Search

Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma samples of GBM patients were significantly lower than normal healthy volunteers. Thus, our study demonstrates for the first time that PYR can be repurposed against GBM with a novel mechanism of action involving Par-4. Herewith, we discuss the role of upregulated Par-4 in a highly interconnected signaling network thereby advocating its importance as a therapeutic target.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 μg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 μg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen–Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

Background High Risk Human Papilloma Viruses (HR-HPV) recurrently infect women having Human Immunodeficiency Virus − 1 (HIV-1)infection. Transforming HPV E6 and E7 genes promote invasive cancers and interact with Notch-1receptor. Concomitantly, HIV-1 Tat binds to EGF motifs within the Notch-1extracellular domain. HR-HPV infection activates Notch-1 signalling. Permissive HIV-1entry into the cervix is allowed. Notch-1 inhibitors may offer solace to the aggressive cancer phenotype in HIV-1 positive women. Still, the molecular cross talk between different oncogenes within the Notch-1pathway during HIV-1/HPV-16 + co-infections has not been elucidated. Methods Adherent cervical tumor derived cell lines-CaSki cell line (with inherent HPV16+ sequence and endogenous Notch-1 activity) and C33A cell line (cervical cancer phenotype, HPV -ve for HPV DNA and RNA) were used. Plasmids (pLEGFPN1 encoding HIV-1 Tat and pNL 4 − 3 encoding HIV-1(full HIV-1 genome), were transfected at 600 ng/mL into the above mentioned cell lines, in order to examine independent effects of HIV-1 Tat and HIV-1 transfection in HPV-16+ cervical cancer cells. We performed western blotting, cell cycle analysis and RT-qPCR post transfection. Three sets of independent experiments were analyzed by Graph Pad Prism 5. Statistical significance was calculated using Student t -test. Data expressed as mean ± standard deviation (SD). p values ≤ 0.05* were considered statistically significant. Results HIV-1 Tat and HIV-1 inhibited Notch-1expression, with differential effects on EGFR when comparing C33A and CaSki cell lines. CDK2 induction in Tat transfected CaSki cells, showed concomitant G0/G1 phase accumulation favouring cancer progression. Notch-1 inhibition shut off significant Cyclin D expression with a significant p21 induction and increased G2-M cell population in CaSki cells. On the contrary, HIV-1 infection utilized Hes-1-EGFR-CyclinD-p21axis, G2-M arrest, DDR response and cancer progression. Conclusion Our study highlights for the first time that HIV-1 Tat and/or HIV-1 driven cancers in HPV-16+ CaSki cells, show Notch-1 suppression and CDK2 dependent activity. HIV-1 Tat activates Hes-1 amplifying the EGFR gene which improves the aggressive state possibly through irreversible oxidative-stress induced senescence. HIV-1, favours cancer progression through its CXCR4 receptor, responsible for unbridled mitosis, G2-M arrest and damaged DNA response (DDR). Treatment of CaSki cells with a Notch-1 inhibitor, DAPT showed marginal recovery in p21expression with Go/G1 and S phase recovery.

Introduction: Radiation exposure to brain is known to cause cognitive deficits. Aim of the current study was to investigate whether lithium protects against radiation. It was further investigated whether this protective effect is associated with neuritogenesis and altered Neural Cell Adhesion Molecule (NCAM) expression. Materials and Methods: Human neuroblastoma cell line, SK-N-MC was exposed to gamma radiation alone and after 24 h pretreatment with lithium. Cells were scored for viability by MTT assay and checked for apoptotic changes by flow cytometry (FCM). Geimsa stained cells were scored for number of cells bearing neuritis and length of neuritis. Real time PCR was used to measure levels of NCAM transcript in the cells. Results: Radiation exposure of 5 Gray resulted in significant decrease in cell viability. Pretreatment with lithium rescued cells from radiation- induced toxicity as indicated by MTT assay and by FCM analysis. Morphological observations suggested radiation caused increase in neurite lengths and decrease in number of cells bearing neurites. This was reversed by lithium treatment. Several fold decrease in NCAM expression was observed post radiation exposure which was reversed by lithium pretreated cells. Conclusion: Lithium protects neuronal cells against radiation damage. The protective effect of lithium is associated with change in differentiation status of cells as indicated by neuritogenesis and with alteration in expression of NCAM, a molecule known to be associated with neurite formation.

