Human Glioma Stem Cells Research Articles

Prosaposin is a biomarker of mesenchymal glioblastoma and regulates mesenchymal transition through the TGF-β1/Smad signaling pathway.

Mesenchymal glioblastoma (GBM) is the most aggressive subtype of GBM. Our previous study found that neurotrophic factor prosaposin (PSAP) is highly expressed and secreted in glioma and can promote the growth of glioma. The role of PSAP in mesenchymal GBM is still unclear. In this study, bioinformatic analysis, western blotting and RT-qPCR were used to detect the expression of PSAP in different GBM subtypes. Human glioma cell lines and patient-derived glioma stem cells were studied in vitro and in vivo, revealing that mesenchymal GBM expressed and secreted the highest level of PSAP among four subtypes of GBM, and PSAP could promote GBM invasion and epithelial-mesenchymal transition (EMT)-like processes in vivo and in vitro. Bioinformatic analysis and western blotting showed that PSAP mainly played a regulatory role in GBM invasion and EMT-like processes via the TGF-β1/Smad signaling pathway. In conclusion, the overexpression and secretion of PSAP may be an important factor causing the high invasiveness of mesenchymal GBM. PSAP is therefore a potential target for the treatment of mesenchymal GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Visualization of spatiotemporal dynamics of human glioma stem cell invasion

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

ATP1A1 Integrates AKT and ERK Signaling via Potential Interaction With Src to Promote Growth and Survival in Glioma Stem Cells.

Glioma stem cells (GSCs) have been considered to be responsible for treatment failure due to their self-renewal and limitless proliferative property. Recently, the Na+/K+-ATPase a1 (ATP1A1) subunit was described as a novel therapeutic target for gliomas. Interestingly, our previous proteomics study revealed that ATP1A1 is remarkably overexpressed in GSCs. In the current study, we investigated the role of ATP1A1 in regulating growth, survival, and tumorigenicity of primary human GSCs and the underlying molecular mechanism. We tested RNA and protein expression of ATP1A1 in glioma tissues and GSCs. In addition, we knocked down ATP1A1 in GSCs and assessed the effects thereof on growth, survival, and apoptosis. The role of ATP1A1 in signaling pathways was investigated in vitro. We found that the ATP1A1 expression level was associated with the grade of glioma. Knockdown of ATP1A1 in GSCs in vitro inhibited cell proliferation and survival, increased apoptosis, and halted cell-cycle progression at the G1 phase. Cell proliferation and survival were resumed upon rescue of ATP1A1 expression in ATP1A1-knockdown GSCs. The ERK1/2 and AKT pathways were inhibited through suppression of Src phosphorylation by ATP1A1 knockdown. Collectively, our findings suggest that ATP1A1 overexpression promotes GSC growth and proliferation by affecting Src phosphorylation to activate the ERK1/2 and AKT signaling pathways.

ShRNA-mediated PPARα knockdown in human glioma stem cells reduces in vitro proliferation and inhibits orthotopic xenograft tumour growth.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD‐SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα‐expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c‐Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα‐target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self‐renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

3-bromopyruvic acid inhibits the proliferation of malignant transformed dendritic cells

