MB Cell Research Articles

Effects of Daidzein and Vincristine on Medulloblastoma Cell Proliferation

Medulloblastoma – the most common type of childhood brain cancer – is a malignant tumor of the cerebellum that can spread throughout the central nervous system. Currently, the primary treatment for MB patients is chemotherapy, but its use is limited due to its negative side effects, which include cognitive and neurological dysfunctions, growth impairment of the brain, and immune system compromise. Previous studies with the isoflavone genistein determined that a diet high in soy could potentially reduce the amount of chemotherapy drugs necessary to treat MB, which would consequently reduce the risk of harmful side effects while maintaining or improving disease control. Unlike genistein, daidzein – another key soy isoflavone with high bioavailability – has not been studied. Therefore, we sought to detail the effects of vincristine coupled with daidzein on MB cell lines. For this, HTB‐186 cells were treated with daidzein and varied concentrations of vincristine. Cells in all treatment groups were incubated for 1 week. After the study period, the viable cells in each group were enumerated via a dye exclusion assay.Grant Funding Source: Saluki Scholars Research Opportunity

Abstract 288: PDGFR alpha and PDGFR beta differentially regulate cell proliferation and migration/invasion in medulloblastoma cells

Abstract Medulloblastoma (MB) is the most common brain tumor in children and often spread to the spinal cord though the cerebrospinal fluid. Understanding the biological mechanism of this disease would provide a better care for these patients. Recently, overexpression of platelet derived growth factor receptor (PDGFR) alpha and beta has been linked to a metastatic stage of the disease. To comprehensively analyze the functions of PDGFRs and find their potential downstream targets in MB, we utilized Daoy cells, a metastatic MB cell line, as a MB model to assess the roles of PDGFR alpha and beta in this disease. The expression levels of PDGFR alpha and beta were determined by real time PCR and immunobloting. It was shown that Daoy cells express both PDGFR alpha and PDGFR beta. By using siRNAs to knockdown PDGFR alpha or beta specifically, and neutralizing antibody specific to PDGFR alpha and PDGFR beta respectively, it was demonstrated that PDGFR alpha signaling hinders cell proliferation and cell migration/invasion while PDGFR beta signaling promotes cell proliferation and migration/invasion. To elucidate the mechanism and find their potential downstream targets, total RNAs from the PDGFR knockdown cells were further analyzed by real time PCR and our results showed that PDGFR alpha and PDGFR beta signaling differentially modulate a set of genes which are important for cell death/surviving and proliferation such as NF kappa B, ATF3, Txnip and cMYC. To elucidate which pathway might govern the different roles of PDGFR alpha and beta in tumor cell proliferation/surviving, an NF kappa B report plasmid was co-transfected with siRNAs of PDGFRs into the MB cells and then the NF kappa B activities in the co-transfected cells were determined using a luciferase assay system. It was demonstrated that the NF kappa B activity was largely reduced in PDGFR beta knockdown cells, which suggested that PDGFR alpha and PDGFR beta may balance the cell proliferation/surviving signaling via cMYC and NF kappa B pathway. Furthermore, by analyzing the expression of cell surface protein on the PDGFR knockdown cells using immunohistochemistry staining, we demonstrated that CD44, a hall marker for cell migration/invasion was down regulated by PDGFR beta knockdown but not by PDGFR alpha knockdown, which implicated PDGFRs balance their migration/invasion signaling via CD44. Taken together, these data suggest that PDGFR alpha and PDGFR beta play different roles in cell proliferation and migration/invasion in MB brain tumor cells and these studies also highlight the potential of targeting PDGFRs signaling therapy in MB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 288.

