which should reduce Ras activity in tumor OPCs. We found that the expression of WT but not the “GAP-dead” NF1-GAP led to a decrease in proliferation and an increase in differentiation of glioma cells. To test pharmacological compounds, we explore the possibility of targeting downstream effectors of Ras signaling, specifically mTOR inhibition because phospho-Akt level is elevated in tumor OPCs compared to WT OPCs. When we inhibited mTOR activity in MADM tumor mice at an age that is critical for mutant OPC expansion prior to transformation, we found that it blocked the ability of mutant OPCs to over-expand, suggesting that mTOR inhibition could potentially prevent gliomagenesis. In summary our data demonstrates that while NF1 loss increases OPC proliferation and decreases differentiation, p53 loss is critical for transformation and that restoring the function of either of these pathways has profound effects on tumor OPC biology that could be translated into future therapeutic strategies. Citation Format: Phillippe P. Gonzalez, Hui Zong. Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B16.

RACK1 affects glioma cell growth and differentiation through the CNTN2-mediated RTK/Ras/MAPK pathway

Receptor for activated C kinase 1 (RACK1) and contactin-2 (CNTN2) are known to be abnormally expressed in gliomas; however, the association between RACK1 and CNTN2, and the effects of RACK1 and CNTN2 on glioma cell differentiation and the related molecular mechanisms remain largely unknown. The present study aimed to investigate the interaction between RACK1 and CNTN2, and to examine whether RACK1/CNTN2/receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) axis plays a role in glioma growth and differentiation. The results from western blot analysis revealed that the protein expression levels of RACK1 and CNTN2 were higher in high‑grade glioma tissues and cells, and lower in low-grade glioma tissues and cells. A co-immunoprecipitation assay demonstrated that RACK1 interacts with CNTN2, and RACK1 upregulated the expression of CNTN2. Gain-of‑function and loss-of‑function experiments indicated that both RACK1 and CNTN2 promoted glioma cell proliferation, inhibited glioma cell differentiation and activated the RTK/Ras/MAPK pathway. However, the effects of RACK1 on glioma cell proliferation, differentiation and the activation of the RTK/Ras/MAPK signaling pathway were abolished by the knockdown of CNTN2 using siRNA. In Therefore, the findings of this study firstly demonstrate that RACK1 interacts with CNTN2, and that the effects of RACK1 on glioma cell growth and differentiation are mediated by CNTN2. The RACK1/CNTN2/RTK/Ras/MAPK axis exists in glioma cells, and it may be a potential therapeutic target in gliomas.

ATPS-04REPURPOSING PHENFORMIN FOR THE TREATMENT OF GLIOBLASTOMA: ROLE OF THE H19/LET-7/STAT3 AXIS

Glioblastoma (GBM) are characterized by an infiltrative nature and high resistance to radio- and chemotherapy. Despite advances in novel therapeutic approaches, the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need for the identification of alternative therapeutic approaches. The anti-diabetic biguanide drug metformin has been reported to exert anti-tumor effects. Phenformin, a metformin analog, is a more potent inhibitor of tumor growth, especially for Liver Kinase B1-deficient tumors. Our study demonstrates, for the first time, the effect of phenformin on the stemness and apoptosis of glioma cells and glioma stem cells (GSCs). We found that phenformin inhibited the self-renewal of GSCs, decreased the expression of the stemness markers, CD44 and Sox2 and the mesenchymal transformation of these cells. Phenformin at higher concentrations also induced apoptosis in both GSCs and differentiated glioma cells. The expression of AMPK and the phosphorylation of this kinase in response to phenformin were relatively low in GSCS, suggesting that this drug impacted the self-renewal and apoptosis of these cells mostly in an AMPK-independent manner. Phenformin inhibited the STAT3 pathway and its effects on GSCs were further enhanced by STAT3 inhibitors. In addition, phenformin decreased the expression of the long non-coding RNA, H19 and increased the expression of miR-124 and Let-7 and silencing of these miRNAs abrogated phenformin effects. Combined treatment of phenformin with radiation and temozolomide exerted a synergistic anti-tumor effects on GSCs which were more significant than those exerted by combined treatments with metformin. Using GSC-derived xenografts we demonstrated that phenformin inhibited tumor growth and angiogenesis, increased cell apoptosis and prolonged the overall survival of mice bearing GSC-derived xenografts. Our results demonstrate that phenformin can target GSCs via the STAT3/H19/Let7 axis and can be efficiently combined with current therapies for the treatment of GBM and the eradication of GSCs.

