An exo-cell assay for examining real-time γ-secretase activity and inhibition

γ-Secretase is an aspartyl protease that cleaves multiple substrates that are involved in broad biological processes ranging from stem cell development to neurodegeneration. The investigation of γ-secretase has been limited by currently available assays that require genetic or biochemical manipulation in the form of substrate transfection or membrane preparation. Here we report an exo-cell assay that is capable of characterizing γ-secretase activity in any cellular system without limitation. Using a highly active, recombinant substrate this assay can quickly and easily ascertain the status of γ-secretase activity in cell systems and patient samples. We have applied this method to determine the activity of γ-secretase in primary cell samples where transfection and/or membrane isolation are not viable options. Importantly, it allows for the detection of real time γ-secretase activity after inhibitor or drug treatment. The application of this assay to determine the role of γ-secretase in physiological and pathological conditions will greatly facilitate our characterization of this complex protease and help in the development and evaluation of γ-secretase-targeted therapies in Alzheimer's disease or a variety of neoplasms

Colorectal Cancer Prognosis Following Obesity Surgery in a Population-Based Cohort Study

Background: Obesity surgery involves mechanical and physiological changes of the gastrointestinal tract that might promote colorectal cancer progression. Thus, we hypothesised that obesity surgery is associated with poorer prognosis in patients with colorectal cancer. Methods: This nationwide population-based cohort study included all patients with an obesity diagnosis who subsequently developed colorectal cancer in Sweden from 1980 to 2012. The exposure was obesity surgery, and the main and secondary outcomes were disease-specific mortality and all-cause mortality, respectively. Cox proportional hazard survival models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs), adjusted for sex, age, calendar year and education level. Results: The exposed and unexposed cohort included 131 obesity surgery and 1332 non-obesity surgery patients with colorectal cancer. There was a statistically significant increased rate of colorectal cancer deaths following obesity surgery (disease-specific HR 1.50, 95% CI 1.00–2.19). When analysed separately, the mortality rate was more than threefold increased in rectal cancer patients with prior obesity surgery (disease-specific HR 3.70, 95% CI 2.00–6.90), while no increased mortality rate was found in colon cancer patients (disease-specific HR 1.10, 85% CI 0.67–1.70). Conclusion: This population-based study among obese individuals found a poorer prognosis in colorectal cancer following obesity surgery, which was primarily driven by the higher mortality rate in rectal cancer

Single atom Cu(I) promoted mesoporous titanias for photocatalytic Methyl Orange depollution and H 2 production

Tailoring the physicochemical properties and hence reactivity of semiconductor photocatalysts in a predictable fashion, remains a challenge to their industrial application. Here we demonstrate the striking promotional effect of incorporating single Cu(I) atoms, on aqueous phase photocatalytic dye degradation and H2 production over surfactant-templated mesoporous TiO2. X-ray absorption spectroscopy reveals that ultra-low concentrations of copper (0.02-0.1 wt%) introduced into the mesoporous TiO2 surface create isolated Cu (I) species which suppress charge recombination, and confer a six-fold photocatalytic promotion of Methyl Orange degradation and four-fold enhancement of H2 evolution. The impact of mesopore structure and photophysical properties on photocatalytic activity is also quantified for the first time: calcination increases mesopore size and nanocrystalline order, and induces an anatase to rutile phase transition that is accompanied by a decrease in the optical band gap, increased charge carrier lifetime, and a concomitant significant activity enhancement

Tailored mesoporous silica supports for Ni catalysed hydrogen production from ethanol steam reforming

Mesoporous silica supported Ni nanoparticles have been investigated for hydrogen production from ethanol steam reforming. Ethanol reforming is structure-sensitive over Ni, and also dependent on support mesostructure; three-dimensional KIT-6 possessing interconnected mesopores offers superior metal dispersion, steam reforming activity, and on-stream stability against deactivation compared with a two-dimensional SBA-15 support

La confiance mutuelle dans l'espace pénal européen/Mutual Trust in the European Criminal Area

L'ouvrage constitue une réflexion approfondie sur la problématique de la confiance mutuelle dans l'espace pénal européen, un thème d’une brûlante actualité dès lors que l'importance de la confiance mutuelle dans cet espace va croissant

Abstract A75: Synergy between conventional chemotherapy and Cdc7 inhibition as a novel approach for cancer therapy.

