Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation

<p>Abstract</p> <p>Background</p> <p>Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis.</p> <p>Methods</p> <p>cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation.</p> <p>Results</p> <p>PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression.</p> <p>Conclusion</p> <p>Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.</p

Augmented intracellular glutathione inhibits fas-triggered apoptosis of activated human neutrophils

Q1Q1Agonist signals delivered through cell surface Fas induce apoptosis. However, the apoptotic program can be modulated by signals from the environment, and in particular, by signals delivered through adhesion molecules. Because neutrophil functional activity in inflammation is contingent on cell survival, and because circulating neutrophils normally die rapidly through a constitutively expressed apoptotic program, we evaluated Fas-mediated apoptosis in resting and inflammatory human neutrophils. We show that normal neutrophils respond to Fas engagement with accelerated rates of apoptosis, but cross-linking of β2 integrins or priming with bacterial lipopolysaccharide (LPS) prevents this increase. Adhesion molecule cross-linking results in increased intracellular glutathione (GSH). Augmentation of intracellular GSH with exogenous GSH or N-acetylcysteine is sufficient to reduce the Fas-triggered increase in apoptotic rates. Prevention of the activation induced GSH increase by buthionine sulfoximine, a cell permeable inhibitor of GSH biosynthesis, restored Fas responsiveness in activated neutrophils, an effect that could be blocked with exogenous GSH. Taken together, these data show that Fas-induced signaling for neutrophil apoptosis is blocked in a redox sensitive manner by costimulatory signals delivered through β2 integrins or activation by LPS, and provide a biologic explanation for sustained neutrophil survival in the inflammatory environment.Revista Nacional - Indexad

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

Considerations for design of source apportionment studies

This report recommends procedures for source and ambient sampling and analysis in source apportionment studies. The recommendations are based on the results of receptor model studies of atmospheric particles in urban areas, especially a recent study of Houston, TX, undertaken as part of the Mathematical and Empirical Receptor Models Workshop (Quail Roost II). The recommendations are presented at three levels of increasing cost and detail of information obtained. Existing mass emissions inventories combined with chemically resolved test data from similar sources (not necessarily in the same locale) can be used to initially estimate the sources of elements present on ambient particles. To aid local users in construction of chemically resolved emission estimates, the U.S. Environmental Protection Agency (EPA) is compiling a library of compositions and size distributions of particulate emissions from major source types. More reliable source characterization can be achieved if the actual sources are tested directly. EPA should develop and publish detailed procedures for source sampling that would be more appropriate for receptor model use than are existing standard methods. Source and ambient sampling should be conducted by similar methods. If possible, particles from sources should be collected in a way that simulates changes that would normally occur before they reach distant receptors (e.g. by diluting and cooling the particles from hot sources). It is recommended that particulate samples be routinely collected in two size fractions by use of virtual impactors and that all samples be subjected, at a minimum, to mass and X-ray fluorescence analyses. Additional measurements are suggested for obtaining more detailed information: neutron activation analysis; X-ray diffraction; automated particle classification by electron microscopy; analyses for classes of organic species, ^(14)C and thermally released carbonaceous species; and real-time observation of several gases during sample collection. Methods for collecting meteorological data in parallel with ambient samples are described, as are methods for incorporating such data into the source identification process

Spitzer observations of HH54 and HH7-11: mapping the H2 ortho-to-para ratio in shocked molecular gas

We report the results of spectroscopic mapping observations carried out toward the Herbig-Haro objects HH7-11 and HH54 over the 5.2 - 37 micron region using the Infrared Spectrograph of the Spitzer Space Telescope. These observations have led to the detection and mapping of the S(0) - S(7) pure rotational lines of molecular hydrogen, together with emissions in fine structure transitions of Ne+, Si+, S, and Fe+. The H2 rotational emissions indicate the presence of warm gas with a mixture of temperatures in the range 400 - 1200 K, consistent with the expected temperature behind nondissociative shocks of velocity ~ 10 - 20 km/s, while the fine structure emissions originate in faster shocks of velocity 35 - 90 km/s that are dissociative and ionizing. Maps of the H2 line ratios reveal little spatial variation in the typical admixture of gas temperatures in the mapped regions, but show that the H2 ortho-to-para ratio is quite variable, typically falling substantially below the equilibrium value of 3 attained at the measured gas temperatures. The non-equilibrium ortho-to-para ratios are characteristic of temperatures as low as ~ 50 K, and are a remnant of an earlier epoch, before the gas temperature was elevated by the passage of a shock. Correlations between the gas temperature and H2 ortho-to-para ratio show that ortho-to-para ratios < 0.8 are attained only at gas temperatures below ~ 900 K; this behavior is consistent with theoretical models in which the conversion of para- to ortho-H2 behind the shock is driven by reactive collisions with atomic hydrogen, a process which possesses a substantial activation energy barrier (E_A/k ~ 4000 K) and is therefore very inefficient at low temperature.Comment: 45 pages, including 16 figures. Accepted for publication in Ap