Induction of neural differentiation in rat C6 glioma cells with taxol.

Glioblastoma is a common and aggressive type of primary brain tumor. Several anticancer drugs affect GBM (glioblastoma multiforme) cells on cell growth and morphology. Taxol is one of the widely used antineoplastic drugs against many types of solid tumors, such as breast, ovarian, and prostate cancers. However, the effect of taxol on GBM cells remains unclear and requires further investigation. Survival rate of C6 glioma cells under different taxol concentrations was quantified. To clarify the differentiation patterns of rat C6 glioma cells under taxol challenge, survived glioma cells were characterized by immunocytochemical, molecular biological, and cell biological approaches. After taxol treatment, not only cell death but also morphological changes, including cell elongation, cellular processes thinning, irregular shapes, and fragmented nucleation or micronuclei, occurred in the survived C6 cells. Neural differentiation markers NFL (for neurons), β III-tubulin (for neurons), GFAP (for astrocytes), and CNPase (for oligodendrocytes) were detected in the taxol-treated C6 cells. Quantitative analysis suggested a significant increase in the percentage of neural differentiated cells. The results exhibited that taxol may trigger neural differentiation in C6 glioma cells. Increased expression of neural differentiation markers in C6 cells after taxol treatment suggest that some anticancer drugs could be applied to elimination of the malignant cancer cells as well as changing proliferation and differentiation status of tumor cells.

Antitumorigenic effect of interferon-β by inhibition of undifferentiated glioblastoma cells.

Glioma stem-like cells (GSCs) are undifferentiated cells that are considered to be an origin of glioblastomas. Furthermore, they may contribute to treatment resistance and recurrence in glioblastomas. GSCs differentiate into differentiated glioma cells (non-glioma stem-like cells: non-GSCs), and interconversion might occur between GSCs and non-GSCs. We investigated whether interferon-beta (IFN-β) could exert any efficacy towards GSCs or such interconversion processes. The neural stem cell marker CD133 and pluripotency marker Nanog in GSCs were analyzed to evaluate their differentiation levels. GSCs were considered to undergo differentiation into non-GSCs upon serum exposure, since the expression of CD133 and Nanog in the GSCs was negatively affected. Furthermore, the cells regained their undifferentiated features upon removal of the serum. However, we verified that IFN-β reduced cell proliferation and tumor sphere formation in GSCs, and induced suppression of the restoration of such undifferentiated features. In addition, we also confirmed that IFN-β suppressed the acquisition process of undifferentiated features in human malignant glioma cell lines. Our data thus suggest that IFN-β could be an effective agent not only through its cell growth inhibitory effect on GSCs but also as a means of targeting the interconversion between GSCs and non-GSCs, indicating the possibility of IFN-β being used to prevent treatment resistance and recurrence in glioblastomas, via the inhibition of undifferentiated features.

Nootropic potential of Ashwagandha leaves: Beyond traditional root extracts

Rapidly increasing aging population and environmental stressors are the two main global concerns of the modern society. These have brought in light rapidly increasing incidence of a variety of pathological conditions including brain tumors, neurodegenerative & neuropsychiatric disorders, and new challenges for their treatment. The overlapping symptoms, complex etiology and lack of full understanding of the brain structure and function to-date further complicate these tasks. On the other hand, several herbal reagents with a long history of their use have been asserted to possess neurodifferentiation, neuroregenerative and neuroprotective potentials, and hence been recommended as supplement to enhance and maintain brain health and function. Although they have been claimed to function by holistic approach resulting in maintaining body homeostasis and brain health, there are not enough laboratory studies in support to these and mechanism(s) of such beneficial activities remain largely undefined. One such herb is Ashwagandha, also called “Queen of Ayurveda” for its popular use in Indian traditional home medicine because of its extensive benefits including anticancer, anti-stress and remedial potential for aging and neurodegenerative pathologies. However, active principles and underlying mechanism(s) of action remain largely unknown. Here we provide a review on the effects of Ashwagandha extracts and active principles, and underlying molecular mechanism(s) for brain pathologies. We highlight our findings on the nootropic potential of Ashwagandha leaves. The effects of Ashwagandha leaf extracts are multidimensional ranging from differentiation of neuroblastoma and glioma cells, reversal of Alzheimer and Parkinson's pathologies, protection against environmental neurotoxins and enhancement of memory.

