Get Healthy after Breast Cancer - examining the feasibility, acceptability and outcomes of referring breast cancer survivors to a general population telephone-delivered program targeting physical activity, healthy diet and weight loss.

PURPOSE: This pilot study assessed the feasibility, acceptability and outcomes of referring breast cancer survivors to the 'Get Healthy Service' (GHS), a state health-funded 6-month telephone-delivered lifestyle program. METHODS: Pre-post study with eligible and consenting women following treatment for stages I-III breast cancer referred by nurses in a cancer treatment centre to the GHS. Feasibility was assessed via GHS uptake and completion; acceptability was assessed via patient satisfaction and nurse feedback. Changes in weight, physical activity, diet, quality of life (QoL) and fatigue from baseline to 6 months were examined. RESULTS: Fifty-three women (mean ± SD body mass index, 31.0 ± 5.5 kg/m2; age, 57.3 ± 10.0 years; 14.0 ± 7.1 months post-diagnosis; 43.4% born outside Australia, 49% high school or less education, 32.1% English as a second language) took up the GHS, with 62% completing the program. Almost all (92%) completers had high satisfaction ratings and breast nurses provided positive feedback. Findings from GHS completers (n = 33) show a statistically significant effect from baseline to 6 months for weight loss (mean ± SE; -2.4 ± 0.7 kg; p = 0.002) and total physical activity minutes per week (55 ± 18 min/week; p = 0.006). No significant changes in fruit or vegetable servings per day or takeaways and fast food frequency per week were observed. A significant improvement in mental QoL was observed (3.5 ± 1.6; p = 0.041), but not for physical QoL or fatigue. CONCLUSION: GHS referral appeared feasible, acceptable and effective for a diverse group of women following completion of treatment for breast cancer, yet more remains to be done to fully integrate GHS screening and referral into usual care.

In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

BACKGROUND: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. METHODS: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. RESULTS: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. CONCLUSIONS: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Effectiveness of lifestyle-based weight loss interventions for adults with type 2 diabetes: a systematic review and meta-analysis.

AIMS: To provide a systematic review and meta-analysis of recent evidence on the effectiveness of lifestyle-based weight loss interventions for adults with type 2 diabetes. METHODS: A search of the literature from January 2003 to July 2013 was conducted (PubMed, Embase, CINAHL and Web of Science). The studies considered eligible were randomized controlled trials evaluating weight loss interventions (diet and physical activity, with or without behavioural strategies) of ≥12 weeks duration, compared with usual care or another comparison intervention. Ten studies were included for review. Some heterogeneity was present in the sample, therefore, random-effects models were used to calculate pooled effects. RESULTS: Intervention duration ranged from 16 weeks to 9 years, with all but one delivered via individual or group face-to-face sessions. From six studies comparing lifestyle intervention with usual care the pooled effect on weight (n = 5795) was -3.33 kg [95% confidence interval (CI) -5.06, -1.60 kg], and on glycated haemoglobin (HbA1c; n = 5784) was -0.29% (95% CI -0.61, 0.03%), with both attenuated in sensitivity analyses. The pooled within-group effect on weight (n = 3063) from all 10 lifestyle intervention groups was -5.33 kg (95% CI -7.33, -3.34 kg), also attenuated in sensitivity analyses. None of the participant or intervention characteristics examined explained the heterogeneity. Only one study assessed whether intervention effects were maintained after the end of the intervention. CONCLUSIONS: Lifestyle-based weight loss intervention trials in type 2 diabetes achieve, on average, modest reductions in weight and HbA1c levels, but results were heavily influenced by one trial. Evidence-based approaches for improving the effectiveness of lifestyle-based interventions in type 2 diabetes are needed, along with future studies reporting on maintenance and cost-effectiveness.

Methylation-specific multiplex ligation-dependent probe amplification identifies promoter methylation events associated with survival in glioblastoma.

