Survival Of Human Glioblastoma Cells Research Articles

P10.30.B COMBINATION TREATMENT WITH ERIBULIN AND TTFIELDS INDUCES APOPTOSIS AND G2/M CELL CYCLE ARREST IN HUMAN GLIOBLASTOMA CELLS

Abstract BACKGROUND TTFields has emerged to a first line treatment in newly diagnosed glioblastoma (GBM) patients. Several preclinical studies showed that the alternating electric fields inhibit proliferation and survival of human glioblastoma cells by disruption of the spindle apparatus and impairment of mitotic segregation. Eribulin is an antimitotic drug, that is currently used in the third line treatment of breast cancer. As it is also able to inhibit the pro-mitotic function of TERT, which is mutated in 80% of human glioblastoma, the drug was also shown to have a radiosensitizing and antiproliferative effect on human glioblastoma and ovarian cancer cell lines. This study aims to evaluate the effect of the combination of Eribulin and TTFields on the survival and cell cycle of glioblastoma cells. MATERIAL AND METHODS U87, A172 and four patient derived glioblastoma cell lines were cultured and plated to 80% confluence. Cells were then treated with TTFields at a frequency of 200kHz for 24 hours and/or received the ED50 and 0.5-fold ED50 of Eribulin. Controls remained untreated respectively. After 24 hours cells were harvested and counted by automatic cell count. Survival and cell cycle status were evaluated by FACS analysis. RESULTS The rate of early apoptotic cells was 82.28% in the group treated with TTFields and Eribulin. With TTFields treatment only 50.99% and with Eribulin alone 14.38% of cells were in an early apoptotic state. Untreated control cells had an apoptosis rate of 4.25%. The combination of TTFields and Eribulin was significantly more effective than the single treatment modalities (p>0.05). Treatment with TTFields increased the rate of cells an G2/M status. This effect was even stronger in the group with combination treatment. CONCLUSION Combination treatment of the antimitotic drug Eribulin with TTFields is able to improve the inhibitory effect of the drugs on cell survival. Furthermore the combination therapy effects the cell cycle of glioblastoma cells inducing G2/M arrest. Future preclinical studies should focus on the antimitotic effect and the influence of the treatment on genome integrity for a more detailed insight into the mechanisms of action.

Wnt-Induced Stabilization of KDM4C Is Required for Wnt/β-Catenin Target Gene Expression and Glioblastoma Tumorigenesis.

Wnt/β-catenin signaling activates the transcription of target genes to regulate stem cells and cancer development. However, the contribution of epigenetic regulation to this process is unknown. Here, we report that Wnt activation stabilizes the epigenetic regulator KDM4C that promotes tumorigenesis and survival of human glioblastoma cells by epigenetically activating the transcription of Wnt target genes. KDM4C protein expression was upregulated in human glioblastomas, and its expression directly correlated with Wnt activity and Wnt target gene expression. KDM4C was essential for Wnt-induced gene expression and tumorigenesis of glioblastoma cells. In the absence of Wnt3a, protein kinase R phosphorylated KDM4C at Ser918, inducing KDM4C ubiquitination and degradation. Wnt3a stabilized KDM4C through inhibition of GSK3-dependent protein kinase R activity. Stabilized KDM4C accumulated in the nucleus and bound to and demethylated TCF4-associated histone H3K9 by interacting with β-catenin, promoting HP1γ removal and transcriptional activation. These findings reveal that Wnt-KDM4C-β-catenin signaling represents a novel mechanism for the transcription of Wnt target genes and regulation of tumorigenesis, with important clinical implications. SIGNIFICANCE: These findings identify the Wnt-KDM4C-β-catenin signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma.

Aurora A plays a dual role in migration and survival of human glioblastoma cells according to the CXCL12 concentration

Primary glioblastoma is the most frequent human brain tumor in adults and is generally fatal due to tumor recurrence. We previously demonstrated that glioblastoma-initiating cells invade the subventricular zones and promote their radio-resistance in response to the local release of the CXCL12 chemokine. In this work, we show that the mitotic Aurora A kinase (AurA) is activated through the CXCL12–CXCR4 pathway in an ERK1/2-dependent manner. Moreover, the CXCL12–ERK1/2 signaling induces the expression of Ajuba, the main cofactor of AurA, which allows the auto-phosphorylation of AurA.We show that AurA contributes to glioblastoma cell survival, radio-resistance, self-renewal, and proliferation regardless of the exogenous stimulation with CXCL12. On the other hand, AurA triggers the CXCL12-mediated migration of glioblastoma cells in vitro as well as the invasion of the subventricular zone in xenograft experiments. Moreover, AurA regulates cytoskeletal proteins (i.e., Actin and Vimentin) and favors the pro-migratory activity of the Rho-GTPase CDC42 in response to CXCL12. Altogether, these results show that AurA, a well-known kinase of the mitotic machinery, may play alternative roles in human glioblastoma according to the CXCL12 concentration.