Mathematical modeling reveals a critical role for cyclin D1 dynamics in phenotype switching during glioma differentiation

Glioma differentiation therapy is a novel modality to increase anti-glioma effects using specific drugs to induce glioma cell differentiation to glia-like cells. However, the molecular mechanisms underlying glioma differentiation remain poorly understood. In this study, we built an experiment-integrated mathematical model for glioma differentiation signaling pathways. Our modeling and experimental analysis revealed that a “one-way-switch” bifurcation of cyclin D1 dynamics was critical for controlling the phenotypic transition of glioma cells. We also quantitatively evaluated drug combinations toward a synergistic therapeutic effect. These results provide insights into the molecular mechanisms underlying glioma differentiation and implications for the design of novel therapeutic targets in anti-cancer therapy.

Programmed death-ligand 1 (PD-L1) may play a role in malignant glioma infiltration

Infiltration is the main cause of death of malignant glioma patients. However, the mechanisms of the infiltration in the development of brain cancer (glioblastoma multiforme) are not completely understood. Here, we hypothesized that, in the development of human brain cancer, infiltration is primarily due to differentiated cancer cells and that the cell surface molecule programmed death-ligand 1 (PD-L1) may play an important role in brain tumor infiltration. PD-L1 is highly expressed in differentiated glioma cells in comparison to undifferentiated ones. As PD-L1 has both pro-survival and immunoinhibitory functions in tumor cells, the potential role of PD-L1 in tumor propagation suggest a new therapeutic target for preventing glioma infiltration and recurrence.

The role of microRNA-30c in the self-renewal and differentiation of C6 glioma cells

BackgroundSphere formation, one method for identifying self-renewal ability, has been used to report that cancer stem-like cells exist in rat C6 glioma cells. Recent studies suggested that cancer stem-like cells share the stem cell properties of self-renewal and multipotent ability of neural stem cells and might be regulated by microRNAs (miRNAs). However, the mechanism of miRNA involvement in the sphere formation and neural differentiation abilities of cancer stem-like cells is poorly understood. ResultsWe found that miRNA-30c could assist in sphere formation of C6 cells under defined conditions in neural stem cell medium DMEM/F12-bFGF-EGF-B27. Moreover, overexpression of miRNA-30c might reduce 3-isobutyl-1-methylxanthine (IBMX)-induced neural differentiation, as the expression of neural markers, especially glial fibrillary acidic protein (GFAP), decreased. Further experiments revealed that miRNA-30c inhibited the IBMX-induced astrocyte differentiation via targeting the upstream genes and inactivating phosphorylation of STAT3 of the JAK-STAT3 pathway. Subsequently, the expression of GFAP was reduced and the number of astrocyte differentiation from C6 cells decreased. ConclusionsOur findings suggest that miRNA-30c could play a regulatory role in self-renewal and neural differentiation in C6 glioma cells.

The expression of SALL4 in patients with gliomas: high level of SALL4 expression is correlated with poor outcome.

The spalt-like transcription factor 4 (SALL4) gene has been demonstrated to be overexpressed in many malignancies, but little is known about its expression in gliomas. To explore the expression of SALL4 in patients with gliomas and the relationship between SALL4 expression and clinicopathologic characteristics, qPCR and immunohistochemical staining were used to investigate the SALL4 expression level in 54 glioma specimens and seven normal brain tissues. In vitro, siRNAs against SALL4 in U251 cell line were constructed and cell proliferation was evaluated by CCK8 assay. The SALL4 expression level in glioma was significantly higher than that in normal brain tissues (P < 0.05). Both qPCR and immunohistochemical analysis found that the expression of SALL4 was tightly correlated with glioma pathology grade (P < 0.05). Analysis using glioma and normal brain tissues revealed that SALL4 was positively proportionated to glioma cell differentiation with high sensitivity (92.59 %) and specificity (85.71 %). Survival analysis indicated the SALL4 expression was an independent prognostic factor. High level of SALL4 expression was correlated with poor outcome in patients with gliomas. This result agreed with the negative correlation between SALL4 expression and overall survival period obtaining in GBM patients from the cancer genome atlas database. The CCK8 experiments demonstrated SALL4 could significantly inhibit cell proliferation in U251 cell line (P < 0.05). The findings of the current study indicated that the SALL4 may play an important role in progression, development and maintenance of glioma.

