Investigation of β-carotene–gelatin composite particles with a multiwavelength UV/vis detector for the analytical ultracentrifuge

A multiwavelength UV/vis detector for the analytical ultracentrifuge (MWL-AUC) has been developed recently. In this work, β-carotene–gelatin composite particles are investigated with MWL-AUC. Band centrifugation with a Vinograd cell is used to ensure maximum sample separation. Spectral changes of the system are observed in dependence of the sedimentation coefficient and are attributed to a previously unknown inhomogeneity of the β-carotene chemical composition with both H- and J-aggregates coexisting in a mixture. In addition, our data suggest that pure H- and J-aggregates exist in a particle while their relative concentrations in a mixture determine the color characteristics of the sample. The unique abilities and properties of MWL-AUC include sedimentation coefficient distributions for all possible wavelengths, full UV/vis spectra of each different species in the mixture and 3D movies of the sedimentation process. These properties significantly extend the scope of the analytical ultracentrifuge technique and show that complex biopolymer multicomponent mixtures can be resolved into their individual species

The novel mTOR inhibitor RAD001 (Everolimus) induces antiproliferative effects in human pancreatic neuroendocrine tumor cells

Background/Aim: Tumors exhibiting constitutively activated PI(3) K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3) K/Akt/mTOR signaling. Methods: BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. Results: RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. Conclusion: In summary, our data demonstrate antiproliferative and apoptotic effects of RAD001 in NET cells in vitro supporting its clinical use in current phase II trials in NET patients. Copyright (c) 2007 S. Karger AG, Basel

Performance of a fast fiber based UV/Vis multiwavelength detector for the analytical ultracentrifuge

The optical setup and the performance of a prototype UV/Vis multiwavelength analytical ultracentrifuge (MWL-AUC) is described and compared to the commercially available Optima XL-A from Beckman Coulter. Slight modifications have been made to the optical path of the MWL-AUC. With respect to wavelength accuracy and radial resolution, the new MWL-AUC is found to be comparable to the existing XL-A. Absorbance accuracy is dependent on the light intensity available at the detection wavelength as well as the intrinsic noise of the data. Measurements from single flashes of light are more noisy for the MWL-AUC, potentially due to the absence of flash-to-flash normalization in the current design. However, the possibility of both wavelength and scan averaging can compensate for this and still give much faster scan rates than the XL-A. Some further improvements of the existing design are suggested based on these findings

Altering Chemosensitivity by Modulating Translation Elongation

BACKGROUND: The process of translation occurs at a nexus point downstream of a number of signal pathways and developmental processes. Modeling activation of the PTEN/AKT/mTOR pathway in the Emu-Myc mouse is a valuable tool to study tumor genotype/chemosensitivity relationships in vivo. In this model, blocking translation initiation with silvestrol, an inhibitor of the ribosome recruitment step has been showed to modulate the sensitivity of the tumors to the effect of standard chemotherapy. However, inhibitors of translation elongation have been tested as potential anti-cancer therapeutic agents in vitro, but have not been extensively tested in genetically well-defined mouse tumor models or for potential synergy with standard of care agents. METHODOLOGY/PRINCIPAL FINDINGS: Here, we chose four structurally different chemical inhibitors of translation elongation: homoharringtonine, bruceantin, didemnin B and cycloheximide, and tested their ability to alter the chemoresistance of Emu-myc lymphomas harbouring lesions in Pten, Tsc2, Bcl-2, or eIF4E. We show that in some genetic settings, translation elongation inhibitors are able to synergize with doxorubicin by reinstating an apoptotic program in tumor cells. We attribute this effect to a reduction in levels of pro-oncogenic or pro-survival proteins having short half-lives, like Mcl-1, cyclin D1 or c-Myc. Using lymphomas cells grown ex vivo we reproduced the synergy observed in mice between chemotherapy and elongation inhibition and show that this is reversed by blocking protein degradation with a proteasome inhibitor. CONCLUSION/SIGNIFICANCE: Our results indicate that depleting short-lived pro-survival factors by inhibiting their synthesis could achieve a therapeutic response in tumors harboring PTEN/AKT/mTOR pathway mutations

GRP78 Knockdown Enhances Apoptosis via the Down-Regulation of Oxidative Stress and Akt Pathway after Epirubicin Treatment in Colon Cancer DLD-1 Cells

INTRODUCTION: The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway. CONCLUSIONS: Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells

NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines

HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27 kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3α/β (GSK-3α/β) as a substrate. To explore the role of Akt signalling in NFV-mediated growth inhibition of NSCLC cells, we blocked this signal pathway by transfection of Akt small interfering RNA (siRNA) in these cells; transient transfection of Akt siRNA in NCI-H460 cells decreased the level of Bcl-2 protein and slowed their proliferation compared to the nonspecific siRNA-transfected cells. Conversely, forced-expression of Akt partially reversed NFV-mediated growth inhibition of these cells, suggesting that Akt may be a molecular target of NFV in NSCLC cells. Also, we found that inhibition of Akt signalling by NFV enhanced the ability of docetaxel to inhibit the growth of NCI-H460 and -H520 cells, as measured by MTT assay. Importantly, NFV slowed the proliferation and induced apoptosis of NCI-H460 cells present as tumour xenografts in nude mice without adverse systemic effects. Taken together, this family of compounds might be useful for the treatment of individuals with NSCLC

Phone and e-mail counselling are effective for weight management in an overweight working population: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>The work setting provides an opportunity to introduce overweight (i.e., Body Mass Index ≥ 25 kg/m²) adults to a weight management programme, but new approaches are needed in this setting. The main purpose of this study was to investigate the effectiveness of lifestyle counselling by phone or e-mail on body weight, in an overweight working population. Secondary purposes were to establish effects on waist circumference and lifestyle behaviours, and to assess which communication method is the most effective.</p> <p>Methods</p> <p>A randomized controlled trial with three treatments: intervention materials with phone counselling (phone group); a web-based intervention with e-mail counselling (internet group); and usual care, i.e. lifestyle brochures (control group). The interventions used lifestyle modification and lasted a maximum of six months. Subjects were 1386 employees, recruited from seven companies (67% male; mean age 43 (SD 8.6) y; mean BMI 29.6 (SD 3.5) kg/m²). Body weight was measured by research personnel and by questionnaire. Secondary outcomes fat, fruit and vegetable intake, physical activity and waist circumference were assessed by questionnaire. Measurements were done at baseline and after six months. Missing body weight was multiply imputed.</p> <p>Results</p> <p>Body weight reduced 1.5 kg (95% CI -2.2;-0.8, p < 0.001) in the phone group and 0.6 kg (95% CI -1.3; -0.01, p = 0.045) in the internet group, compared with controls. In completers analyses, weight and waist circumference in the phone group were reduced with 1.6 kg (95% CI -2.2;-1.0, p < 0.001) and 1.9 cm (95% CI -2.7;-1.0, p < 0.001) respectively, fat intake decreased with 1 fatpoint (1 to 4 grams)/day (95% CI -1.7;-0.2, p = 0.01) and physical activity increased with 866 METminutes/week (95% CI 203;1530, p = 0.01), compared with controls. The internet intervention resulted in a weight loss of 1.1 kg (95% CI -1.7;-0.5, p < 0.001) and a reduction in waist circumference of 1.2 cm (95% CI -2.1;-0.4, p = 0.01), in comparison with usual care. The phone group appeared to have more and larger changes than the internet group, but comparisons revealed no significant differences.</p> <p>Conclusion</p> <p>Lifestyle counselling by phone and e-mail is effective for weight management in overweight employees and shows potential for use in the work setting.</p> <p>Trial registration</p> <p>ISCRTN04265725.</p

Expression and localisation of Akt-1, Akt-2 and Akt-3 correlate with clinical outcome of prostate cancer patients

We investigated the correlation between the expression and localisation of Akt-1, Akt-2, Akt-3, phospho-Akt proteins and the clinicopathological parameters in 63 prostate cancer specimens. More than 60% of cancerous tissues overexpressed Akt-1, Akt-2 or Akt-3. Cytoplasmic Akt-1 expression was correlated with a higher risk of postoperative prostate-specific antigen (PSA) recurrence and shorter PSA recurrence interval. Cytoplasmic Akt-2 did not show any significant correlation with clinicopathological parameters predicting outcomes. Cytoplasmic Akt-3 was associated with hormone-refractory disease progression and extracapsular invasion. Nuclear Akt-1 and Akt-2 expression were correlated with favourable outcome parameters such as absence of lymph node and perineural invasion. Kaplan–Meier analysis and Cox regression model also showed that Akt-1 and Akt-2, but not Akt-3 or phospho-Akt was associated with a significantly higher risk of PSA recurrence. In contrast, nuclear Akt-1 was significantly associated with a lower risk of PSA recurrence. Multivariate analysis revealed that clinical stage, Gleason score and the combined cytoplasmic nuclear Akt-1 marker in cancerous tissues were significant independent prognostic factors of PSA recurrence. This is the first report demonstrating in patients with prostate cancer and the particular role of Akt-1 isoform expression as a prognostic marker depending of its localisation

mTOR signaling: implications for cancer and anticancer therapy

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option