A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [ 18 F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Deathswitch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [ 18 F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [ 18 F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers. © 2013 Macmillan Publishers Limited. All rights reserved

Design and synthesis of imidazoline derivatives active on glucose homeostasis in a rat model of type II diabetes.2. Syntheses and biological activities of 1,4-dialkyl-,1,4-dibenzyl and 1-benzyl-4-alkyl-2-(4',5'-dihydro-1' H-imidazol-2'yl)piperazines and isosteric analogues of imidazoline

International audienc

Drug-target interactions: only the first step in the commitment to a programmed cell death?

The search for novel antitumour drugs has reached a plateau phase. The carcinomas remain almost as intractable as they did 40 years ago and the need for effective therapy is pressing. There is an argument that the current pharmacopoeia is sufficient but, to be effective, the biochemical mechanisms of drug resistance must be circumvented. In tackling the question of why certain cancer cells are resistant, the converse question of why others are sensitive still remains to be answered fully. Asking the fundamental question of why and how a cell dies may provide clues as to what avenues lie open for improved chemotherapy. In this review we survey the recent literature on cell death and we argue that it is possible that the outcome of chemotherapy may be determined by the response of the cell to the formation of the drug-target complex, and/or its sequellae, rather than to the biochemical changes brought about by the drug alone. One of these responses, determined by the phenotype of the cell, may be activation of a genetic programme for cell death

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines

Circulating biomarkers during treatment in patients with advanced biliary tract cancer receiving cediranib in the UK ABC-03 trial

BACKGROUND: Advanced biliary tract cancer (ABC) has a poor prognosis. Cediranib, in addition to cisplatin/gemcitabine [CisGem], improved the response rate, but did not improve the progression-free survival (PFS) in the ABC-03 study. Minimally invasive biomarkers predictive of cediranib benefit may improve patient outcomes. METHODS: Changes in 15 circulating plasma angiogenesis or inflammatory-related proteins and cytokeratin-18 (CK18), measured at baseline and during therapy until disease progression, were correlated with overall survival (OS) using time-varying covariate Cox models (TVC). RESULTS: Samples were available from n=117/124 (94%) patients. Circulating Ang1&2, FGFb, PDGFbb, VEGFC, VEGFR1 and CK18 decreased as a result of the therapy, independent of treatment with cediranib. Circulating VEGFR2 and Tie2 were preferentially reduced by cediranib. Patients with increasing levels of VEGFA at any time had a worse PFS and OS; this detrimental effect was attenuated in patients receiving cediranib. TVC analysis revealed CK18 and VEGFR2 increases correlated with poorer OS in all patients (P< 0.001 and P=0.02, respectively). CONCLUSIONS: Rising circulating VEGFA levels in patients with ABC, treated with CisGem, are associated with worse PFS and OS, not seen in patients receiving cediranib. Rising levels of markers of tumour burden (CK18) and potential resistance (VEGFR2) are associated with worse outcomes and warrant validation

Systematic Analysis of Circulating Soluble Angiogenesis-Associated Proteins in ICON7 Identifies Tie2 as a Biomarker of Vascular Progression on Bevacizumab

background: There is a critical need for predictive/resistance biomarkers for VEGF inhibitors to optimise their use. methods: Blood samples were collected during and following treatment and, where appropriate, upon progression from ovarian cancer patients in ICON7, a randomised phase III trial of carboplatin and paclitaxel with or without bevacizumab. Plasma concentrations of 15 circulating angio-biomarkers were measured using a validated multiplex ELISA, analysed through a novel network analysis and their relevance to the PFS then determined. results: Samples (n=650) were analysed from 92 patients. Bevacizumab induced correlative relationships between Ang1 and Tie2 plasma concentrations, which reduced after initiation of treatment and remained decreased until progressive disease occurred. A 50% increase from the nadir in the concentration of circulating Tie2 (or the product of circulating Ang1 and Tie2) predicted tumour progression. Combining Tie2 with GCIG-defined Ca125 data yielded a significant improvement in the prediction of progressive disease in patients receiving bevacizumab in comparison with Ca125 alone (74.1% vs 47.3%, P<1 × 10−9). conclusions: Tie2 is a vascular progression marker for bevacizumab-treated ovarian cancer patients. Tie2 in combination with Ca125 provides superior information to clinicians on progressive disease in patients with VEGFi-treated ovarian cancers