Plasmodium falciparum malaria is the most severe form of malaria with several complications. The malaria pigment‐hemozoin (Hz) is associated with severe anemia, cytokine dysfunction, and immunosuppression, thus making it an interesting target for developing new strategies for antimalarial therapy. Monocytes (MO) in circulation actively ingest Hz released by Plasmodium parasites and secrete pro‐ and anti‐inflammatory cytokines. M1 and M2 types represent the two major forms of MO/macrophages (MQ) with distinct phenotypes and opposing functions. Imbalance in the polarization of these types is reported in many infectious diseases. Though the association of Hz with immunosuppression is well documented, its role in activation of MO in context of M1/M2 phenotypes remains to be addressed. We report here that natural Hz drives human MO toward M2‐like phenotype as evidenced by the expression of M2 signature markers. Hz‐fed MO showed elevated transcript and secreted level of IL‐10, CCL17, CCL1, expression of mannose‐binding lectin receptor (CD206), and arginase activity. Hz attenuated HLA‐DR expression, nitric oxide, and reactive oxygen species production, which are the features of M1 phenotype. Our data also implicate the involvement of p38 MAPK, PI3K/AKT, and NF‐κB signaling pathways in skewing of Hz‐fed MO toward M2‐like type and suppression of mitogen‐stimulated lymphocyte proliferation. Importantly, antimalarial drugs—chloroquine and artemisinin—partially reversed activation of Hz‐induced MO toward M2‐like phenotype. Considering the limitations in the current therapeutic options for malaria, we propose that these drugs may be re‐examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria.

The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors—rapamycin, temsirolimus, torin 1, and PP242—suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as “stemness-inhibiting drugs” and a promising therapeutic approach to target glioma stem cells.

The mechanisms underlying the differentiation of Mesenchymal stem cells (MSCs) toward neuronal cell type are not clearly understood. Earlier, we reported that laminin-1 induces neurite outgrowth in human MSCs via c-Jun/AP-1 activation through ERK, JNK, and Akt pathways. In this study, we demonstrate that laminin-1 increases the expression of proneural gene, neuroD1 and induces the expression of immediate-early biomarkers of neuronal cell-programming-Egr1, Egr3, PC3, and PC4. Gene expression profiling of MSCs cultured on laminin-1 and Poly-l-lysine for 12 h revealed differential regulation of 267 genes (>1.5 fold, p < 0.05), predominantly in the category of nervous system development and affected the pathways involved in TGF-β/TNF-α signaling, regulation of MAPK and JNK cascade. Data for 11 selected genes related to nervous system development was validated by real time PCR. Transcriptional regulatory network analysis revealed c-Jun as the key transcription factor regulating majority of differentially expressed genes and identified Disrupted in schizophrenia 1, as a novel target of c-Jun. Modeling and analysis of biological network showed selective induction of Growth Arrest and DNA damage 45 (GADD45B) and repression of NF-κB inhibitor A (NFκBIA). Collectively, our findings provide the basis for understanding the molecular mechanisms associated with laminin-1-induced neurogenic expression in MSCs.

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti-cancer agent. The anti-cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti-tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti-cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK-N-MC, SH-SY5Y, and SK-N-SH, we show that non-toxic dose (2 mM) of VPA enhanced staurosporine (STS)-induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti-apoptotic protein crucial in resistance to STS-mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS-induced apoptosis. Moreover, MS-275, a structurally distinct class I HDAC inhibitor did not affect STS-mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS-induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer.

Glioblastoma (GBM) is the most aggressive type of brain tumors in adults with survival period

Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes—mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways—Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells. © 2010 Wiley-Liss, Inc.

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins-fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3h, and by 24h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against alpha6 and beta1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin alpha6beta1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin alpha6beta1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.

