Anjali Shiras

Spontaneous Transformation of Human Adult Nontumorigenic Stem Cells to Cancer Stem Cells Is Driven by Genomic Instability in a Human Model of Glioblastoma

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.

MiR-145 functions as a tumor-suppressive RNA by targeting Sox9 and adducin 3 in human glioma cells

BACKGROUND MicroRNAs (miRNAs) are increasingly being recognized as being involved in cancer development and progression in gliomas. METHODS Using a model cell system developed in our lab to study glioma progression comprising human neuroglial culture (HNGC)-1 and HNGC-2 cells, we report here that miR-145 is one of the miRNAs significantly downregulated during malignant transformation in glioblastoma multiforme (GBM). In a study using tumor samples derived from various glioma grades, we show that expression of miR-145 is decreased in a graded manner, with GBM patients showing lowest expression relative to lower-grade gliomas (P < .05) and normal brain tissues (P < .0001). Functional studies involving ectopic expression of miR-145 in glioma cells had a negative impact on cell proliferation and tumor development, as well as invasion and induced apoptosis, providing further support to the concept that inactivation of miR-145 is important for glioma disease pathogenesis. More notably, these growth-suppressive effects of miR-145 are mediated through its target proteins Sox9 and the cell adhesion-associated molecule adducin 3 (ADD3). RESULTS Inhibiting Sox9 and ADD3 rescued effects of miR-145 loss. Interestingly, miR-145 loss in glioma cells led to overexpression of molecules involved in cell proliferation, like cyclin D1, c-myc, and N-myc, as well as enhanced expression of cell adhesion- and invasion-related molecules N-cadherin and E-cadherin, an effect which was again restored upon miR-145 overexpression in glioma cells. The miR-145 promoter was methylated at its cytosine-phosphate-guanine (CpG) islands in the glioma cell lines studied. CONCLUSION Our study demonstrates that miR-145 has a tumor-suppressive function in glioblastoma in that it reduces proliferation, adhesion, and invasion of glioblastoma cells, apparently by suppressing the activity of oncogenic proteins Sox9 and ADD3. Reduced levels of miR-145 may lead to neoplastic transformation and malignant progression in glioma due to unregulated activity of these proteins.

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK–MEK/ERK signaling pathways

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.

Tumor suppressive miRNA-34a suppresses cell proliferation and tumor growth of glioma stem cells by targeting Akt and Wnt signaling pathways

MiRNA‐34a is considered as a potential prognostic marker for glioma, as studies suggest that its expression negatively correlates with patient survival in grade III and IV glial tumors. Here, we show that expression of miR‐34a was decreased in a graded manner in glioma and glioma stem cell‐lines as compared to normal brain tissues. Ectopic expression of miR‐34a in glioma stem cell‐lines HNGC‐2 and NSG‐K16 decreased the proliferative and migratory potential of these cells, induced cell cycle arrest and caused apoptosis. Notably, the miR‐34a glioma cells formed significantly smaller xenografts in immuno‐deficient mice as compared with control glioma stem cell‐lines. Here, using a bioinformatics approach and various biological assays, we identify Rictor, as a novel target for miR‐34a in glioma stem cells. Rictor, a defining component of mTORC2 complex, is involved in cell survival signaling. mTORC2 lays downstream of Akt, and thus is a direct activator of Akt. Our earlier studies have elaborated on role of Rictor in glioma invasion (Das et al., 2011). Here, we demonstrate that miR34a over‐expression in glioma stem cells profoundly decreased levels of p‐AKT (Ser473), increased GSK‐3β levels and targeted for degradation β‐catenin, an important mediator of Wnt signaling pathway. This led to diminished levels of the Wnt effectors cyclin D1 and c‐myc. Collectively, we show that the tumor suppressive function of miR‐34a in glioblastoma is mediated via Rictor, which through its effects on AKT/mTOR pathway and Wnt signaling causes pronounced effects on glioma malignancy.