Beyond severity: Utility as a criterion for setting the scope of RGCS

Reproductive genetic carrier screening (RGCS) allows prospective parents to identify and act upon their chances of having a child with a genetic condition. In deciding which genetic conditions to include in RGCS, severity is often used as a criterion. However, the concept is inherently complex, subjective and multidimensional, and determinations of severity will remain intractably contested. We propose the concept of utility as a criterion for setting the scope of RGCS, and put forward two central arguments for doing so. First, utility is a more appropriate and effective concept as it responds to context and makes an explicit connection between the purpose of RGCS and the value of information obtained for that purpose: namely, to facilitate reproductive decision-making. Utility comprises both clinical and personal utility, and varies according to the availability and accessibility of reproductive options, including pre-implantation genetic testing, prenatal genetic diagnosis, and termination of pregnancy. Second, there are ethical reasons for preferring utility over severity. Utility is a property of the information gleaned from RGCS, while severity is a property of a genetic condition or of an instance of this condition in a person. While consideration of the severity of genetic conditions is not lost when focusing on utility, the need to rely on value judgements regarding the quality of life of people who live with genetic conditions is circumvented. Therefore, utility should replace severity as justification for the inclusion of genetic conditions in RGCS programmes

STUDIES ON EFFECT OF COW DUNG ASH IN AQUEOUS MEDIUM AS FOLIAR SPRAY ON Zea mays (POACEAE)

Present work deals with study on effect of cow dung ash in aqueous medium as foliar spray on commercial crop and experimental crop plant Zea mays (Poaceae). We discovered that using cow dung ash as a foliar spray in an aqueous medium improves plant development. In comparison to 1% to 3% and 6% to 10%, the 4% and 5% cow dung ash sprouts are more successful, exhibiting robust development, dark green colored leaves, and pest-free healthy plants.&nbsp; Cow dung ash sprouts between 1% and 3% demonstrate a steady rise in plant height and color. While 6% to 10% of the ash from cow manure is less effective or withering

Beyond platitudes: a qualitative study of Australian Aboriginal people's perspectives on biobanking.

BACKGROUND: Biobanks are vital resources for genetics and genomics, and it is broadly recognised that for maximal benefit it is essential that they include samples and data from diverse ancestral groups. The inclusion of First Nations people, in particular, is important to prevent biobanking research from exacerbating existing health inequities, and to ensure that these communities share in the benefits arising from research. AIMS: To explore the perspectives of Australian Aboriginal people whose tissue - or that of their family members - has been stored in the biobank of the National Centre for Indigenous Genomics (NCIG). METHODS: Semi-structured interviews with 42 Aboriginal people from the Titjikala, Galiwinku, Tiwi Islands, Yarrabah, Fitzroy Crossing, Derby, One Arm Point and Mulan communities, as well as a formal discussion with A. Hermes, an Indigenous Community Engagement Coordinator at the NCIG who had conducted the interviews. The interviews and the structured discussion were double coded using a procedure informed by Charmaz's outline of grounded theory analysis and Morse's outline of the cognitive basis of qualitative research. RESULTS: In this article, we report on A. Hermes' interviews with members from the above Aboriginal communities, as well as on her personal views, experiences, and interpretations of the interviews she conducted with other community members. We found that participation in the NCIG biobank raised issues around broken trust, grief and loss, but also - somewhat unexpectedly - was perceived as a source of empowerment, hope and reconnection. CONCLUSIONS: This research reminds us (again) of the need to engage deeply with communities in order to respond appropriately with respect for their cultural values and norms, and to develop culturally relevant policies and processes that enhance the benefits of biobank participation and minimise potential harms