Effects of all‑trans retinoic acid on VEGF and HIF‑1α expression in glioma cells under normoxia and hypoxia and its anti‑angiogenic effect in an intracerebral glioma model.

All‑trans retinoic acid (ATRA) is one of the most potent inducers of differentiation and is capable of inducing differentiation and apoptosis in glioma cells. However, the effect of ATRA on glioma angiogenesis is yet to be elucidated. The present study investigated the effects of ATRA on the expression of vascular endothelial growth factor (VEGF) and hypoxia‑inducible factor‑1α (HIF‑1α) in various glioma cell lines under normoxia and hypoxia. The effect of ATRA on angiogenesis in a rat intracerebral glioma model was also investigated, with the aim of revealing the effect of ATRA on glioma angiogenesis. In the present study, U‑87MG and SHG44 glioma cells were treated with ATRA at various concentrations (0, 5, 10, 20 and 40µmol/l) under normoxia or hypoxia. Quantitative polymerase chain reaction and western blot analysis were used to investigate VEGF and HIF‑1α mRNA and protein expression, respectively. An intracerebral glioma model was generated using intracerebral implantation of C6glioma cells into rats. Tumor‑bearing rats were treated with ATRA at different doses (0, 5 and 10mg/kg/day) for two weeks, and immunohistochemical assays were performed to detect the cluster of differentiation34‑positive cells in order to evaluate the microvessel density (MVD) in each group. Following ATRA treatment, the expression of VEGF and HIF‑1α was found to vary among the different concentration groups. In the glioma cells in the lower concentration groups (5and 10µmol/l ATRA), a significant increase in VEGF and HIF‑1α expression was observed. Conversely, a significant decrease in VEGF and HIF‑1α expression was found in the glioma cells in the high ATRA concentration group (40µmol/l), compared with that in the cells in the control group. Furthermore, in the rat intracerebral glioma model, ATRA decreased glioma MVD, particularly in the high‑dose group (10mg/kg/day), compared with the control group. These results suggest that ATRA may exhibit a dose‑dependent effect on glioma angiogenesis and may inhibit glioma angiogenesis invivo.

Control of glioma cell death and differentiation by PKM2–Oct4 interaction

Glioma stem cells are highly resistant to cell death and as such are supposed to contribute to tumor recurrence by eluding anticancer treatments. Here, we show that spheroids that contain rat neural stem cells (NSCs) or rat glioma stem cells (cancer stem cells, CSCs) express isoforms 1 and 2 of pyruvate kinase (PKM1 and PKM2); however, the expression of PKM2 is considerably higher in glioma spheroids. Silencing of PKM2 enhances both apoptosis and differentiation of rat and human glioma spheroids. We establish that PKM2 was implicated in glioma spheroid differentiation through its interaction with Oct4, a major regulator of self-renewal and differentiation in stem cells. The small molecule Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, increases the amount of PKM2/Oct4 complexes and thus inhibited Oct4-dependent gene expression. Taken together, our results highlight a new molecular pathway through which PKM2 can manage gliomagenesis via the control of glioma stemness by Oct4.