Glucose regulated protein 78 (GRP78) has recently been suggested to be associated with drug resistance in breast cancer patients. However, the precise role of GRP78 in drug resistance and the involved signaling pathways are not clearly understood. In the present study, we show that among a panel of drugs, namely Paclitaxel (TAX), Doxorubicin (DOX), 5-fluorouracil (5-FU), UCN-01 and Tamoxifen (TAM) used, TAM alone up-regulated the expression of GRP78 significantly and induced apoptosis in MCF-7 and MDA-MB-231 cells. Interestingly, inhibition of GRP78 by a specific pharmacological inhibitor, VER-155008 augmented TAM-induced apoptosis, and overexpression of GRP78 rendered the cells resistant to TAM-induced cell death suggesting a role for GRP78 in TAM-induced cytotoxicity. Mechanistically, the expression of phosphorylated AKT as determined by Western blot analyses revealed that TAM selectively upregulated phosphorylation of AKT at Thr308 but not at Ser473, and siRNA silencing of GRP78 resulted in inhibition of AKT phosphorylation at Thr308 but not at Ser473. Further, a GRP78 inhibitor, VER155008 inhibited TAM-induced phosphorylation of GSK3β, a downstream substrate of AKT. These results, thus suggests a role for GRP78 in TAM-induced AKT activation. Additionally, co-localization studies by immunofluorescence, and immunoprecipitation experiments demonstrated a complex formation of AKT and GRP78. Furthermore, in glucose-free medium, the cells were sensitized to TAM-induced cell death that was associated with reduced AKT phosphorylation at Thr308, thus strengthening the association of AKT regulation with drug response. Collectively, our findings identify a role of GRP78 in AKT regulation in response to TAM in breast cancer cells.

Tumor associated macrophages (TAMs) constitute a substantial mass in gliomas. The activated macrophages secrete various cytokines that affect diverse functions of tumors. The aim of this study was to elucidate the role of Akt and NF-kappaB pathways in resistance to TNF-alpha mediated cell death in human gliomas using monolayers and multicellular spheroids (MCS) as in vitro models. Akt and NF-kappaB are constitutively expressed and intimately involved in progression of gliomas. The activation of these pathways also renders the tumors resistant to conventional treatments including chemotherapy. While PI3K/Akt is shown to regulate the NF-kappaB activation in diverse systems, other studies place NF-kappaB upstream of Akt activation. Using a stable IkappaBalpha mutant LN-18 cell line and pharmacological inhibitors to PI3K/Akt (LY294002) and Akt (Akt2), we provide evidence that Akt and NF-kappaB are activated independently on stimulation with TNF-alpha and both the pathways contribute towards resistance to TNF-alpha mediated cell death. TNF-alpha-induced NF-kappaB activation independent of PI3K/Akt pathway was also confirmed in human glioma cell lines-LN-229 and U373MG. We also show that NF-kappaB and Akt are activated during spheroidogenesis and their expression is further enhanced on stimulation with TNF-alpha implicating their involvement in resistance to cell death. The findings thus underscore the relevance of spheroids as appropriate in vitro models for studying the signaling pathways in drug induced resistance.

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycledependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-NMC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells.

Multi-cellular spheroids (MCS) generated from tumor cells serve as excellent in vitro models for understanding the mechanisms of tumor progression and micro-metastasis. We have compared the expression of molecular markers with reference to their growth as conventional adherent monolayers (2-D) and anchorage independent cultures (3-D) using two mouse melanoma cell lines, B16F10 and Clone M3. The two cell lines differed in their ability to form spheroids with respect to their aggregation potential, with B16F10 forming large clusters compared to Clone M3. A panel of molecular markers comprising cell adhesion molecules, cyclin dependent kinase inhibitors and members of the cadherin–catenin complex were analyzed by flow cytometry in 2-D and 3-D cultures. There was a distinct difference in the patterns of expression of CD44(S) and variant isoforms v3,v10 in spheroids compared to cells grown as monolayers in both cell lines. Also, there was an increase in cells positive for CDK inhibitor p27 in 3-D cultures from the B16F10 cell line. The expression of alpha and gamma catenin was down regulated in spheroids. As these molecules are implicated in the regulation of cell proliferation, alterations in the expression of these molecules in 3-D cultures compared to their 2-D counterparts suggests the importance of spheroids as experimental model for tumorigenesis.