Wnt3a mediated activation of Wnt/β-catenin signaling promotes tumor progression in glioblastoma.

Presence of a distinct population of cells that drives tumor progression supports the hierarchical model of tumor development in Glioblastoma (GBM) and substantiates the cancer stem cell hypothesis. Amongst the various developmental signaling pathways that are aberrantly activated, we here show that activated Wnt/β-catenin signaling pathway plays a critical role in malignant transformation and tumor progression in gliomas. We demonstrate that Wnt ligands - Wnt1 and Wnt3a are expressed in a graded manner in these tumors as well as over-expressed in glioma stem cell-lines. A selective inhibition of Wnt signaling pathway by selective knock-down of its ligands Wnt1 and Wnt3a in glioma-derived stem-like cells led to decreased cell proliferation, cell migration and chemo-resistance. Furthermore, Wnt silencing in glioma cells reduced the capacity to form intra-cranial tumors in vivo. Taken together, our study indicates Wnt/β-catenin signaling pathway as an essential driver of glioma tumorigenesis, recognizing role of Wnt3a as an oncogene and thereby offering novel therapeutic strategies for management of these tumors.

Porphyran capped gold nanoparticles as a novel carrier for delivery of anticancer drug: In vitro cytotoxicity study

In the present study, we have explored porphyran as a reducing agent for one pot size controlled green synthesis of gold nanoparticles (AuNps) and further investigated its application as a carrier for the delivery of an anticancer drug. The prepared AuNps showed surface plasmon resonance centered at 520 nm with average particle size of 13±5 nm. FTIR spectra suggested that the sulfate moiety is mainly responsible for reduction of chloroauric acid. The capping of the AuNps with porphyran was evident from the negative zeta potential value responsible for the electrostatic stability. Thus, porphyran acts as reducing as well as capping agent. These AuNps are highly stable in a wide range of pH and electrolyte concentration. Porphyran capped AuNps exhibited enhanced cytotoxicity on human glioma cell line (LN-229) as compared to native porphyran. Consequently, these AuNps have been utilized as a carrier for delivery of the anticancer drug doxorubicin hydrochloride (DOX). Spectroscopic examination revealed that DOX conjugated onto AuNps via hydrogen bonding. The release of DOX from DOX loaded AuNps was found to be sixfold higher in acetate buffer (pH 4.5) as compared to physiological buffer (pH 7.4). Further, the DOX loaded AuNps demonstrated higher cytotoxicity on LN-229 cell line as compared with an equal dose of native DOX solution. This established the potential of these AuNps as a carrier for anticancer drug delivery.

Biocompatible gellan gum-reduced gold nanoparticles: cellular uptake and subacute oral toxicity studies.

Currently gold nanoparticles are being explored for drug delivery and other biomedical applications; therefore it is necessary to study the fate of such nanoparticles inside the body. The objective of the present study was to investigate the cellular uptake and toxicity of the gold nanoparticles synthesized using a microbial polysaccharide, gellan gum, as a capping and reducing agent. The cellular uptake of gold nanoparticles was studied on mouse embryonic fibroblast cells, NIH3T3 and human glioma cell line, LN‐229. The cellular uptake study indicated that the gellan gum‐reduced gold nanoparticles were located in cancer cells (LN‐229) while no uptake was observed in normal mouse embryonic fibroblast cells (NIH3T3). The toxicity of the gold nanoparticles was evaluated by carrying out subacute 28 day oral toxicity studies in rats. Subacute administration of gum‐reduced gold nanoparticles to the rats did not show any hematological or biochemical abnormalities. The weight and normal architecture of various organs did not change compared with control. The current findings, while establishing the specific uptake of nanoparticles into cancerous cells, also demonstrates that the gellan gum‐reduced gold nanoparticles are devoid of toxicity in animals following oral administration. Copyright