Differential patterns of NOTCH1-4 receptor expression are markers of glioma cell differentiation

Background Notch signaling is deregulated in human gliomas and may play a role in their malignancy. However, the role of each Notch receptor in glioma cell differentiation and progression is not clear. We examined the expression pattern of Notch receptors and compared it with differentiation markers in glioma cell lines, primary human cultures, and biopsies of different grades. Furthermore, the effects of a γ-secretase inhibitor (GSI) on cell survival were assessed. Methods Notch receptors and markers of cellular differentiation were analyzed by reverse transcriptase PCR, Western blotting, immunohistochemistry, and immunocytochemistry. GSI sensitivity was assessed in both cell lines and primary cultures grown as monolayers or tumorspheres, by MTT assay. Results In cell lines, Notch1 and Notch2/4 levels paralleled those of glial fibrillary acidic protein (GFAP) and vimentin, respectively. In human gliomas and primary cultures, Notch1 was moderate/strong in low-grade tumors but weak in glioblastoma multiforme (GBM). Conversely, Notch4 increased from astrocytoma grade II to GBM. Primary GBM cultures grown in serum (monolayer) showed moderate/high levels of CD133, nestin, vimentin, and Notch4 and very low levels of GFAP and Notch1, which were reduced in tumorspheres. This effect was drastic for Notch4. GSI reduced cell survival with stronger effect in serum, whilst human primary cultures showed different sensitivity. Conclusion Data from cell lines and human gliomas suggest a correlation between expression of Notch receptors and cell differentiation. Namely, Notch1 and Notch4 are markers of differentiated and less differentiated glioma cells, respectively. We propose Notch receptors as markers of glioma grading and possible prognostic factors.

Identification and Characterization of Human MIBP1 Gene in Glioma Cell Differentiation

Malignant gliomas are the most common and lethal intracranial tumors; differentiation therapy is a promising candidate for their treatment. In order to reveal the mechanisms related to glioma differentiation, after confirming that differentiation was induced by sodium phenylbutyrate in SHG-44 human glioma cells, RNA arbitrary primer differential display was used to screen differentially expressed genes. One gene was found to be upregulated by differential display, and this was also confirmed by reverse northern blot and quantitative real-time PCR analysis. After it was cloned and sequenced, the 505-bp fragment was identified as the MIBP1 (c-myc intron-binding protein 1) gene, also named Hivep2/MBP-2/Schnurri-2. Quantitative real-time PCR analysis of 30 human tissue samples revealed that the expression of MIBP1 tended to decrease with increasing WHO grade and was significantly depressed in the high malignancy gliomas group (WHO grade IV). We cloned and sequenced the MIBP1 gene, which was accepted by GenBank as number DQ231041. Finally, transfection of MIBP1 in a reverse transcription vector into glioma cells inhibited cell growth, induced differentiation, and blocked the cell cycle. Here, we identify and describe the structure and function of a differentiation-related gene, human MIBP1, in human glioma.

Aqueous Ethanolic Extract of Tinospora cordifolia as a Potential Candidate for Differentiation Based Therapy of Glioblastomas

Glioblastomas are the most aggressive primary brain tumors and their heterogeneity and complexity often renders them non responsive to various conventional treatments. Search for herbal products having potential anti-cancer activity is an active area of research in the Indian traditional system of medicine i.e., Ayurveda. Tinospora cordifolia, also named as ‘heavenly elixir’ is used in various ayurvedic decoctions as panacea to treat several body ailments. The current study investigated the anti-brain cancer potential of 50% ethanolic extract of Tinospora cordifolia (TCE) using C6 glioma cells. TCE significantly reduced cell proliferation in dose-dependent manner and induced differentiation in C6 glioma cells, resulting in astrocyte-like morphology as indicated by phase contrast images, GFAP expression and process outgrowth data of TCE treated cells which exhibited higher number and longer processes than untreated cells. Reduced proliferation of cells was accompanied by enhanced expression of senescence marker, mortalin and its translocation from perinuclear to pancytoplasmic spaces. Further, TCE showed anti-migratory and anti-invasive potential as depicted by wound scratch assay and reduced expression of plasticity markers NCAM and PSA-NCAM along with MMP-2 and 9. On analysis of the cell cycle and apoptotic markers, TCE treatment was seen to arrest the C6 cells in G0/G1 and G2/M phase, suppressing expression of G1/S phase specific protein cyclin D1 and anti-apoptotic protein Bcl-xL, thus supporting its anti-proliferative and apoptosis inducing potential. Present study provides the first evidence for the presence of anti-proliferative, differentiation-inducing and anti-migratory/anti-metastatic potential of TCE in glioma cells and possible signaling pathways involved in its mode of action. Our primary data suggests that TCE and its active components may prove to be promising phytotherapeutic interventions in gliobalstoma multiformae.