Abstract 763: A systematic screening identify new drugs impairing medulloblastoma tumorigenesis

Abstract Medulloblastoma (MB) is the most common Central nervous system (CNS) neoplasia accounting for 25% of all childhood brain tumors. The Wnt, Sonic and Notch developmental cascades all regulate CNS cell dynamics, and are mutationally activated in a subset of MBs. So far the main limitation of the current treatment of MB is the lack of drug specificity, we aim to identify new specific impairment pathway drugs to offer new therapeutic tools in order to improve the clinical management of this neoplasia. The Wnt signaling pathway ends in the regulation of the levels of β-catenin which accumulates in the nucleus and interacts with the LEF/TCF family of transcription factors; in the absence of Wnt stimulation, β-catenin is phosphorylated by several kinases, as a complex comprising GSK3-β, Axin and APC, and it is targeted for degradation in the cytoplasm via the ubiquitin-proteasome pathway. Wnt activation includes key regulators of proliferation, among which c-Myc and cyclin D1 play a crucial role. We screened a drug library including almost 1200 compounds which are not well characterized on their activity against cancer cells. The library contains a repository of Natural Products, that derive from marine and plant sources collected worldwide, and a Synthetic library Products. We tested the efficacy of the Natural Products to interfere with Wnt pathway, using the TOP/FOP luciferase assay. From this large screening, we focused our attention on three natural compounds (Cantharidin, Parthenolide, Homoharringotnine) which positively inhibit Wnt pathway in three independent MB cell lines: Daoy (a desmoplastic medulloblastoma), ONS-76 and UW288(classic medulloblastoma cell lines). We determined their ability to turn-off Wnt signaling by western blotting detecting β-catenin activated protein and by RT-PCR for controlling of mRNA levels of WNT target genes. Cantharidin (CTD) has an anticancer activity in leukemic cells and melanoma cells, but it is still to be identified which is the basis of its mechanism. CTD is a potent inhibitor of phosphatase PP1 and PP2A which play a positive role in regulating Wnt signalling since it dephosphorylates AXIN, by impairing APC complex. We screened CTD as the most effective drug to inhibit Wnt pathway in MB cells. By immunofluorescence, we observed that the nuclear amount of β-catenin was found reduced when the cells were exposed to CTD. We found its effectiveness in vitro and in vivo on tumor growth assays in nude and strengthen those with additional orthotopic cerebellum xenografs assays to further assign its molecular mechanism of action in MB thus suggesting its use for future therapeutic applications in mouse MB genetic models. Altogether these results are suggesting a new therapeutic strategy to treat MB to be further applied to other solid tumors which are mainly Wnt driven. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 763.

MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

BackgroundThrough negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies.Methodology/Principal FindingsIn a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept.Conclusions/SignificanceDespite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

An Epigenetic Genome-Wide Screen Identifies SPINT2 as a Novel Tumor Suppressor Gene in Pediatric Medulloblastoma

Medulloblastoma (MB) is a malignant cerebellar tumor that occurs primarily in children. The hepatocyte growth factor (HGF)/MET pathway has an established role in both normal cerebellar development as well as the development and progression of human brain tumors, including MB. To identify novel tumor suppressor genes involved in MB pathogenesis, we performed an epigenome-wide screen in MB cell lines, using 5-aza-2'deoxycytidine to identify genes aberrantly silenced by promoter hypermethylation. Using this technique, we identified an inhibitor of HGF/MET signaling, serine protease inhibitor kunitz-type 2 (SPINT2/HAI-2), as a putative tumor suppressor silenced by promoter methylation in MB. In addition, based on single nucleotide polymorphism array analysis in primary MB samples, we identified hemizygous deletions targeting the SPINT2 locus in addition to gains on chromosome 7 encompassing the HGF and MET loci. SPINT2 gene expression was down-regulated and MET expression was up-regulated in 73.2% and 45.5% of tumors, respectively, by quantitative real-time PCR. SPINT2 promoter methylation was detected in 34.3% of primary MBs examined by methylation-specific PCR. SPINT2 reexpression in MB cell lines reduced proliferative capacity, anchorage independent growth, cell motility in vitro, and increased overall survival times in vivo in a xenograft model (P<0.0001). Taken together, these data support the role of SPINT2 as a putative tumor suppressor gene in MB, and further implicate dysregulation of the HGF/MET signaling pathway in the pathogenesis of MB.