The influence of extracellular matrix (ECM) on monocyte-macrophage (mo-mφ) differentiation was investigated using an in vitro model with human peripheral blood mononuclear cells (PBMC) maintained on different matrix protein substrata. Macrophage specific markers associated with differentiation studied were, (a) endocytosis of modified proteins; (b) appearance of mφ specific matrix metalloproteinases (MMPs); (c) activities of myeloperoxidase (MPO) and β-D-glucuronidase; (d) changes in the expression of cell surface antigens. As the duration of monocytes in culture increased, a progressive increase in the rate of differentiation was seen as evidenced by mφ specific functions such as endocytosis of 125[I] acetyl BSA and the appearance of gelatinases A and B. Significantly higher rate of endocytosis and production of MMPs were found in monocytes maintained on fibronectin (FN) and COL I than on COL IV (FN > COL I > COL IV) indicating that cells in contact with stromal components differentiate at a faster rate. FACS analysis done on cells maintained in vitro for phenotypic profile characteristic to mo-mφ differentiation showed downregulation of CD14 occurring in a substratum dependent manner viz, (FN > COL IV > COL I) and upregulation of CD71 was high in cells maintained on COL I and COL IV. Intracellular enzymatic activities such as MPO significantly decreased irrespective of matrix substrata, while β-D-glucuronidase activity increased in a substratum dependent manner (FN > COL I > COL IV). Pretreatment of cells with genistein significantly decreased the secretion of MMPs, particularly MMP 9 in cells maintained on ECM protein (FN) indicating a phosphorylation dependent signalling process in mediating matrix effect. The results of these in vitro studies on mφ specific markers suggest that apart from the diffusible factors, the microenvironment as provided by various matrix proteins particularly FN can modulate mo-mφ differentiation.

We have previously reported that a fungal lectin, Rhizoctonia bataticola lectin (RBL), stimulates proliferation and secretion of Th1/Th2 cytokines in human peripheral blood mononuclear cells (PBMC). In the present study, we evaluated the ability of RBL to differentiate human monocytes to macrophages. RBL induced morphological changes indicative of differentiation in primary monocytes and THP-1 cells. Stimulation with RBL resulted in significant up-regulation of differentiation markers - CD54, HLA-DR, CD11b and CD11c and secretion of proinflammatory cytokines - IL-1β, TNF-α and IL-6. Functionally, RBL profoundly increased phagocytic activity in monocytes. In THP-1 cells, RBL-induced phagocytosis was higher compared to the effect induced by combination of phorbol-12-myristate-13-acetate (PMA) and lipopolysaccharide (LPS). RBL induced a significant increase in matrix metalloproteinase-9 (MMP-9) activity in comparison with a combined treatment of PMA+LPS. Mechanistic studies revealed the involvement of the NF-κB pathway in RBL-induced differentiation of monocytes. The data suggest that RBL mimics the combined action of PMA and LPS to induce morphological and functional differentiation in human monocytes and monocytic cell line - THP-1 to macrophages. Human monocytes differentiated to macrophages with RBL have the potential as an in vitro model to study macrophage biology.

Neuroblastoma (NB) cell lines are transformed, neural crest derived cells, capable of unlimited proliferation in vitro. These cell lines retain the ability of differentiation into neuronal cell types on treatment with various agents. This ability of NB cells to proliferate as well as to differentiate makes it an excellent in vitro system for various studies. This review article focuses on the applications and potential uses of murine and human NB cell lines. NB cells are extensively used for testing neurotoxicity of putative drugs such as antimalarial or anticancer agents. NB cell lines have wide applications in virus research to understand various aspects of virus-host cell interactions at the molecular and cellular levels. They have been used to dissect the relationships between proliferation, differentiation and apoptosis. This feature has been useful in understanding the pediatric cancer--neuroblastoma and for development of newer therapies.

