The Apoptosome Pathway to Caspase Activation in Primary Human Neutrophils Exhibits Dramatically Reduced Requirements for Cytochrome c

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the “apoptosome” as a result of the cell stress–associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell–free extracts readily supported Apaf-1–dependent caspase activation, suggesting that these cells may assemble cytochrome c–independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1–dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1–dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation

Predicting the cell death responsiveness and sensitization of glioma cells to TRAIL and temozolomide.

Genotoxic chemotherapy with temozolomide (TMZ) is a mainstay of treatment for glioblastoma (GBM); however, at best, TMZ provides only modest survival benefit to a subset of patients. Recent insight into the heterogeneous nature of GBM suggests a more personalized approach to treatment may be necessary to overcome cancer drug resistance and improve patient care. These include novel therapies that can be used both alone and with TMZ to selectively reactivate apoptosis within malignant cells. For this approach to work, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified first. Here, we describe the first proof-of-principle study that merges quantitative protein-based analysis of apoptosis signaling networks with data- and knowledge-driven mathematical systems modeling to predict treatment responsiveness of GBM cell lines to various apoptosis-inducing stimuli. These include monotherapies with TMZ and TRAIL, which activate the intrinsic and extrinsic apoptosis pathways, respectively, as well as combination therapies of TMZ+TRAIL. We also successfully employed this approach to predict whether individual GBM cell lines could be sensitized to TMZ or TRAIL via the selective targeting of Bcl-2/Bcl-xL proteins with ABT-737. Our findings suggest that systems biology-based approaches could assist in personalizing treatment decisions in GBM to optimize cell death induction

Activation of executioner caspases is a predictor of progression-free survival in glioblastoma patients: a systems medicine approach.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. GBM cells are highly resistant to apoptosis induced by antitumor drugs and radiotherapy resulting in cancer progression. We assessed whether a systems medicine approach, analysing the ability of tumor cells to execute apoptosis could be utilized to predict the response of GBM patients to treatment. Concentrations of the key proapoptotic proteins procaspase-3, procaspase-9, Smac and Apaf-1 and the antiapopotic protein XIAP were determined in a panel of GBM cell lines and GBM patient tumor resections. These values were used as input for APOPTO-CELL, a systems biological based mathematical model built to predict cellular susceptibility to undergo caspase activation. The modeling was capable of accurately distinguishing between GBM cells that die or survive in response to treatment with temozolomide in 10 of the 11 lines analysed. Importantly the results obtained using GBM patient samples show that APOPTO-CELL was capable of stratifying patients according to their progression-free survival times and predicted the ability of tumor cells to support caspase activation in 16 of the 21 GBM patients analysed. Calculating the susceptibility to apoptosis execution may be a potent tool in predicting GBM patient therapy responsiveness and may allow for the use of APOPTO-CELL in a clinical setting

The effectiveness of a stratified group intervention using the STarTBack screening tool in patients with LBP--a non randomised controlled trial

Background: Low back pain (LBP) is costly to society and improving patient outcomes is a priority. Stratifying LBP patients into more homogenous groups is advocated to improve patient outcome. The STarT Back tool, a prognostic screening tool has demonstrated efficacy and greater cost effectiveness in physiotherapy settings. The management of LBP patients in groups is common but to date the utility of the STarT Back tool in group settings has not been explored. The aim of this study is to determine if the implementation of ‘stratified care’ when delivered in a group setting will lead to significantly better physical and psychological outcomes and greater cost effectiveness in LBP patients compared to a bestcare historical control group. Methods/Design: This study is a non randomised controlled trial. Low back pain patients recruited from the Waterford Primary Care area (population = 47,000) will be stratified into low, medium or high risk of persisting symptoms using the STarT Back Tool. Low risk patients will be offered a single one off education/exercise class offering positive messages on LBP management in line with recommended guidelines. Medium risk patients will be offered a 12 week group exercise/education intervention addressing their dominant physical obstacles to recovery. A 12 week group cognitive behavioural approach will be delivered to the high risk patients, characterised by the presence of high levels of psychosocial prognostic factors. These patients will be compared with a historical control group where therapists were blinded as to the risk stratification of patients and a generic group intervention was delivered to all patients, irrespective of their initial risk stratification. The primary outcome measure will be disability (Roland Morris Disability Questionnaire). Secondary outcomes will include back pain intensity (Visual Analogue Scale), distress (Distress and Risk Assessment Method), back beliefs (Back Beliefs Questionnaire), health status (Euroqol), global benefit (7 point likert scale), satisfaction (7 point likert scale), cost effectiveness and functional status. Outcome will be measured at baseline, 12 weeks and 6 months. Discussion: This paper details the rationale, design, methods, planned analysis and operational aspects of a study examining the utility of the STarT Back Tool as a 'stratification tool for targeted treatment' in a group intervention.Pfizer Healthcare IrelandIrish Pain Societ

Platinum(iv) oxaliplatin-peptide conjugates targeting memHsp70+ phenotype in colorectal cancer cells.

Novel Pt(iv) tumour penetrating peptide (TPP) conjugates are reported. They are the first example of metallodrugs to target a membrane bound heat shock protein 70 positive (memHSP70+) phenotype in cancer cells. The conjugates exhibit superior cytotoxicity as compared to oxaliplatin alone in Pt resistant colorectal cancer cells with relatively high memHSP70+ expression. Substitution of TPP in Pt(iv) peptide conjugates with scrambled peptide (ScP) essentially abolishes the observed cytotoxicity

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activitie

Transcriptional cdk inhibitors cyc065 and thz1 induce apoptosis in glioma stem cells derived from recurrent gbm

Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment re-sistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin‐dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA‐GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non‐tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in glio-maspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non‐stem tumour cells resulted in sphere disruption. Collec-tively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation

Ni(II), Pd(II), and Pt(II) Complexes of the Hedgehog Pathway Inhibitor GANT61-D

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Partial Cleavage of RasGAP by Caspases Is Required for Cell Survival in Mild Stress Conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions

System-based approaches as prognostic tools for glioblastoma

Background: The evasion of apoptosis is a hallmark of cancer. Understanding this process holistically and overcoming apoptosis resistance is a goal of many research teams in order to develop better treatment options for cancer patients. Efforts are also ongoing to personalize the treatment of patients. Strategies to confirm the therapeutic efficacy of current treatments or indeed to identify potential novel additional options would be extremely beneficial to both clinicians and patients. In the past few years, system medicine approaches have been developed that model the biochemical pathways of apoptosis. These systems tools incorporate and analyse the complex biological networks involved. For their successful integration into clinical practice, it is mandatory to integrate systems approaches with routine clinical and histopathological practice to deliver personalized care for patients. Results: We review here the development of system medicine approaches that model apoptosis for the treatment of cancer with a specific emphasis on the aggressive brain cancer, glioblastoma. Conclusions: We discuss the current understanding in the field and present new approaches that highlight the potential of system medicine approaches to influence how glioblastoma is diagnosed and treated in the future