Insulin-like Growth Factor 2 Is Required for Progression to Advanced Medulloblastoma in patched1 Heterozygous Mice

Medulloblastoma (MB) can arise in the cerebellum due to genetic activation of the Sonic Hedgehog (Shh) signaling pathway. During normal cerebellum development, Shh spurs the proliferation of granule neuron precursors (GNP), the precursor cells of MB. Mutations in the Shh receptor gene patched1 (ptc1+/-) lead to increased MB incidence in humans and mice. MB tumorigenesis in mice heterozygous for ptc1+/- shows distinct steps of progression. Most ptc1+/- mice form clusters of preneoplastic cells on the surface of the mature cerebellum that actively transcribe Shh target genes. In approximately 15% of mice, these preneoplastic cells will become fast-growing, lethal tumors. It was previously shown that the loss of function of insulin-like growth factor 2 (igf2) suppresses MB formation in ptc1+/- mice. We found that igf2 is not expressed in preneoplastic lesions but is induced as these lesions progress to more advanced MB tumors. Igf2 is not required for formation of preneoplastic lesions but is necessary for progression to advanced tumors. Exogenous Igf2 protein promoted proliferation of MB precursor cells (GNP) and a MB cell line, PZp53(MED). Blocking igf2 signaling inhibited growth of PZp53(MED) cells, implicating igf2 as a potential clinical target.

Effects of sertoli cell-conditioned medium on ventral midbrain neural stem cells: A preliminary report

The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.

Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27(neg) B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.

Aberrant promoter methylation of previously unidentified target genes is a common abnormality in medulloblastomas--implications for tumor biology and potential clinical utility.

Medulloblastomas exhibit an array of diverse cytogenetic abnormalities. To evaluate the significance of epigenetic rather than genetic lesions in medulloblastomas and other primitive neuroectodermal tumors (PNETs) of the childhood CNS we performed a systematic analysis of gene specific and global methylation. Methylation-specific PCR detected no methylation for p15(INK4B), von Hippel Lindau and TP53 and only limited methylation for E-Cadherin and p16(INK4A) in tumors. The cell lines Daoy and MHH-PNET-5 in which the p16(INK4A) promoter was methylated did not express the gene, but demonstrated abnormalities by SSCP. Immunohistochemistry for p16 was negative in all examined normal cerebella and medulloblastomas. Using the technique of Restriction Landmark Genomic Scanning we detected methylation affecting up to 1% of all CpG islands in primary MB/PNETs and 6% in MB cell lines. Methylation patterns differed between medulloblastomas and PNETs. Examination of several methylated sequences revealed homologies to known genes and expressed sequences. Analysis of survival data identified seven of 30 hypermethylated sequences significantly correlating with poor prognosis. We suggest that DNA hypermethylation has an outstanding potential for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in MBs and other PNETs of the CNS.

Sensitivity in vitro of herpes simplex virus isolates to human fibroblast interferon.

Sensitivity of herpes simplex virus (HSV) isolates, from patients with recurrent infections, to human fibroblast beta interferon (IFN), was tested in vitro. Among 25 HSV strains, 12 were HSV-1, isolated from facial and labial lesions, and 13 were HSV-2 isolated from genital lesions. Viral sensitivity to IFN was examined on epithelial MB cell line by yield reduction and plaque reduction and close correspondence between the two methods was observed. Most of HSV-1 isolates were in the same range of sensitivity to IFN, while HSV-2 isolates varied in sensitivity and differences approached statistical significance (P = 0.14). No correlation was found between various biological properties, such as plaque size, virulence in baby mice, growth at different temperatures, thymidine kinase activity in the presence or absence of IFN and sensitivity to IFN. Application of beta IFN containing cream reduced clinical symptoms in most of the patients. However, larger numbers of patients should be evaluated in order to conclude whether in vitro sensitivity correlates to clinical improvement.