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 mean 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.

Investigating the role of the IGF axis as a predictor of biochemical recurrence in prostate cancer patients post-surgery.

BACKGROUND: Between 20% and 35% of prostate cancer (PCa) patients who undergo treatment with curative intent (ie, surgery or radiation therapy) for localized disease will experience biochemical recurrence (BCR). Alterations in the insulin-like growth factor (IGF) axis and PTEN expression have been implicated in the development and progression of several human tumors including PCa. We examined the expression of the insulin receptor (INSR), IGF-1 receptor (IGF-1R), PTEN, and AKT in radical prostatectomy tissue of patients who developed BCR post-surgery. METHODS: Tissue microarrays (TMA) of 130 patients post-radical prostatectomy (65 = BCR, 65 = non-BCR) were stained by immunohistochemistry for INSR, IGF-1R, PTEN, and AKT using optimized antibody protocols. INSR, IGF1-R, PTEN, and AKT expression between benign and cancerous tissue, and different Gleason grades was assessed. Kaplan-Meier survival curves were used to examine the relationship between proteins expression and BCR. RESULTS: INSR (P < 0.001), IGF-1R (P < 0.001), and AKT (P < 0.05) expression was significantly increased and PTEN (P < 0.001) was significantly decreased in cancerous versus benign tissue. There was no significant difference in INSR, IGF-1R, or AKT expression in the cancerous tissue of non-BCR versus BCR patients (P = 0.149, P = 0.990, P = 0.399, respectively). There was a significant decrease in PTEN expression in the malignant tissue of BCR versus non-BCR patients (P = 0.011). Combinational analysis of the tissue proteins identified a combination of decreased PTEN and increased AKT or increased INSR was associated with worst outcome. We found that in each case, our hypothesized worst group was most likely to experience BCR and this was significant for combinations of PTEN+INSR and PTEN+AKT but not PTEN+IGF-1R (P = 0.023, P = 0.028, P = 0.078, respectively). CONCLUSIONS: Low PTEN is associated with BCR and this association is strongly modified by high INSR and high AKT expression. Measurement of these proteins could help inform appropriate patient selection for postoperative adjuvant therapy and prevent BCR.

Serial cytokine alterations and abnormal neuroimaging in newborn infants with encephalopathy.

AIM: Inflammatory cytokines may play a role in the final common pathway in the pathogenesis of hypoxic-ischaemic injury in experimental models. We aimed to profile the systemic pro-and anti-inflammatory response over the first week of life in term infants at risk of neonatal encephalopathy. METHOD: In a tertiary referral university neonatal intensive care unit, serial blood samples were analysed from 41 term infants (requiring resuscitation at birth) in this prospective observational pilot study. Serum levels of 10 pro-and anti-inflammatory cytokines were evaluated including interleukin(IL)-1α, IL-1ß, IL-6, IL-8, IL-10, tumour necrosis factor(TNF)-α, interferon (IFN)-γ, vascular endothelial growth factor (VEGF), granulocyte/colony-stimulating factor (G-CSF) and granulocyte macrophage/colony-stimulating factor (GM-CSF). RESULTS: Infants with neonatal encephalopathy and abnormal neuroimaging (n = 15) had significantly elevated granulocyte macrophage/colony-stimulating factor at 0-24 h and interleukin-8, interleukin-6 and interleukin-10 at 24-48 hour. Tumour necrosis factor-α and vascular endothelial growth factor levels were lower at 72-96 hour (p < 0.05). Significantly elevated levels of interleukin-10 were associated with mortality. CONCLUSION: Serum cytokine changes and innate immune dysregulation in the first week of life may be indicators of outcome in neonatal encephalopathy but require validation in larger studies.

Improving multivariable prostate cancer risk assessment using the Prostate Health Index.

OBJECTIVES: To analyse the clinical utility of a prediction model incorporating both clinical information and a novel biomarker, p2PSA, in order to inform the decision for prostate biopsy in an Irish cohort of men referred for prostate cancer assessment. PATIENTS AND METHODS: Serum isolated from 250 men from three tertiary referral centres with pre-biopsy blood draws was analysed for total prostate-specific antigen (PSA), free PSA (fPSA) and p2PSA. From this, the Prostate Health Index (PHI) score was calculated (PHI = (p2PSA/fPSA)*√tPSA). The men's clinical information was used to derive their risk according to the Prostate Cancer Prevention Trial (PCPT) risk model. Two clinical prediction models were created via multivariable regression consisting of age, family history, abnormality on digital rectal examination, previous negative biopsy and either PSA or PHI score, respectively. Calibration plots, receiver-operating characteristic (ROC) curves and decision curves were generated to assess the performance of the three models. RESULTS: The PSA model and PHI model were both well calibrated in this cohort, with the PHI model showing the best correlation between predicted probabilities and actual outcome. The areas under the ROC curve for the PHI model, PSA model and PCPT model were 0.77, 0.71 and 0.69, respectively, for the prediction of prostate cancer (PCa) and 0.79, 0.72 and 0.72, respectively, for the prediction of high grade PCa. Decision-curve analysis showed a superior net benefit of the PHI model over both the PSA model and the PCPT risk model in the diagnosis of PCa and high grade PCa over the entire range of risk probabilities. CONCLUSION: A logical and standardized approach to the use of clinical risk factors can allow more accurate risk stratification of men under investigation for PCa. The measurement of p2PSA and the integration of this biomarker into a clinical prediction model can further increase the accuracy of risk stratification, helping to better inform the decision for prostate biopsy in a referral population.