Possible Uses of Animal Databases for Further Statistical Evaluation and Modeling

Many studies have been performed in animals which mimic potential exposures of people in order to understand how factors modify radiation dose-response relationships. Cooperative analyses by investigators in different laboratories have a large potential for strengthening the conclusions that can be drawn from individual studies. When information on each animal is combined, then formal tests can be made to demonstrate that apparent consistencies or inconsistencies are statistically significant. Statistical methods must be carefully chosen so that differences between laboratories or studies can be controlled or described as part of the analysis in the interpretation of the conclusions. In this report, the example of bone cancer of the large number of studies of modifying factors for bone cancer available from studies in US and European laboratories

Reverse Shock Emission Revealed in Early Photometry in the Candidate Short GRB 180418A

We present observations of the possible short GRB 180418A in $\gamma$-rays, X-rays, and in the optical. Early optical photometry with the TAROT and RATIR instruments show a bright peak ($\approx$ 14.2 AB mag) between $T+28$ and $T+90$ seconds that we interpret as the signature of a reversal shock. Later observations can be modeled by a standard forward shock model and show no evidence of jet break, allowing us to constrain the jet collimation to $\theta_j> 7^\circ$. Using deep late-time optical observations we place an upper limit of $r>24$ AB mag on any underlying host galaxy. The detection of the afterglow in the \textit{Swift} UV filters constrains the GRB redshift to $z<1.3$ and places an upper bound on the $\gamma$-ray isotropic equivalent energy $E_{\rm{\gamma,iso}} < 3 \times 10^{51}$ erg. The properties of this GRB (e.g. duration, hardness ratio, energetic, and environment) lie at the intersection between short and long bursts, and we can not conclusively identify its type. We estimate that the probability that it is drawn from the population of short GRBs is 10\%-30\%.Comment: Accepted por publication in Ap

Feature Fusion of Raman Chemical Imaging and Digital Histopathology using Machine Learning for Prostate Cancer Detection

The diagnosis of prostate cancer is challenging due to the heterogeneity of its presentations, leading to the over diagnosis and treatment of non-clinically important disease. Accurate diagnosis can directly benefit a patient's quality of life and prognosis. Towards addressing this issue, we present a learning model for the automatic identification of prostate cancer. While many prostate cancer studies have adopted Raman spectroscopy approaches, none have utilised the combination of Raman Chemical Imaging (RCI) and other imaging modalities. This study uses multimodal images formed from stained Digital Histopathology (DP) and unstained RCI. The approach was developed and tested on a set of 178 clinical samples from 32 patients, containing a range of non-cancerous, Gleason grade 3 (G3) and grade 4 (G4) tissue microarray samples. For each histological sample, there is a pathologist labelled DP - RCI image pair. The hypothesis tested was whether multimodal image models can outperform single modality baseline models in terms of diagnostic accuracy. Binary non-cancer/cancer models and the more challenging G3/G4 differentiation were investigated. Regarding G3/G4 classification, the multimodal approach achieved a sensitivity of 73.8% and specificity of 88.1% while the baseline DP model showed a sensitivity and specificity of 54.1% and 84.7% respectively. The multimodal approach demonstrated a statistically significant 12.7% AUC advantage over the baseline with a value of 85.8% compared to 73.1%, also outperforming models based solely on RCI and median Raman spectra. Feature fusion of DP and RCI does not improve the more trivial task of tumour identification but does deliver an observed advantage in G3/G4 discrimination. Building on these promising findings, future work could include the acquisition of larger datasets for enhanced model generalization.Comment: 19 pages, 8 tables, 18 figure