Glioblastoma cell secretome: Analysis of three glioblastoma cell lines reveal 148 non-redundant proteins

Glioblastoma multiforme (GBM) is the most aggressive among human gliomas with poor prognosis. Study of tumor cell secretome - proteins secreted by cancer cell lines, is a powerful approach to discover potential diagnostic or prognostic biomarkers. Here we report, for the first time, proteins secreted by three GBM cell lines, HNGC2, LN229 and U87MG. Analysis of the conditioned media of these cell lines by LC-MS/MS using ESI-IT mass spectrometer (LTQ) resulted in the confident identification of 102, 119 and 64 proteins, respectively. Integration of the results from all the three cell lines lead to a dataset of 148 non-redundant proteins. Subcellular classification using Genome Ontology indicated that 42% of the proteins identified belonged to extracellular or membrane proteins, viz. Vinculin, Tenascin XB, SERPIN F1 and TIMP-1. 52 proteins matched with the secretomes of 11 major cancer types reported earlier whereas remaining 96 are unique to our study. 25 protein identifications from the dataset represent proteins related to GBM or other cancer tissues as per Human Protein Atlas; at least 22 are detectable in plasma, 11 of them being reported even in cerebrospinal fluid. Our study thus provides a valuable resource of GBM cell secretome with potential for further investigation as GBM biomarkers.

Differentiation-inducing ability of sophorolipids of oleic and linoleic acids using a glioma cell line.

Sophorolipids are biosurfactants produced by non‐pathogenic yeasts. They show structural similarity with the membrane components of mammalian cells, i.e., glycosphingolipids and gangliosides, which are involved in processes such as signaling, oncogenesis, and differentiation. Sophorolipids have been reported to induce differentiation in several leukemic cell lines, cell death via apoptosis in a human liver cancer cell line, and necrosis in a pancreatic adenocarcinoma cell line. Here we report, for the first time, the effects of precursor fatty acids and sophorolipids of oleic and linoleic acids in pure acidic and crude forms on LN‐229, a glioma cell line. In response to different sophorolipid forms, various morphological changes were observed, such as formation of long thread‐like extensions arising from the ends of the cells, cell alignment, cell elongation and bundle formation in a dose‐dependent manner. In this study we present the morphological evidence of the potential of sophorolipids as differentiation inducers.

Dlxin-1, a MAGE family protein, induces accelerated neurite outgrowth and cell survival by enhanced and early activation of MEK and Akt signalling pathways in PC12 cells.

Dlxin-1 (also known as NRAGE or MAGED1) is a member of Type II melanoma-associated antigen (MAGE) family of proteins characterized by presence of a unique region of about 200 amino acids known as the MAGE homology domain (MHD). Dlxin-1 is associated with a large number of diverse cellular functions ranging from transcriptional regulation, cell cycle progression and differentiation to developmental apoptosis. While there are numerous studies reporting the role of NRAGE in facilitating cell death by interaction with p75NTR, we found varied effects of Dlxin-1 over-expression on PC12 cells grown in presence of NGF. These include induction of increased cell survival in presence of NGF and accelerated neuronal differentiation. We here categorically demonstrate that the effects on neuritogenesis are promoted through interactions of Dlxin-1 with the neurotrophin receptor TrkA. Further, using pharmacological inhibitors to specific pathways, we delineate the effects on enhanced neuritogenesis to the early and sustained activation of MEK pathway whereas the effects on cell survival to the early activation of Akt pathway. Next, we demonstrate a physical interaction of necdin with Dlxin-1 in PC12 cells. Our results establish that Dlxin-1 is an enhancer of neuronal differentiation and suggests that its possible interaction with NGF and necdin is critical in mediating pathways involved in neuronal survival and differentiation. Further in-depth analyses of the activation of various signalling pathways mediated through interaction with Dlxin-1 may provide valuable insight on the mechanisms that govern decisions regarding neuronal survival, growth, differentiation or apoptosis.