Abstract Cell division cycle 7-related protein kinase (Cdc7) is a heterodimer of a kinase and its activator (Dbf4) and is essential for the activation of the minichromosome maintenance complex (MCM2–7), the helicase that unwinds the strands of DNA during replication. This kinase as well as its substrate have been shown to be overexpressed in many different tumors including the majority of both solid tumors and hematologic malignancies. For this reason, it represents a potential novel target for a cytotoxic chemotherapeutic agent. Previous work in our laboratory used a high throughput screen to identify compounds that inhibit Cdc7 kinase activity. MSK-747 and 3 of its naturally occurring derivatives, including MSK-777, were identified as lead compounds based on their potency, and MSK-777 was chosen as the lead compound for further preclinical and clinical development. While we are completing the FDA required studies needed to move MSK-777 to a Phase I clinical trial, we have begun work to determine an optimal Phase II regimen. Time course experiments have been performed to determine if synergy exists between known anti-leukemic chemotherapies such as etoposide, cytarabine, daunorubicin, or hydroxyurea and MSK-777. Cells were collected at time points for viable cell count, fluorescence-activated cell sorting (FACS) for DNA content, Western blotting, and caspase-3 activation assay. Multiple drug titration experiments were performed using daunorubicin, etoposide, cytarabine, and hydroxyurea in order to determine the optimal dosing of the drugs. Experiments using daunorubicin and cytarabine were performed where the standard chemotherapies were given prior to, concurrent with, and following MSK-777. Based on FACS and cell viability, it did not appear that either daunorubicin or cytarabine were synergistic with MSK-777. Etoposide caused the cells to arrest in S phase, and this arrest persisted for at least 48 hours. Based on the mechanism of action of MSK-777, etoposide was administered as a 12 hour pulse and then the cells were released into MSK-777. On FACS, at 24 hours the cells that had been treated with this combination continued to demonstrate the presence of a G1 population that was not seen with either agent alone, but by 48 hours this population no longer existed as the cells began to undergo apoptotic cell death. By 60 hours, cell death was evident by both FACS analysis and cell viability. Significant caspase-3 activation was seen at 36 hours in the cells that had received the combination therapy as compared with the controls. Given that hydroxyurea interferes with the G1/S phase progression, it was hypothesized to potentially be a more synergistic combination. Hydroxyurea was also administered as a 12 hour pulse prior to exposing the cells to MSK-777. On FACS, G1/S phase arrest is evident at 24 hours. This arrest persists with significant cell death at 60 hours being evident by cell viability, FACS analysis, and caspase-3 activation assay. This regimen demonstrated the highest degree of potential synergy. We will present data on the mechanism of this synergy and its use in primary patient samples of leukemia. Based on these results, the combination of hydroxyurea and MSK-777 is a promising regimen to be explored in further pre-clinical experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A75.</jats:p

Abstract 2153: Cdc7 Inhibition as a novel approach for pancreas cancer therapy.

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, about 43,920 people will be diagnosed with pancreatic cancer and about 37,390 people will die of this disease. Unfortunately, because of high fatality rates, pancreatic cancer incidence rates are almost equal to mortality rates. About 90% of pancreatic tumors bharbor activating mutations in the KRAS2 oncogene. Furthermore, 95% of pancreatic tumors display inactivation of the CDKN2A gene (encoding the protein inhibitor of cyclin-dependent kinase 4), resulting in the loss of p16 protein, an important regulator of the G1 to S phase cell-cycle transition. Cdc7 is a highly conserved serine-threonine kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint pathway, and has an important role in promoting a proper response to DNA damage in eukaryotic organisms. Downregulation or inhibition of Cdc7 kinase activity resulted in slowing of S-phase progression and cell cycle arrest followed by accumulation of nuclear damage. This kinase has been shown to be over-expressed in many different tumors including the majority of solid tumors and hematologic malignancies. In our laboratory a novel natural product small molecule Cdc7 kinase inhibitor (MSK-777) has been identified, developed and shown to be efficacious in standard cell based cytotoxicity assays (solid and liquid tumors) and multiple different animal models of cancer. We have begun to examine to efficacy of Cdc7 kinase inhibition as a possible therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in three different human pancreatic carcinoma cell lines, Capan-1, BxPC3, and PANC-1. These cells were treated at different time points with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS) of DNA, and protein studies using western blotting. Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells and will be presenting these results. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease. Citation Format: Lucia Regales, Ruth Santos, Diana Carrillo, Mark G. Frattini. Cdc7 Inhibition as a novel approach for pancreas cancer therapy. [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 2153. doi:10.1158/1538-7445.AM2013-2153</jats:p