Objective To explore the possible inhibiting effect of 3-bromopyruvic acid (3-BrPA) on proliferation of malignant transformed dendritic cells by blocking the pathway of glucose metabolism. Methods In vitro, co-cultured mice myeloid dendritic cells (DC) with human glioma stem cells (SU3) were used to induce the malignant transformation of DC. Among DC monoclonal for malignant transformation, i. e., glioma stem/progenitor cells induced by malignant transformation of dendritic cells (GSPC-MTDCs), proliferative abilities of SU3, normal DC and GSPC-MTDCs were detected by CCK-8 and flat clone formation assay. Cell invasiveness was detected by Transwell chamber invasion assay. Subcutaneous inoculation of GSPC-MTDCs was performed in 20 non-fluorescent nude mice. At 11 d post inoculation, 10 nude mice with uniform tumorigenesis were divided into intervention group and control group with 5 mice in each group. The intervention group was given 3-BrPA (5 mg·kg-1·d-1) intraperitoneal treatment, while the control group was given the same volume of normal saline. After 24 days of intervention, the tumorigenesis was compared between 2 groups. Results (1) GSPC-MTDCs had immortalized characteristics. Compared with SU3, the proliferation rate of GSPC-MTDCs was faster and the clone formation rate was significantly higher (38.1±1.9% vs. 30.2±2.7%, P<0.01). Transwell chamber invasion test showed that GSPC-MTDCs had stronger invasive ability than SU3 (1 042±49 vs. 782±38, P<0.01). (2) GSPC-MTDCs inoculated into the subcutaneous and caudal veins of non-fluorescent nude mice all formed tumors. At 24 days after 3-BrPA intervention, the tumor masses in the intervention group and the control group were 1.5±0.6 g and 3.3±1.0 g, respectively. The difference was statistically significant (P=0.0107). Pathological observation showed that compared with the intervention group, the control group had more abnormal nuclei, more mitosis, abundant blood vessels, obvious infiltration of inflammatory cells and higher proportion of c-myc positive cells. Conclusions Glioma stem cells have the potential to induce malignant transformation of DC cells. 3-BrPA could inhibit the proliferation of GSPC-MTDCs. Key words: Glioma; Dendritic cells; Neoplasm transplantation; Cell proliferation; 3-bromopyruvic acid; Tumor suppressor; Mice

Relevant molecular characteristics analysis on malignant transformation of interstitial cells induced by tumor stem cells in glioma microenvironment

Objective: A variety of interstitial cells in tumor microenvironment (TME) based on glioma stem cells(GSC) have the function to promote malignant progression of tumors, but whether these interstitial cells have already undergone malignant transformation and their related molecular characteristics are still poorly understood. Methods: Human SU3-RFP glioma stem cells (GSC) stably transfected with red fluorescent protein (RFP) and interstitial cells from enhanced green fluorescent protein (EGFP) transgenic nude mice were co-cultured in vitro. SU3-RFP cells were also inoculated in different tissues of EGFP-Balb/c nude mice. Immortal EGFP(+) cells were monocloned either from co-culture cells in vitro, or from their xenografts in vivo. These immortal EGFP(+) cells were confirmed to bear characteristics of tumor cell via chromosomal analysis and tumorigenicity assay. Related molecular phenotypes of these cells were further detected through RT-PCR, flow cytometry and immunochemistry(IHC) techniques. Results: (1) Two EGFP(+) cell lines were obtained in vitro, and 5 EGFP(+) cell lines were obtained in vivo tumorigenic experiments. Seven EGFP(+) cell lines all have characteristics of self-renewal, heteroploid of chromosomes and 100% tumorigenicity. (2) Cell surface marker analysis showed cell origin of these cell lines were macrophages (tMΦ1 and tMΦ2 ), dendritic cells (tDC1 and tDC2), fibroblasts (tFB), oligodendrocytes (tOG) and BMSC cells (tBMSC), respectively. (3)All of these seven cell lines co-expressed Sca-1 and c-myc, and have Sox-2 or Nanog expression also, which suggest that they may bear molecular characteristics of mesenchymal stem cells or pluripotent stem cells. Conclusions: (1) Tumor stromal cells in TME have undergone malignant transformation, which is related to the tissue remodeling of TME by GSCs, and not limit to the specific type of their parasitic tissues. (2) Tumor cells originated from GSC and tumor interstitial cells, respectively, are two major types of tumor cells with different origins in glioma parenchyma, can not be simply regarded as tumor heterogeneity, transformed interstitial cells of TME may have the potential to serve as new targets for target diagnosis and therapy.

Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.

Fsk and IBMX inhibit proliferation and proapoptotic of glioma stem cells via activation of cAMP signaling pathway.