We studied the effect of staurosporine (SSP), a broad-spectrum protein kinase inhibitor, on the levels of protein kinase C (PKC) activity and proliferation in two murine neuroblastoma cell lines, Neuro2a and its clone NB41A3. The PKC activity was examined in whole cell lysate, cytosolic and particulate fractions. A differential response to SSP treatment in the enzyme activity in whole cell lysate and particulate fractions was demonstrated in the two cell lines. The data on proliferation indicated that Neuro2a cells were more sensitive to the SSP treatment with significant inhibition in DNA synthesis in a time course study. Our findings suggest that the data on basal levels of PKC activity in tumors will be of significance in studies using PKC inhibitors as an approach for therapeutic intervention.

Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line - HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers - Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC.

The mechanisms underlying the differentiation of Mesenchymal stem cells (MSCs) toward neuronal cell type are not clearly understood. Earlier, we reported that laminin-1 induces neurite outgrowth in human MSCs via c-Jun/AP-1 activation through ERK, JNK, and Akt pathways. In this study, we demonstrate that laminin-1 increases the expression of proneural gene, neuroD1 and induces the expression of immediate-early biomarkers of neuronal cell-programming-Egr1, Egr3, PC3, and PC4. Gene expression profiling of MSCs cultured on laminin-1 and Poly-l-lysine for 12 h revealed differential regulation of 267 genes (1.5 fold, p0.05), predominantly in the category of nervous system development and affected the pathways involved in TGF-β/TNF-α signaling, regulation of MAPK and JNK cascade. Data for 11 selected genes related to nervous system development was validated by real time PCR. Transcriptional regulatory network analysis revealed c-Jun as the key transcription factor regulating majority of differentially expressed genes and identified Disrupted in schizophrenia 1, as a novel target of c-Jun. Modeling and analysis of biological network showed selective induction of Growth Arrest and DNA damage 45 (GADD45B) and repression of NF-κB inhibitor A (NFκBIA). Collectively, our findings provide the basis for understanding the molecular mechanisms associated with laminin-1-induced neurogenic expression in MSCs.

We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins- Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia.

Background: A lectin from the plant pathogenic fungus Rhizoctonia bataticola (RBL) has shown unique sugarbinding specificity towards the complex N-glycans, which are known to be expressed in many cancers, including ovarian cancer. Previous studies have demonstrated that RBL exerts cytotoxic effects on human ovarian cancer PA-1 cells. In the present study, we determined the signaling mechanism of RBL-induced cytotoxicity in PA-1 cells upon binding to cell surface receptors and also assessed the anti-tumor activity of RBL in NOD-SCID mice bearing ovarian xenografts. Methods: Cytotoxicity was determined by MTT assay, Cell cycle analysis, Annexin V staining, and expression of caspase-3 was determined by flow cytometry using specific fluorescent detection reagents. Western blotting was carried out to observe the intermediates of apoptotic pathway. Results: Treatment of PA-1 cells with RBL decreased their viability in a dose- and time- dependent manner. Cell cycle analysis revealed an increase in the hypo diploid population in response to lectin treatment. RBL-treated cells stained with Annexin V and PI showed an increase in both the early and late apoptotic populations. The mechanism of RBL-induced apoptosis in PA-1 cells was assessed by the observed decrease in MMP (ΔΨm), release of cytochrome-c, activation of caspase-9 and -3, and cleavage of PARP. Specific pharmacological inhibitors revealed the involvement of caspase-9 and -3 but not caspase-8. RBL at a subtoxic concentration (50 μg/mouse) suppressed tumor growth in NOD-SCID mice bearing PA-1 xenografts. Conclusions: The present study demonstrates that, RBL induced cytotoxicity in PA-1 cells is mediated by intrinsic apoptotic pathway. The present study strengthens the merit of RBL for its potential application in ovarian cancer research.