DNA methylation plays an important role in cancer biology and methylation events are important prognostic and predictive markers in many tumor types. We have used methylation-specific multiplex ligation-dependent probe amplification to survey the methylation status of MGMT and 25 tumor suppressor genes in 73 glioblastoma cases. The data obtained was correlated with overall survival and response to treatment. The study revealed that methylation of promoter regions in TP73 (seven patients), THBS1 (eight patients) and PYCARD (nine patients) was associated with improved outcome, whereas GATA5 (21 patients) and WT1 (24 patients) promoter methylation were associated with poor outcome. In patients treated with temozolomide and radiation MGMT and PYCARD promoter methylation events remained associated with improved survival whereas GATA5 was associated with a poor outcome. The identification of GATA5 promoter methylation in glioblastoma has not previously been reported. Furthermore, a cumulative methylation score separated patients into survival groups better than any single methylation event. In conclusion, we have identified specific methylation events associated with patient outcome and treatment response in glioblastoma, and these may be of functional and predictive/prognostic significance. This study therefore provides novel candidates and approaches for future prospective validation.

Comparative effectiveness of antinociceptive gene therapies in animal models of diabetic neuropathic pain.

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15 months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33 days for Pt(IV)-M13 vs 24 days for Pt(IV) prodrug, 22.5 days for cisplatin and 22 days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Determination of type I receptor specificity by the type II receptors for TGF-beta or activin.

Transforming growth factor-beta (TGF-beta) and activin signal primarily through interaction with type I and type II receptors, which are transmembrane serine-threonine kinases. Tsk 7L is a type I receptor for TGF-beta and requires coexpression of the type II TGF-beta receptor for ligand binding. Tsk 7L also specifically bound activin, when coexpressed with the type IIA activin receptor. Tsk 7L could associate with either type II receptor and the ligand binding specificity of Tsk 7L was conferred by the type II receptor. Tsk 7L can therefore act as type I receptor for both activin and TGF-beta, and possibly other ligands.

Cloning of a type I TGF-beta receptor and its effect on TGF-beta binding to the type II receptor.

Transforming growth factor-beta (TGF-beta) affects cellular proliferation, differentiation, and interaction with the extracellular matrix primarily through interaction with the type I and type II TGF-beta receptors. The type II receptors for TGF-beta and activin contain putative serine-threonine kinase domains. A murine serine-threonine kinase receptor, Tsk 7L, was cloned that shared a conserved extracellular domain with the type II TGF-beta receptor. Overexpression of Tsk 7L alone did not increase cell surface binding of TGF-beta, but coexpression with the type II TGF-beta receptor caused TGF-beta to bind to Tsk 7L, which had the size of the type I TGF-beta receptor. Overexpression of Tsk 7L inhibited binding of TGF-beta to the type II receptor in a dominant negative fashion. Combinatorial interactions and stoichiometric ratios between the type I and II receptors may therefore determine the extent of TGF-beta binding and the resulting biological activities.

Microtubule dynamics: if you need a shrink try stathmin/Op18.

Recent studies show that stathmin/Op18 may be an important physiological regulator of microtubule dynamics; the activity of stathmin/Op18 is controlled by the actions of several signalling pathways, allowing it to play a central role in coordinating microtubule behaviour.

Regulation of actin dynamics: The LIM kinase connection.

A signalling pathway has recently been delineated that connects Rho-family GTPases to the cytoskeleton via LIM kinase and the F-actin depolymerising protein cofilin. The existence of this pathway helps to explain some of the effects of LIM kinase and cofilin in the control of actin dynamics.

Synergistic activation of SAPK1/JNK1 by two MAP kinase kinases in vitro.

Mitogen-activated protein kinases (MAPKs) mediate many of the cellular effects of growth factors, cytokines and stress stimuli. Their activation requires the phosphorylation of a threonine and a tyrosine residue located in a Thr-X-Tyr motif (where X is any amino acid) [1]. This phosphorylation is catalysed by MAPK kinases (MKKs), which are all thought to be 'dual specificity' enzymes that phosphorylate both the threonine and the tyrosine residue of the Thr-X-Tyr motif [2]. Here, we report that the MAPK family member known as stress-activated protein kinase-1c (SAPK1c, also known as JNK1) [3] is activated synergistically in vitro by MKK4 ([4] [5] [6]; also called SKK1 and JNKK1) and MKK7 ([7] [8] [9]; also called SKK4 and JNKK2). We found that MKK4 had a preference for the tyrosine residue, and MKK7 for the threonine residue, within the Thr-X-Tyr motif. These observations suggest that the full activation of SAPK1c in vivo may sometimes require phosphorylation by two different MKKs, providing the potential for integrating the effects of different extracellular signals. They also raise the possibility that other MAPK family members may be activated by two or more MKKs and that some MKKs may have gone undetected because they phosphorylate the tyrosine residue only, and therefore do not induce any activation unless the threonine has first been phosphorylated by another MKK.