Cdc7 inhibition as a novel approach for pancreas cancer therapy.

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease. </jats:p

Direct Comparison of in-Vitro Growth of Primary Human Acute Myeloid Leukemia (AML) Cells in Stromal and Stromal-Free Conditions

Abstract Background: Acute myeloid leukemia (AML) is a very aggressive bone marrow malignancy which carries a poor prognosis despite intensive chemotherapy. The treatment of relapsed and refractory AML remains suboptimal. Although more novel therapies are being introduced for AML, there is a limitation of appropriate, predictive, preclinical models available to identify and test novel therapies. In-vitro drug sensitivity testing of patient-derived AML cells is increasingly being used to facilitate treatment options. However, these tests are often done in suboptimal conditions with difficulty in the interpretation of results. Accumulating evidence has shown that the long-term, in-vitro, survival of primary AML cells can be supported with stromal co-culture, which would also take into account the influence of the surrounding tumor microenvironment. However, there has been no direct comparison of a stromal co-culture method to non-stromal growth method of primary AML cells. This is the first comprehensive direct comparison of the proliferation and immunophenotypes of primary AML cells under two different stromal conditions and cytokines, the results of which would not only highlight the importance of stromal cells to chemotherapy resistance but also lay the groundwork for the feasibility and importance of comparing it to non-stromal drug sensitivity testing. Methods: The proliferation and immunophenotypes of seven bio-banked, relapsed/refractory primary AML peripheral blood samples were assessed in 6-well plates under four culture conditions: 1) cell culture media only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned media or 4) in a cocktail of four cytokines. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 were determined by flow cytometry weekly for 2 weeks. Results: Our results demonstrated a heterogeneous growth response among the seven patient samples in response to the four growth conditions. However, the end effect was that cytokines, HS-5 conditioned media and HS-5 stromal cells can all support the long-term proliferation and viability of the majority of primary AML samples. These three conditions showed statistically significant higher growth compared to the same primary cells cultured in media alone (p-values &lt; 0.05). The degree of expansion was still significantly higher for those cells with cytokines than those with HS-5 conditioned media or stromal cells. Six of the 7 patient samples expanded with cytokine, up to 4-fold by 2 weeks of culture. In comparison 5 of the 7 patient samples expanded up to 3-fold with HS-5 stromal cells, and 4 of the 7 patient samples also expanded up to 3-fold with HS-5 conditioned media at 2 weeks of culture. For the 4 patients, whose cells were supported by both stromal cells and conditioned media, the degree of expansion was similar. Unexpectedly, one out of 7 patient samples had significantly higher expansion compared to all of the other patients in all growth condition, with a 9-fold expansion in cytokine and conditioned media, and a 12-fold expansion in HS-5 stromal cells. There was also a 3-fold expansion in media alone. Baseline AML immunophenotypes were preserved after 2 weeks of culture for all 4 growth conditions, with minimal differentiation. Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that each experimental condition alone can expand primary AML cells. In addition, cytokine support alone can expand primary AML cells at 2 weeks of culture for the majority of patient samples, independent of stromal support, a finding that has previously not been reported. Consistent with previous studies, our data also supports the conclusion that stromal support maintains long-term in-vitro expansion of primary AML cells. All three experimental conditions showed an overall effect of growth with high viability and maintenance of immunophenotypes at 2 weeks of culture. The direct comparison of drug screening under these three experimental conditions would permit the exploration of the protective effect of the bone marrow microenvironment on AML cells allowing for the identification of individualized therapeutic options, while also allowing for comparison to non-stromal based in-vitro assays. Disclosures No relevant conflicts of interest to declare. </jats:sec