We aimed to find out the underlying mechanism of forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX) on glioma stem cells (GSCs). The expression of cAMP-related protein CREB and pCREB as well as apoptosis-related proteins were detected through Western blot analysis. The level of proliferation and growth rate of human GSCs was measured through thiazolyl blue tetrazolium bromide assay and stem cells forming sphere assay. The apoptosis-related gene expression was measured through reverse transcription-polymerase chain reaction. cAMP signaling pathway was activated in GSCs with Fsk-IBMX administration. Fsk-IBMX could inhibit the proliferation as well as invasion and promote the apoptosis of U87 cells. Besides, U0126 could inhibit MAPK signaling pathway to increase the sensitivity of GSCs to cAMP signaling pathway. As a result, Fsk-IBMX combined with U0126 had more negative effect on GSCs. The relationship of cAMP and MAPK signaling pathway in GSCs may provide a potential therapeutic strategy in glioma.

PO-252 Impaired glucose metabolism in human glioma stem cells upon treatment with a cell-penetrating peptide based on CONNEXIN43

IntroductionConnexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in glioma stem cells (GSCs). Restoring Cx43 in GSCs reverses their phenotype through the inhibition of c-Src and consequently reduces GSCs tumorigenicity. We have developed a cell-penetrating peptide (TAT-Cx43266–283) containing the region of Cx43 that interacts with c-Src that mimics the effect of Cx43 on GSC phenotype. GSCs reprogram their metabolism to compete for glucose resources through HIF-1-alpha, which can in turn be regulated by c-Src. Therefore, the aim of this work was to study the effect of TAT-Cx43266–283 on GSC metabolism.Material and methodsG166 (human GSCs), Wistar rat organotypic brain slices, neurons and astrocytes from primary culture.2-NBDG uptake: Cells were incubated with 146 µM 2-NBDG for 1 hour, lysed, and supernatant fluorescence intensity was measured by spectrofluorimetry and normalised to mg of protein.GSCs-brain organotypic slice co-cultureFluorescently-dyed human GSCs were injected into rat organotypic brain slices and 2-NBDG uptake was analysed by confocal microscopy.Cell energy analysis were performed on an extracellular flux analyser (Agilent Seahorse XF Technology ) using Mito Stress and Glycolysis Stress kits.Results and discussionsBecause TAT-Cx43266–283 inhibits Src activity in GSCs, we analysed the effect of TAT-Cx43266–283 on the rate of glucose uptake in human GSCs. Our results showed that TAT-Cx43266–283 reduced the uptake of a fluorescent glucose analogue (2-NBDG) into GSCs. Interestingly, TAT-Cx43266–283 did not significantly affect the uptake of glucose in neurons or astrocytes from primary culture, suggesting a specific effect on GSCs. Moreover, experiments using 6-NBDG, a fluorescent glucose analogue that cannot be phosphorylated by HK-2 (whose expression is regulated by HIF-1-a), showed that 6-NBDG uptake does not differ between treated and not treated GSCs. Moreover, we analysed 2-NBDG uptake on a GSCs-brain organotypic slice co-culture. Our results revealed that TAT-Cx43266–283 reduced glucose uptake in tumoral cells when they are within the brain parenchyma. More importantly, data obtained with a cell energy analysis platform (Agilent Seahorse XF Technology) showed impaired metabolism in GSCs treated with TAT-Cx43266–283, but not in neurons or astrocytes.ConclusionIn vitro and ex-vivo experiments revealed that TAT-Cx43266–283 reduces the rate of glucose uptake selectively in human glioma stem cells with the subsequent decrease in metabolic activity and survival.

Abstract 142: cdk5 inhibition promotes neurogenesis while reducing self-renewal of brain tumor stem cells