Objectives Sclerotium rolfsii lectin (SRL), isolated from soil born phytopathogenic fungus Sclerotium rolfsii, exhibits exquisite binding specificity to the oncofoetal Thomsen-Friedenreich (Galβ1,3GalNAcα-O-Ser/Thr, T or TF) antigen and associated glycans. In the present study, we report anti-proliferative activity of SRL and investigate underlying mechanisms of SRL-induced apoptosis, in the human ovarian cancer cell line PA-1. Materials and methods SRL-induced anti-proliferative effects were determined using MTT assay and induction of apoptosis was determined by flow cytometry, confocal microscopy and western blot analysis. Results SRL inhibited population growth of PA-1 cells in a dose- and time-dependent manner with maximum inhibition (71.3 ± 1.9%) occurring at concentration of 50 μg/ml after 72 h incubation. Observed effects of SRL could be blocked by competing glycoproteins, asialomucin, mucin and fetuin. Treatment with SRL resulted in increase in hypodiploid cell population as determine by cell cycle analysis. Increase in numbers of annexin V-PI positive cells, and cleavage of PARP confirmed apoptosis-inducing activity of SRL. Involvement of caspases in SRL-mediated apoptosis was determined by cleavage of caspases-3, -8 and -9 in a time-dependent manner, thereby suggesting possible involvement of both intrinsic and extrinsic caspase-dependent pathways. Conclusion The present study demonstrates anti-proliferative and apoptosis-inducing activity of SRL that can be exploited for potential application in ovarian cancer research.

Ethanol mediated free radical generation plays an important role in the pathogenesis of liver injuries and alcoholic liver diseases. In the present study two ellagitannins namely geraniin and amariin isolated from Phyllanthus amarus were examined for their ability to protect mouse liver slices against ethanol induced toxicity and possible mechanism of its protection. Oxidative stress markers such as, lipid peroxidation, protein carbonyl formation, amount of 8-hydroxy-2-deoxyguanosine and antioxidant enzymes levels were measured using specific biochemical assays. Poly (ADP-ribose) polymerase (PARP), Bax and Bcl2 were checked to assess the induction of apoptosis using western blots. The results showed that geraniin and amariin protected mouse liver slices against ethanol induced cytotoxicity. Both compounds inhibited oxidation of lipid, protein and formation of 8-hydroxy-2-deoxyguanosine, all of which were found to be elevated on exposure to ethanol. These compounds restored the antioxidant enzymes altered on ethanol exposure. Compounds also inhibited the cleavage of PARP and bax and restored Bcl2, induced on exposure to ethanol. In summary, both ellagitannins effectively protected mouse liver slices against ethanol induced cytotoxicity and apoptosis by reducing oxidative damage to biological molecules and modulating Bax/Bcl-2 ratio respectively, thus minimizing liver injury.

We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.

In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

We present here the Energetic pharmacophore model representing complementary features of the 1,2,3,4-tetrahydropyrimidine for selective cyclooxygenase-2 (COX-2) inhibition. For the development of pharmacophore hypothesis, a total of 43 previously reported compounds were docked on active site of COX-2 enzyme. The generated pharmacophore features were ranked using energetic terms of Glide XP docking for 1,2,3,4-tetrahydropyrimidine scaffold to optimize its structure requirement for COX-2 inhibition. The thirty new 4,5,6-triphenyl-1,2,3,4-tetrahydropyrimidine derivatives were synthesized and assessed for selective COX-2 inhibitory activity. Two compounds 4B1 and 4B11 were found to be potent and selective COX-2 inhibitors. The molecular docking studies revealed that the newly synthesized compounds can be docked into COX-2 binding site and also provide the molecular basis for their activity.

A new synthesis protocol is described to obtain a CdTe decorated magnetite bifunctional nanosystem via dodecylamine (DDA) as cross linker. High resolution transmission electron microscopy (HRTEM), energy-dispersive x-ray spectroscopy (EDAX), vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FTIR), diffuse reflectance spectroscopy (DRS) and fluorescence microscopy are used to characterize the constitution, size, composition and physical properties of these superparamagnetic–fluorescent nanoparticles. These CdTe decorated magnetite nanoparticles were then functionalized with anti-epidermal growth factor receptor (EGFR) antibody to specifically target cells expressing this receptor. The EGFR is a transmembrane glycoprotein and is expressed on tumor cells from different tissue origins including human leukemic cell line Molt-4 cells. The magnetite–CdTe composite nanosystem is shown to perform excellently for specific selection, magnetic separation and fluorescent detection of EGFR positive Molt-4 cells from a mixed population. Flow cytometry and confocal laser scanning microscopy results show that this composite nanosystem has great potential in antibody functionalized magnetic separation and imaging of cells using cell surface receptor antibody.

