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Glioblastoma (GBM), the most malignant of the brain tumors is classified on the basis of molecular signature genes using TCGA data into four subtypes- classical, mesenchymal, proneural and neural. The mesenchymal phenotype is associated with greater aggressiveness and low survival in contrast to GBMs enriched with proneural genes. The proinflammatory cytokines secreted in the microenvironment of gliomas play a key role in tumor progression. The study focused on the role of Oncostatin-M (OSM), an IL-6 family cytokine in inducing mesenchymal properties in GBM. Analysis of TCGA and REMBRANDT data revealed that expression of OSMR but not IL-6R or LIFR is upregulated in GBM and has negative correlation with survival. Amongst the GBM subtypes, OSMR level was in the order of mesenchymal > classical > neural > proneural. TCGA data and RT-PCR analysis in primary cultures of low and high grade gliomas showed a positive correlation between OSMR and mesenchymal signature genes-YKL40/CHI3L1, fibronectin and vimentin and a negative correlation with proneural signature genes-DLL3, Olig2 and BCAN. OSM enhanced transcript and protein level of fibronectin and YKL-40 and reduced the expression of Olig2 and DLL3 in GBM cells. OSM-regulated mesenchymal phenotype was associated with enhanced MMP-9 activity, increased cell migration and invasion. Importantly, OSM induced mesenchymal markers and reduced proneural genes even in primary cultures of grade-III glioma cells. We conclude that OSM-mediated signaling contributes to aggressive nature associated with mesenchymal features via STAT3 signaling in glioma cells. The data suggest that OSMR can be explored as potential target for therapeutic intervention.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)‐4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par‐4 induces apoptosis in cancer cells, its potential in drug‐resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi‐drug resistance and serve as ideal experimental models. We investigated the role of Par‐4 in Tamoxifen (TAM)‐induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par‐4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par‐4 expression, high level of chemo‐resistance genes and were resistant to TAM‐induced cytotoxicity. In monolayer cells, TAM‐induced cytotoxicity was associated with enhanced expression of Par‐4 and was alleviated by silencing of Par‐4 using specific siRNA. TAM effectively induced secretory Par‐4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM‐induced cell death by exposure to conditioned medium (CM)‐containing Par‐4 (derived from TAM‐treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par‐4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par‐4 induces apoptosis by binding to membrane GRP78, secretory Par‐4 is an attractive candidate for potentially overcoming therapy‐resistance not only in malignant glioma but in broad spectrum of cancers.

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 molecular mechanisms involved in tumor progression from a low-grade astrocytoma to the most malignant glioblastoma multiforme (GBM) have been hampered due to lack of suitable experimental models. We have established a model of tumor progression comprising of two cell lines derived from the same astrocytoma tumor with a set of features corresponding to low-grade glioma (as in HNGC-1) and high-grade GBM (as in HNGC-2). The HNGC-1 cell line is slowgrowing, contact-inhibited, nontumorigenic, and noninvasive, whereas HNGC-2 is a rapidly proliferating, anchorage-independent, highly tumorigenic, and invasive cell line. The proliferation of cell lines is independent of the addition of exogenous growth factors. Interestingly, the HNGC-2 cell line displays a near-haploid karyotype except for a disomy of chromosome 2. The two cell lines express the neuronal precursor and progenitor markers vimentin, nestin, MAP-2, and NFP160, as well as glial differentiation protein S100μ. The HNGC-1 cell line also expresses markers of mature neurons like Tuj1 and GFAP, an astrocytic differentiation marker, hence contributing toward a more morphologically differentiated phenotype with a propensity for neural differentiation in vitro. Additionally, overexpression of epidermal growth factor receptor and c-erbB2, and loss of fibronectin were observed only in the HNGC-2 cell line, implicating the significance of these pathways in tumor progression. This in vitro model system assumes importance in unraveling the cellular and molecular mechanisms in differentiation, transformation, and gliomagenesis.

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins–Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.

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.

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

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.

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

Abstract: In this study, we demonstrated that laminin-1 (LM-1) induces neurite outgrowth and enhances the expression of neurofilament-L and MAP2 in human bone marrow mesenchymal stem cells (MSCs). The c-Jun transcription factor was strongly activated by LM-1 during neurite induction. Suppression of c-Jun inhibited the expression of the c-Jun target genes α6 integrin and neurofilament-L, resulting in the loss of neurite outgrowth. Additionally, we found that the LM-1-α6 integrin interaction stimulated phosphorylation of FAK, leading to the activation of JNK and Akt. Pharmacological inhibition of these pathways blocked c-Jun activation and neurite outgrowth. Collectively, our findings suggest that c-Jun/AP-1 activity mediated by JNK, PI3K/Akt and ERK pathways is required for LM-1-induced neurite outgrowth in human bone marrow MSCs. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]