Radiosensitizing effect of 5-aza-2'-deoxycytidin in human glioblastoma cells

Glioblastoma multiforme represents the most aggressive brain tumor. Its high infiltration rate and resistance to current treatment protocols results in a median survival of only 15 months following surgical removal and standard therapy. Epigenetic modulators have been shown to inhibit the growth of IDH-mutant glioma cells and may modulate DNA repair. Here, we analyzed the combined effect of the methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and ionizing radiation (IR) on the clonogenic survival of human glioblastoma cells.

Cell density modulates SHC3 expression and survival of human glioblastoma cells through Fak activation.

Shc3 protein levels are high in human glioblastoma but they decrease in vitro. We found that SHC3 mRNA and protein increased when glioblastoma cells grew as multicellular tumor spheroid (MTS). Shc3 expression was also induced in adherent cultures by increasing cell density. Among the Shc family members, only Shc2 and Shc3 increased with cell density. Shc3 and focal adhesion kinase (Fak) interact as shown by co-immunoprecipitation. Inhibition of Fak activation reduced Shc3 increase and MTS formation and changed Shc3 phosphorylation pattern. Our results suggest that in gliomas cell density modulates Shc3 protein levels and its activity, at least in part, through Fak activation.

Abstract 4104: Netrin-1 promotes the invasiveness and survival of human glioblastoma cells.

Abstract Glioblastoma multiforme (GBM) is the most severe human brain cancer, which has no efficient treatment. The main reason for the lethality of GBM is its resistance to all current therapies and highly invasive. According to microarray data on Oncomine database netrin-1 is upregulated in glioblastoma tumors. Netrin-1 is a secreted laminin related extracellular matrix protein. Its main function is to regulate the axon guidance during embryogenesis. Recently it has been observed to act as a survival factor for different forms of cancer such as aggressive neuroblastoma, metastatic breast cancer and non small cell lung cancer. It also promotes tumorigenesis of colorectal cancer. Based on these observations we explored the possibility whether netrin-1 plays a role in human GBM. We analyzed the effects of netrin-1 expression on human GBM cells. According to Matrigel invasion assays overexpression of netrin-1 increased the invasiveness whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness of human GBM cells. On the other hand, complete knock-down of netrin-1 led to apoptotic death of those cells. To further analyze netrin-1 signaling in human GBM we performed a mass spectrometry screen to identify binding partners of netrin-1. We identified Notch as a novel receptor for netrin-1. In addition, we observed that netrin-1 overexpression increased and knockdown decreased Notch activation. The role of Notch signaling regarding the aggressiveness of various cancers has been controversial. Our results confirm that increased Notch signaling increases glioblastoma cell invasiveness, and that netrin-1 increases Notch signaling. Further, we identified here a recombinant netrin-1 fragment that inhibits Notch signaling by blocking the Notch signaling complex to the cell surface. Current studies provide new mechanistic information on the tumorigenesis of GBM. Activation of Notch signaling pathway has been linked to the initiation of EMT which is essential for cancer cell invasion. Our results suggest that netrin-1 regulates GBM invasion by activating Notch signaling. The inhibition of netrin-1 signaling may prove out to be a treatment option for human glioblastoma. Citation Format: Jorma K. Keski-Oja, Irene Ylivinkka, Yizhou Hu, Tuula Nyman, Marko Hyytiainen. Netrin-1 promotes the invasiveness and survival of human glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4104. doi:10.1158/1538-7445.AM2013-4104

Overexpression of WW domain-containing oxidoreductase WOX1 preferentially induces apoptosis in human glioblastoma cells harboring mutant p53

PurposeHuman WWOX gene encoding WW domain-containing oxidoreductase, named WWOX, FOR, or WOX1, has been studied in various types of cancer cells and shown to be a tumor suppressor with pro-apoptotic properties. Mutation or gain-of-function of p53 in glioma cells is associated with resistance to radiation therapy and poor prognosis. In this study, we overexpressed WOX1 to examine the pro-apoptotic activity against human glioblastoma cells harboring mutant p53. MethodsOverexpression of WOX1 in glioblastoma cell lines and apoptosis-related assays were performed. ResultsOur results showed that overexpressed WOX1 induced apoptosis of glioblastoma U373MG harboring mutant p53 by causing hypoploidy and DNA fragmentation. However, ectopic WOX1 had no effect with U87MG possessing wild type p53. Unlike temozolomide, WOX1 induced apoptosis of U373MG cells via a mitochondria-independent and caspase-3-independent pathway. ConclusionsOverexpression of WOX1 preferentially inhibited viability and induced apoptosis in human glioblastoma cells expressing mutant p53 via a mechanism independent of the intrinsic apoptotic pathway. Conceivably, the survival of human glioblastoma cells depends upon interactions between the gain-of-function of p53 and WOX1. This suggests that modulation of WOX1 expression may be a novel strategy for treating human glioblastoma cells with mutant p53.

High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5

Activating transcription factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichroism analyses indicate the protein exists in the partially α-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation.

Production of angiogenic factors by human glioblastoma cells following activation of the G-protein coupled formylpeptide receptor FPR

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells.