Differential sensitivity of Glioma stem cells to Aurora kinase A inhibitors: implications for stem cell mitosis and centrosome dynamics

Glioma stem-cell-like cells are considered to be responsible for treatment resistance and tumour recurrence following chemo-radiation in glioblastoma patients, but specific targets by which to kill the cancer stem cell population remain elusive. A characteristic feature of stem cells is their ability to undergo both symmetric and asymmetric cell divisions. In this study we have analysed specific features of glioma stem cell mitosis. We found that glioma stem cells appear to be highly prone to undergo aberrant cell division and polyploidization. Moreover, we discovered a pronounced change in the dynamic of mitotic centrosome maturation in these cells. Accordingly, glioma stem cell survival appeared to be strongly dependent on Aurora A activity. Unlike differentiated cells, glioma stem cells responded to moderate Aurora A inhibition with spindle defects, polyploidization and a dramatic increase in cellular senescence, and were selectively sensitive to Aurora A and Plk1 inhibitor treatment. Our study proposes inhibition of centrosomal kinases as a novel strategy to selectively target glioma stem cells

Hypoxia-inducible factor 1 alpha is required for the tumourigenic and aggressive phenotype associated with Rab25 expression in ovarian cancer

The small GTPase Rab25 has been functionally linked to tumour progression and aggressiveness in ovarian cancer and promotes invasion in three-dimensional environments. This type of migration has been shown to require the expression of the hypoxia-inducible factor 1 alpha (HIF-1α). In this report we demonstrate that Rab25 regulates HIF-1α protein expression in an oxygen independent manner in a panel of cancer cell lines. Regulation of HIF-1α protein expression by Rab25 did not require transcriptional upregulation, but was dependent on de novo protein synthesis through the Erbb2/ERK1/2 and p70S6K/mTOR pathways. Rab25 expression induced HIF-1 transcriptional activity, increased cisplatin resistance, and conferred intraperitoneal growth to the A2780 cell line in immunocompromised mice. Targeting HIF1 activity by silencing HIF-1β re-sensitised cells to cisplatin in vitro and reduced tumour formation of A2780-Rab25 expressing cells in vivo in a mouse ovarian peritoneal carcinomatosis model. Similar effects on cisplatin resistance in vitro and intraperitoneal tumourigenesis in vivo were obtained after HIF1b knockdown in the ovarian cancer cell line SKOV3, which expresses endogenous Rab25 and HIF-1α at atmospheric oxygen concentrations. Our results suggest that Rab25 tumourigenic potential and chemoresistance relies on HIF1 activity in aggressive and metastatic ovarian cancer. Targeting HIF-1 activity may potentially be effective either alone or in combination with standard chemotherapy for aggressive metastatic ovarian cancer

Cucurbit [7] uril encapsulated cisplatin overcomes resistance to cisplatin induced by Rab25 overexpression in an intraperitoneal ovarian cancer model

Background Ovarian cancer is the most fatal of gynaecological malignancies, usually detected at a late stage with intraperitoneal dissemination. Appropriate preclinical models are needed that recapitulate both the histopathological and molecular features of human ovarian cancer for drug-efficacy analysis. Methods Longitudinal studies comparing cisplatin performance either alone or in a novel cisplatin-based delivery-system, cucurbit[7]uril-encapsulated cisplatin (cisplatin@CB[7]) were performed on subcutaneous (s.c.) and intraperitoneal (i.p.) xenografts using the human ovarian cancer cell line A2780 stably expressing the small GTPase Rab25, which allows A2780 intraperitoneal growth; and luciferase, to allow tumour load measurement by non-invasive bioluminescent imaging. Results Rab25 expression induced cisplatin resistance compared to the parental cell line as assessed by the MTT assay in vitro. These findings did not translate in vivo, where cisplatin resistance was determined by the microenvironment. Subcutaneous xenografts of either parental A2780 or cisplatin-resistant Rab25-expressing A2780 cells presented similar responses to cisplatin treatment. In contrast, increased cisplatin resistance was only detected in i.p. tumours. Treatment of the cisplatin-resistant i.p. model with the novel cisplatin@CB[7] delivery system resulted in a substantial reduction of i.p. tumour load and increased necrosis. Conclusions Poor clinical performance of novel chemotherapeutics might reflect inappropriate preclinical models. Here we present an ovarian i.p. model that recapitulates the histopathological and chemoresistant features of the clinical disease. In addition, we demonstrate that the novel cisplatin-delivery system, cisplatin@CB[7] may have utility in the treatment of drug-resistant ovarian human cancers

OS1.4 Induction of mitotic cell death : a novel therapeutic strategy for glioblastoma

Background Glioblastoma (GBM) is the most common and lethal adult brain tumour. Tumours typically contain large numbers of binuclear and multinucleated cells, and a subpopulation of relatively quiescent glioma stem-like cells (GSC) that are thought to be responsible for treatment resistance and tumour recurrence. GSC display robust G2/M arrest following ionising radiation (IR) yet are highly prone to aberrant cell division and are sensitive to mitotic spindle checkpoint inhibitors. The aim of this study was to investigate the therapeutic activity of mitotic inducers in preclinical models of GBM. Material and Methods Bioinformatic analysis of mRNA expression data was used to confirm the relevance of mitotic activity following irradiation of GSC in a 3D patient-derived GBM stem cell model. Immunofluorescence, clonogenic survival and cell viability assays were used to evaluate the therapeutic potential of mitotic inducers (ME-344; Wee1 inhibitor AZ1775) in 2D and 3D U87MGLuc and E2, G1, G7, S2 and R15 patient-derived GSC cell culture models. either alone or in combination with IR. In vivo validation was determined in U87-MGLuc orthotopic GBM mouse model. Results Radiation induced downregulation of mRNA expression of several mitotic genes was observed in G7 and E2 cell lines confirming mitotic relevance. In cell viability and clonogenic survival assays, AZ1775 and ME-344 showed potent cytotoxicity against all GSC cell lines in both 2D and 3D, with EC50 values of 0.2 to 0.4 μM for AZ1775 and 0.003 to 0.02 μM for ME-344. ME-344 and AZ1775 triggered profound morphological and cell cycle effects including mitotic induction and arrest, increased mitotic fraction, reduced nuclear tubulin in mitotic cells and, induced mitotic catastrophe in the most sensitive cell lines U87MGLuc and E2. These events were apoptosis-independent. Combination with IR increased GSC cell death in the two GSC models tested to date. Accumulation of cells in mitosis following ME-344 treatment was recapitulated in orthotopic GBM xenografts in vivo, although few mitotic catastrophe events were observed 24 h after treatment. ME-344 demonstrated therapeutic efficacy as a single agent in U87MGLuc2 orthotopic xenografts by extending mouse survival compared to vehicle (p=0.043). Conclusion Two agents that induce mitosis through different mechanisms have promising single agent activity against all GBM cell lines tested in vitro and in vivo. Further preclinical evaluation in combination with IR and/or temozolomide is underway. Results indicate that this therapeutic strategy for GBM has clinical potential

Coste de la limpieza “cotidiana” de playas

Más de 90.000 buques mercantes al año cruzan el Estrecho de Gibraltar de los que aproximadamente un 5% son petroleros. Ello, conjuntamente con la existencia de varios puertos con refinerías y polígonos industriales petroquímicos y la práctica del bunkering para aprovisionamiento de combustible, hace a la costa gaditana un punto de riesgo para la contaminación por hidrocarburos (Carmona et al., 2009). La Demarcación de Costas de Andalucía-Atlántico (DCAA), dependiente del Ministerio de Agricultura, Alimentación y Medio Ambiente, ha realizado en numerosas ocasiones la limpieza de su litoral debido a la polución producida por ese tipo de vertidos (Carmona et al., 2012), existiendo ya alguna bibliografía sobre cómo abordar la retirada de alquitrán y otros derivados similares de las playas (e.g. DGC 2005, CEPRECO 2006a, CEPRECO 2006b). Sin embargo, además de la fracción no volátil de los hidrocarburos, existe otro tipo de desechos, naturales y/o antrópicos, que llegan a nuestras playas y que, debido sobre todo al carácter turístico de nuestro litoral, deben ser recogidos. Por ley, esta limpieza corresponde a las autoridades locales. No obstante, debido a la escasez de su presupuesto, los municipios suelen atender prioritariamente a la limpieza de las playas más urbanas y de máxima utilización. Es por este motivo que, dentro de un espíritu de colaboración entre Administraciones, la DCAA, mediante su partida de conservación y mantenimiento, ha apoyado las tareas de los Ayuntamientos, reforzando la labor municipal en las playas más concurridas y limpiando aquellas que son menos visitadas ya sea por su lejanía del casco urbano o por su escasez de servicios. En esta ponencia se presentarán los medios humanos y materiales con los que se cuenta para esta tarea, aportando cifras de toneladas de basura retiradas y coste del trabajo, junto con una comparación superficial con los datos de otros organismos y alguna sugerencia de posibles mejoras de la productividad

Elastic properties of graphene suspended on a polymer substrate by e-beam exposure

A method for fabricating multiple free-standing structures on the same sheet of graphene is demonstrated. Mechanically exfoliated mono- and bilayer graphene sheets were sandwiched between two layers of polymethyl-methacrylate. Suspended areas were defined by e-beam exposure allowing precise control over their shape and position. Mechanical characterization of suspended graphene sheets was performed by nanoindentation with an atomic force microscopy tip. The obtained built-in tensions of 12 nN are significantly lower than those in suspended graphene exfoliated on an SiO2 substrate, and therefore permit access to the intrinsic properties of this material system

Cucurbit[7]uril encapsulated cisplatin overcomes cisplatin resistance via a pharmacokinetic effect

The cucurbit[n]uril (CB[n]) family of macrocycles has been shown to have potential in drug delivery where they are able to provide physical and chemical stability to drugs, improve drug solubility, control drug release and mask the taste of drugs. Cisplatin is a small molecule platinum-based anticancer drug that has severe dose-limiting side-effects. Cisplatin forms a host–guest complex with cucurbit[7]uril (cisplatin@CB[7]) with the platinum atom and both chlorido ligands located inside the macrocycle, with binding stabilised by four hydrogen bonds (2.15–2.44 A ˚ ). Whilst CB[7] has no effect on the in vitro cytotoxicity of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant sub-lines A2780/cp70 and MCP1, there is a significant effect on in vivo cytotoxicity using human tumour xenografts. Cisplatin@CB[7] is just as effective on A2780 tumours compared with free cisplatin, and in the cisplatin-resistant A2780/cp70 tumours cisplatin@CB[7] markedly slows tumour growth. The ability of cisplatin@CB[7] to overcome resistance in vivo appears to be a pharmacokinetic effect. Whilst the peak plasma level and tissue distribution are the same for cisplatin@CB[7] and free cisplatin, the total concentration of circulating cisplatin@CB[7] over a period of 24 hours is significantly higher than for free cisplatin when administered at the equivalent dose. The results provide the first example of overcoming drug resistance via a purely pharmacokinetic effect rather than drug design or better tumour targeting, and demonstrate that in vitro assays are no longer as important in screening advanced systems of drug delivery

P08.36 Radioresistance of glioblastoma stem-like cells is associated with DNA replication stress, which is a promising therapeutic target

Introduction: The inevitability of tumour recurrence in glioblastoma (GBM) patients despite multi-modality treatment consisting of surgery, radiotherapy and chemotherapy, is reflected by a median survival of only 14 months. Tumour recurrence is thought to be driven by a small population of glioblastoma stem-like cells (GSCs) that are resistant to conventional therapies. DNA damage response (DDR) pathways have been shown to be up-regulated in GSCs and implicated in radioresistance and treatment failure. However the precise cause of enhanced DDR signalling in GSCs and the extent to which these signalling networks contribute to therapy resistance remains elusive. The objectives of this study were to investigate the underlying cause of DDR upregulation and treatment resistance in GSCs with a view to identifying novel and promising therapeutic targets. Materials and Methods: A panel of primary patient derived GBM cell lines cultured under conditions to enrich for or deplete the tumour stem cell population (GSC vs bulk respectively) were utilised in order to investigate enhanced GSC DDR under basal conditions and in response to ionising radiation. Confirmatory studies were also performed in cells sorted for the putative GSC marker CD133. The effects of a panel of small molecule DDR inhibitor agents on cell survival in GSC and bulk cells were quantified. Results: GSCs exhibited higher levels of total and activated DDR targets ATR, CHK1, ATM and PARP1 under basal conditions and were radioresistant compared to paired bulk populations. This was not due to increased levels of reactive oxygen species (ROS). Instead, we show that RPA is significantly higher in replicating GSCs and confirm by DNA fibre assays that GSCs and CD133+ cells have increased numbers of stalled replication forks, fewer new origins and slower DNA replication compared to bulk or CD133- populations, demonstrating for the first time that replication stress (RS) is a hallmark of GSCs. We identify increased expression of long neural genes as a likely mechanism for RS and DNA double strand breaks (DSBs) in GSCs and show that their radioresistance is reversed by dual inhibition of key RS and DDR proteins ATR and PARP. Conclusions: This study demonstrates the novel finding that replication stress is a hallmark of GSCs and resonates with recently published studies in neural progenitor cells showing that RS preferentially induces DNA DSB in long neural genes. Taken together, we implicate RS as a driver of enhanced DDR in GSCs and identify novel therapeutics with potential to improve clinical outcomes by overcoming the radioresistance of GB

The Herschel Exploitation of Local Galaxy Andromeda (HELGA) II: Dust and Gas in Andromeda

We present an analysis of the dust and gas in Andromeda, using Herschel images sampling the entire far-infrared peak. We fit a modified-blackbody model to ~4000 quasi-independent pixels with spatial resolution of ~140pc and find that a variable dust-emissivity index (beta) is required to fit the data. We find no significant long-wavelength excess above this model suggesting there is no cold dust component. We show that the gas-to-dust ratio varies radially, increasing from ~20 in the center to ~70 in the star-forming ring at 10kpc, consistent with the metallicity gradient. In the 10kpc ring the average beta is ~1.9, in good agreement with values determined for the Milky Way (MW). However, in contrast to the MW, we find significant radial variations in beta, which increases from 1.9 at 10kpc to ~2.5 at a radius of 3.1kpc and then decreases to 1.7 in the center. The dust temperature is fairly constant in the 10kpc ring (ranging from 17-20K), but increases strongly in the bulge to ~30K. Within 3.1kpc we find the dust temperature is highly correlated with the 3.6 micron flux, suggesting the general stellar population in the bulge is the dominant source of dust heating there. At larger radii, there is a weak correlation between the star formation rate and dust temperature. We find no evidence for 'dark gas' in M31 in contrast to recent results for the MW. Finally, we obtained an estimate of the CO X-factor by minimising the dispersion in the gas-to-dust ratio, obtaining a value of (1.9+/-0.4)x10^20 cm^-2 [K kms^-1]^-1.Comment: 19 pages, 18 figures. Submitted to ApJ April 2012; Accepted July 201