Abstract Cancer stem cells are biologically potent and support tumor growth, in part by deregulating asymmetric stem cell division. Asymmetric cell division of stem cells is a type of mitotic division where a stem cell generates one stem and one differentiated cell. Cancer stem cells, however, tend to generate more stem-like dividing cells to promote neoplasm. To uncover mechanisms and potential therapeutic targets relevant to self-renewal in human glioma stem cells (GSCs), we performed a genetic screen for kinases that reverse the tumor phenotype of our novel adult Drosophila brain tumor model and found that knockdown of the brain-specific dCdk5 was highly effective in suppressing the self-renewing stem cell components. A highly specific CDK5 inhibitor is promoting neurogenesis among these fly brain cancer stem cells. These cancer stem cells, although they express glial markers in tumors, do not differentiate into glia upon CDK5 suppression. Additionally, while the tumor generation in adult flies completely disrupts the mushroom body, the main cognitive center in adult fly brain, the CDK5 inhibition partially rescues the mushroom body and extends life span of these flies. dCdk5 and its human ortholog CDK5 (79% identity) are aberrantly activated in some forms of cancer. We demonstrate that GBMs highly express CDK5 mRNA and protein, likely related to copy number gains of chromosome 7, with strong positive correlation with stem cell markers and marked effect on tumor-propagating properties. Using human GSCs isolated from tumor spheres and mouse xenografts, we found that CDK5 directly phosphorylates CREB1 independent of cAMP and stops stem cell renewal. These data together show that CDK5 inhibition not only can attenuate self-renewal properties of brain tumor stem cells, but also can promote neurogenesis, which could potentially become a better treatment strategy for patients and improve their cognitive function as well. Citation Format: Subhas Mukherjee, Cheryl Olson, Daniel Brat. cdk5 inhibition promotes neurogenesis while reducing self-renewal of brain tumor stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 142.

Abstract 169: Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells

Abstract Glioblastoma (GBM) is the most frequent and aggressive primary brain tumour in adults. Despite advances in surgical and medical neuro oncology, prognosis for GBM patients remains dismal, with a median survival of about 15 months. It has been demonstrated that the modest benefit of conventional therapies depends on a small population of cancer stem cells within the tumor, named Glioma Stem Cells (GSCs) that cause tumor relapse and chemoresistance and therefore could play a key role in GBM recurrence. Thus, the identification of new specific ligands for GSCs could be a fundamental challenge for the development of effective glioma therapies. Here, we developed an in vitro evolution based approach, named differential whole cellSELEX; it is used to generate nucleic acid ligands, named aptamers, with high affinity and specificity for GSCs. Aptamers, were obtained through the iterative evolution of a random pool of sequences using human primary GSCs as target. Among different potential candidates we focused on one sequence, named 40L. The 40L aptamer and its truncated form, 40S, were selective for human GSCs distinguishing them from tumor differentiated cells, obtained from the stem cells induced to differentiate. 40L revealed to be functionally active on target cells and able to inhibit stemness, cell growth and migration. 40s preserves binding ability of 40L sequence and it has further proven to strongly reduce tumor proliferation in in vivo experiment. Moreover, both 40L and 40s were able to rapidly internalize upon target binding and therefore may serve as selective vehicle for therapeutics.In conclusion, our results indicate that 40L and its short form 40s can selectively target GSCs both in vitro and in vivo. Given the crucial role of these cells in GBM recurrence and therapy resistance, 40L and 40s represent innovative drug candidates with a great potential in the GBM treatment. Citation Format: Alessandra Affinito, Cristina Quintavalle, Maurizio Albero, Claudia Vilardo, Francesco Palma, Giuseppina Roscigno, Lucia Ricci Vitiani, Roberto Pallini, Carla Lucia Esposito, Vittorio de Franciscis, Gerolama Condorelli. Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 169.

PO-291 A cell-penetrating peptide based on the connexin43-Src interacting sequence reduces glioma stem cell migration and proliferation by recruiting Src, Csk and PTEN

IntroductionThe expression of connexin43 (Cx43), the main gap junction channel-forming protein, is down-regulated in glioma stem cells (GSC). Our previous studies showed that a cell-penetrating peptide containing the region of Cx43 that interacts with the oncoprotein c-Src (TAT-Cx43266–283), inhibits its oncogenic activity and up-regulates the tumour suppressor PTEN. Through this pathway, TAT-Cx43266–283 reduces glioma cell proliferation and reverses the GSC phenotype. Our next goal was to uncover the mechanism of action and the effects of TAT-Cx43266–283 on the migration and invasion of human GSCs.Material and methodsHuman primary GSCs (G9, G12, G13, G15 and G16) were obtained from glioblastoma patients’ biopsies from the Neurosurgery Service of the Hospital Universitario de Salamanca, Spain. The migration was analysed in these primary GSCs by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging and the invasion with Matrigel-treated transwell inserts. The same tumour biopsies were used to study the effect of TAT-Cx43266–283 on fresh undissociated tumour blocks by time-lapse microscopy. The molecular interactions were studied in G166 GSCs by pull-down assays of biotinylated TAT-Cx43266–283 (TAT-Cx43266–283-B) and Western blot analysis.Results and discussionsOur results confirmed that TAT-Cx43266–283 strongly reduces the migration and invasion of human primary GSCs by inhibiting c-Src activity and up-regulating PTEN that consequently, lowers the levels of phosphorylation of focal adhesion kinase tyrosines 397, 576 and 577. In addition, fresh undissociated tumour blocks revealed a dramatic reduction on the survival of these glioblastoma cells when exposed to TAT-Cx43266–283. By pull-down assays, we showed that TAT-Cx43266–283-B recruits c-Src together with its inhibitors PTEN and Csk, confirming the proposed mechanism for Cx43.ConclusionIn conclusion, TAT-Cx43266–283 exerts an important antitumor effect by inhibiting c-Src and up-regulating PTEN with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration, proliferation and survival. This inhibition is mediated by the recruitment of the c-Src intrinsic inhibitors, PTEN and Csk. All together, these results reinforce the relevance of this sequence of Cx43 that interacts with c-Src for the development of new therapies against glioblastoma.

Isoliquiritigenin can inhibit migration and invasion of human glioma stem cells by down-regulating matrix metalloproteinases

To investigate the effects of isoliquiritigenin on the migration and invasion of human glioma stem cells and the underlying mechanism. The stem cell markers CD133 and Nestin in SHG44 human glioma stem cells were examined with immunofluorescence microscopy. The migration and invasion ability of glioma stem cells was determined by transwell method. The mRNA and protein expression of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by real-time RT-PCR and Western blot, respectively. CD133 and Nestin were positive in SHG44 cells. The number of migrated cells in SHG44 cells treated with 20 and 80 μmol/L isoliquiritigenin for 48 h were significantly lower than that in control group (76±5 and 42±4 vs. 85±6, all P<0.01), and the number of migrated cells in 80 μmol/L isoliquiritigenin group was lower than that in 20 μmol/L isoliquiritigenin group (P<0.01). The numbers of cells crossing through membrane in 20 and 80 μmol/L isoliquiritigenin groups were 190±13 and 130±9, respectively, which were significantly lower than that in control group (230±14, all P<0.01), and the number of crossed cells in the 80 μmol/L isoliquiritigenin group was lower than that in 20 μmol/L isoliquiritigenin group (P<0.01). The mRNA and protein expression levels of MMP-2 and MMP-9 were decreased compared with control group (P<0.05 or P<0.01), and the expression levels in 80 μmol/L isoliquiritigenin group were lower than those in 20 μmol/L isoliquiritigenin group (P<0.05 or P<0.01). Isoliquiritigenin exhibits antitumor effects on glioma stem cells by inhibiting cell migration and invasion, which may be related to the down-regulation of MMP-2 and MMP-9.

Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft Models for Glioma Stem Cell Targeted Drug Screening.

Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin, and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold, and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, Temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo, and the same can be used as an effective platform for screening drugs against glioma stem cells.

Novel nor-monoterpenoid indole alkaloids inhibiting glioma stem cells from fruits of Alstonia scholaris

BackgroundGlioblastoma multiforme (GBM) is a highly aggressive and frequently recurrent malignant brain tumor, and to date, the clinically effective drugs against GBM remain scarce. Natural products play an important role in drug discovery, and might be the resource of antitumor agents for GSCs. Alstonia scholaris (L.) R. Br. is rich in monoterpenoid indole alkaloids (MIAs) and used extensively for treatment of tumor in the traditional medicine system of Asia. PurposeTo search for new MIAs with antitumor activity against glioma stem cells from clinical patients and explore their mechanism. MethodsCompounds were obtained from the fruits of A. scholaris by chromatographic separation, including silica gel, Sephadex LH-20 and recrystallization. Their structures were elucidated by the use of UV, IR, NMR and MS spectra. The antitumor activity of the compounds against the glioma stem cells (GSC-3#, GSC-12#, GSC-18#) were investigated by phenotypic screening and MTS assays. Cell proliferation assay by BrdU immunofluorescence staining, and apoptosis assay by cleaved-caspase-3 immunofluorescence staining and real-time PCR assay. The soft-agar clonal formation assay was performed to determine the antitumor efficacy of the compounds in vitro. ResultsTwo new nor-monoterpenoid indole alkaloids were isolated from the fruits of A. scholaris. They exhibited selective antitumor activity against glioma stem cells (GSC-3#, GSC-12#, GSC-18#) with IC50 values of 15–25 µg/ml. Furthermore, they inhibited GSCs proliferation, induced GSCs apoptosis by increasing the expression of TNF-α and cleavage of caspase-3, and significantly damaged colony forming capacity of GSCs. ConclusionNew nor-monoterpenoid indole alkaloids from the fruits of A. scholaris provide new type promising molecule for the selective killing of human glioma stem cells.

Antibodies targeting human endothelin-1 receptors reveal different conformational states in cancer cells.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Effects of valproic acid on the susceptibility of human glioma stem cells for TMZ and ACNU.

To investigate the effect of valproic acid (VPA) on the susceptibility of glioma stem cells to temozolomide (TMZ) and nimustine (ACNU), the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and its expression of MGMT were examined. A total of 3 glioma cell populations were isolated from human glioma tissues, and immunocytochemistry was used to detect the expression of MGMT. VPA inhibition on the growth of the 3 glioma cell populations exposed to various concentrations of TMZ and ACNU was evaluated. Flow cytometry was applied to detect the apoptosis of glioma cells, and a methylation-specific polymerase chain reaction was used to identify methylation of MGMT promoter. Immunocytochemistry results indicated that MGMT was negatively expressed in the G1 population, but positively expressed in the G2 and G3 populations. Cell growth inhibition assays demonstrated that the survival rate in the VPA + TMZ or ACNU groups was decreased compared with that of the TMZ or ACNU alone groups (P<0.05). As for the apoptotic rate, those in the VPA alone group were increased compared with the control group (P<0.05), and the rates in the VPA + TMZ or ACNU groups were increased compared with TMZ or ACNU alone groups (P<0.05). The expression of MGMT remained negative in the G1 population following treatment with VPA, but MGMT expression became negative in the 2 MGMT-positive cell populations (G2 and G3) following VPA treatment. The MGMT promoter in the G1 population was partially methylated in the control group, but was fully methylated following VPA treatment, while the promoters of G2, G3 were unmethylated in the control group and became partially methylated in the VPA treatment group. Taken together, TMZ and ACNU may suppress the growth of glioma stem cells in vitro in a dose-dependent manner. VPA may enhance the inhibitory effects of various concentrations of TMZ and ACNU on the growth of MGMT-negative/positive cells, particularly on MGMT-positive cell populations. VPA itself may induce the apoptosis of glioma cells, and VPA combined with TMZ or ACNU may enhance TMZ/ACNU-induced apoptosis of glioma stem cells. Furthermore, VPA may also promote the methylation of the MGMT promoter to silence MGMT expression in glioma cells, which may be an important mechanism through which VPA enhances the efficacy of TMZ and ACNU in targeting glioma stem cells.

Unprecedented sugar bridged bisindoles selective inhibiting glioma stem cells

Unlike reported bisindoles linked by single bond directly, alstoniasidines A (1) and B (2), from Alstonia scholaris featuring unprecedented skeleton with two indole moieties bridged by a sugar, represented a novel bisindole type having strictosamide-glucopyranose-picraline scaffold. Both compounds exhibited selective cytotoxicity against human glioma stem cells (GSCs) and induced caspase-3 dependent extrinsic apoptosis by increasing the expression of interleukin 1 (IL-1), tumor necrosis factor (TNF-α), and the cleaved caspase-3, while damaged the unlimited proliferation and self-renewal capacity of GSCs. This finding might provide new type of leads for the selective killing of human glioma stem cells.

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.