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes-mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways-Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells.

Background Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. Methods Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. Results The binding and mitogenic activities of RBL were inhibited by glycoproteins– mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines– IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1 h and 3 h respectively. Significant phosphorylation of STAT-5 (tyr 694 ) was observed at 12 h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. Conclusions RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. General significance RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.

Tumor associated macrophages (TAMs) constitute a substantial mass in gliomas. The activated macrophages secrete various cytokines that affect diverse functions of tumors. The aim of this study was to elucidate the role of Akt and NF-kappaB pathways in resistance to TNF-alpha mediated cell death in human gliomas using monolayers and multicellular spheroids (MCS) as in vitro models. Akt and NF-kappaB are constitutively expressed and intimately involved in progression of gliomas. The activation of these pathways also renders the tumors resistant to conventional treatments including chemotherapy. While PI3K/Akt is shown to regulate the NF-kappaB activation in diverse systems, other studies place NF-kappaB upstream of Akt activation. Using a stable IkappaBalpha mutant LN-18 cell line and pharmacological inhibitors to PI3K/Akt (LY294002) and Akt (Akt2), we provide evidence that Akt and NF-kappaB are activated independently on stimulation with TNF-alpha and both the pathways contribute towards resistance to TNF-alpha mediated cell death. TNF-alpha-induced NF-kappaB activation independent of PI3K/Akt pathway was also confirmed in human glioma cell lines-LN-229 and U373MG. We also show that NF-kappaB and Akt are activated during spheroidogenesis and their expression is further enhanced on stimulation with TNF-alpha implicating their involvement in resistance to cell death. The findings thus underscore the relevance of spheroids as appropriate in vitro models for studying the signaling pathways in drug induced resistance.

BackgroundThe role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21cip/waf1and p27kip1in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) asin vitromodels.ResultsTNF-α induced inhibition of proliferation and enhanced the expression of p21cip/waf1and p27kip1in LN-18 cells. p21 was induced on exposure to TNF-α, localized exclusively in the nucleus and functioned as an inhibitor of cell cycle but not as an antiapoptotic protein. In contrast, p27 was constitutively expressed, localized predominantly in the cytoplasm and was not involved in arrest of proliferation. Our data using IκBα mutant LN-18 cells and PI3K/Akt inhibitor-LY294002 revealed that the expression of p21 is regulated by NF-κB. Loss of IκBα function in LN-229 cells (p53 positive) did not influence TNF-α induced accumulation of pp53 (Ser-20 p53) suggesting that p53 was not down stream of NF-κB. Spheroidogenesis enhanced p27 expression and p21 induced by TNF-α was significantly increased in the MCS compared to monolayers.ConclusionThis study demarcates the functional roles for CDKIs-p21cip/waf1and p27kip1during TNF-α stimulated responses in LN-18 glioma cells. Our findings provide evidence that TNF-α-induced p21 might be regulated by NF-κB or p53 independently. p21 functions as an inhibitor of cell proliferation and does not have a direct role in rendering the cells resistant to TNF-α mediated cytotoxicity.

The presence of a CD133+/nestin+ population in brain tumors suggests that a normal neural stem cell may be the cell of origin for gliomas. We have identified human CD133-positive NSCs from adult glioma tissue and established them as long-term in vitro cultures human neuroglial culture (HNGC)-1. Replicative senescence in HNGC-1 led to a high level of genomic instability and emergence of a spontaneously immortalized clone that developed into cell line HNGC-2 with features of cancer stem cells (CSCs), which include the ability for self-renewal and the capacity to form CD133-positive neurospheres and develop intracranial tumors. The data from our study specify an important role of genomic instability in initiation of transformed state as well as its progression into highly tumorigenic CSCs. The activated forms of Notch and Hes isoforms were expressed in both non-neoplastic neural stem cells and brain tumor stem cells derived from it. Importantly, a significant overexpression of these molecules was found in the brain tumor stem cells. These findings suggest that this model comprised of HNGC-1 and HNGC-2 cells would be a useful system for studying pathways involved in self-renewal of stem cells and their transformation to cancer stem cells. Disclosure of potential conflicts of interest is found at the end of this article.