Genetic strategies for brain tumor therapy.

Gene therapy is a potentially useful approach in the treatment of human brain tumors, which are notoriously refractory to conventional approaches. Most human clinical trials to date have been unsuccessful in terms of improving patient outcome. Recent improvements in viral vectors, the development of stem cell technology, and increased understanding of the mechanism of action of therapeutic transgenes provide hope that the next generation of gene therapeutics may show increased efficacy in treatment of this devastating disease.

Significance of chromosome abnormalities in leukemia.

The review deals with technical considerations in relation to culturing and studying the chromosomes of leukemic cells. Specific and non-random chromosomal changes are described by the historic approach. Then follows a description of the application of chromosomal studies to diagnosis, progress and followup in the chronic and acute leukemias. A section is devoted to the application of chromosomal studies in bone-marrow transplantation. Finally, lines of research for leukemia using a genetic approach are suggested.

SKK4, a novel activator of stress-activated protein kinase-1 (SAPK1/JNK).

A cDNA was cloned and expressed that encodes human stress-activated protein kinase kinase-4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues. SKK4 activated SAPK1/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38beta, SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPK1/JNK in Drosophila. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPK1/JNK) were both activated by stressful stimuli, but only SKK4 was activated by proinflammatory cytokines. The identification of SKK4 explains why the major SAPK1/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.

Stathmin overexpression in 293 cells affects signal transduction and cell growth.

Stathmin is a ubiquitous cytoplasmic protein whose phosphorylation state changes markedly in response to extracellular signals, and during the cell cycle. To clarify the function of stathmin, its four phosphorylation sites were mutated to either alanines (4A-stathmin) or glutamates (4E-stathmin). In transfected cells, 4A-stathmin caused a strong G2/M block and also inhibited the responsiveness of a co-transfected fos promoter/ luciferase reporter plasmid to serum stimulation, whereas wild type and 4E-stathmin had relatively minor effects. These results support the idea that stathmin plays a role in multiple cellular processes and indicate that the regulation of the phosphorylation state of stathmin is likely to determine its action.

Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases.

The paired helical filament, which comprises the major fibrous element of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated microtubule-associated protein tau. Many of the hyperphosphorylated sites in tau are serine/threonine-prolines. Here we show that the stress-activated protein (SAP) kinases SAPK1gamma (also called JNK1), SAPK2a (also called p38, RK, CSBPs, Mpk2 and Mxi2), SAPK2b (also called p38beta), SAPK3 (also called ERK6 and p38gamma) and SAPK4 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Based on initial rates of phosphorylation, tau was found to be a good substrate for SAPK4 and SAPK3, a reasonable substrate for SAPK2b and a relatively poor substrate for SAPK2a and SAPK1gamma. Phosphorylation of tau by SAPK3 and SAPK4 resulted in a marked reduction in its ability to promote microtubule assembly. These findings double the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.

Lymphocytotoxic antibodies in leukemic patients treated with bone marrow transplantation.

Lymphocytotoxic antibodies were studied sequentially in a series of 42 patients with leukemia who received a bone marrow graft. Of these patients, 38% had cytotoxic antibodies before bone marrow transplantation (BMT). After BMT the antibody status changed with time, but 62% of the patients had antibodies at some time after BMT. During the first 10 weeks after BMT, 40% of the patients had antibodies. Thereafter the frequency rose to 50% and remained at that level beyond one year after BMT. In successful grafts the gamma globulins are of donor origin six months after BMT; thus donor B cells are capable of forming lymphocytotoxic antibodies even when the immune system is suppressed by cyclosporine. The antibodies had recognizable HLA specificity in about half the cases before and after BMT. When donor and patient were HLA-identical, HLA specificity did not correspond to donor/recipient antigens. In two cases in which the donor was matched for only one haplotype, antibodies formed by recipient cells, active against donor HLA antigens, were found.