The analysis of serum response factor expression in bone and soft tissue prostate cancer metastases.

BACKGROUND: Castration-resistant prostate cancer (CRPC) represents a challenge to treat with no effective treatment options available. We recently identified serum response factor (SRF) as a key transcription factor in an in vitro model of castration resistance where we showed that SRF inhibition resulted in reduced cellular proliferation. We also demonstrated an association between SRF protein expression and CRPC in a cohort of castrate-resistant transurethral resections of the prostate (TURPS). The mechanisms regulating the growth of CRPC bone and visceral metastases have not been explored in depth due to the paucity of patient-related material available for analysis. In this study, we aim to evaluate SRF protein expression in prostate cancer (PCa) metastases, which has not previously been reported. METHODS AND RESULTS: We evaluated the nuclear tissue expression profile of SRF by immunohistochemistry in 151 metastatic sites from 42 patients who died of advanced PCa. No relationship between SRF nuclear expression and the site of metastasis was observed (P = 0.824). However, a negative association between SRF nuclear expression in bone metastases and survival from (a) diagnosis with PCa (P = 0.005) and (b) diagnosis with CRPC (P = 0.029) was seen. These results demonstrate that SRF nuclear expression in bone metastases is associated with survival, with patients with the shortest survival showing high SRF nuclear expression and patients with the longest survival having low SRF nuclear expression. CONCLUSION: Our study indicates that SRF is a key factor determining patients' survival in metastatic CRPC and therefore may represent a promising target for future therapies.

EuroSCORE and neutrophil adhesion molecules predict outcome post-cardiac surgery.

BACKGROUND: This study tested the hypothesis that surgical stress and the host response to this trauma trigger an inflammatory cascade in which the neutrophil plays a central role. We hypothesised that pre-operative neutrophil migratory responses will correlate with post-operative clinical outcome in our shock model of open-heart surgery patients. We also tested the hypothesis that surface expression of adhesion molecules involved in the migratory process - CD11b, CD47 and CD99 - could be used to predict outcome. We believe that combining neutrophil migratory response, CD11b, CD47 and CD99 with the logistic European System for Cardiac Operative Risk Evaluation (EuroSCORE) will strengthen the power of the EuroSCORE not only in predicting post-operative mortality but also other clinical endpoints. MATERIALS AND METHODS: Neutrophils were isolated pre-operatively from n = 31 patients undergoing open-heart surgery and allowed to migrate across endothelial monolayers in response to N-formyl-methionine-leucine-phenylalanine (fMLP). Isolated neutrophils were also assessed for surface expression of CD11b, CD47 and CD99 in response to fMLP by flow cytometry. Post-operative clinical parameters collected included days 1-5 white cell count and creatinine levels as well as intensive care unit (ICU) and post-operative hospital stay. RESULTS: Pre-operative surface expression of CD99 and CD47 correlates with post-operative creatinine levels (P < 0·05), a measurement of renal injury. We also show that while the logistic EuroSCORE alone can be used as a predictor of ICU stay, when combined with pre-operative CD99 surface expression, it improves its AUC value (0·794). CONCLUSION: Immunological markers, specifically the ability of the neutrophil to migrate, combined with the logistic EuroSCORE lead to improved sensitivity and specificity to predict patient outcome.

Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in neonatal sepsis.

BACKGROUND: Efficient and accurate diagnosis of neonatal sepsis is challenging. The potential impact for a reduction in morbidity and mortality as well as antibiotic usage has stimulated the ongoing search for biomarkers of early sepsis. The objective of this pilot study was to quantify the levels of sTREM-1 and correlate with blood cultures and inflammatory markers in neonates evaluated for sepsis. METHODS: Neonates with suspected sepsis were enrolled (n = 83; Preterm n = 35; Term n = 48). Routine bloods for sepsis evaluation were included and plasma sTREM-1 levels were quantified by ELISA. RESULTS: Term and preterm neonates (n = 83; Preterm n = 35; Term n = 48) were enrolled and 16 neonates had positive blood cultures (preterm n = 15; term n = 1). sTREM-1 levels were not significantly different in infants with culture-positive or culture-negative sepsis (356 ± 218 pg/mL and 385 ± 254 pg/mL respectively). The immature-to-total granulocyte (I/T) ratio showed a significant positive correlation with sTREM-1 in the preterm group with positive blood cultures. Additionally, sTREM-1 showed a positive correlation with CRP in the preterm group with negative blood cultures. CONCLUSIONS: sTREM-1 was associated with traditional markers of inflammation (I/T ratio and CRP). However, in this cohort sTREM-1 did not improve the early detection of neonatal culture-positive sepsis.

Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation.

Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.

Applying random forests to identify biomarker panels in serum 2D-DIGE data for the detection and staging of prostate cancer.

In recent years, Prostate Specific Antigen (PSA) testing is widespread and has been associated with deceased mortality rates; however, this testing has raised concerns of overdiagnosis and overtreatment. It is clear that additional biomarkers are required. To identify these biomarkers, we have undertaken proteomics and metabolomics expression profiles of serum samples from BPH, Gleason score 5 and 7 using two-dimensional difference in gel electrophoresis (2D-DIGE) and nuclear magnetic resonance spectroscopy (NMR). Panels of serum protein biomarkers were identified by applying Random Forests to the 2D-DIGE data. The evaluation of selected biomarker panels has shown that they can provide higher prediction accuracy than the current diagnostic standard. With careful validation of these serum biomarker panels, these panels may potentially help to reduce unnecessary invasive diagnostic procedures and more accurately direct the urologist to curative surgery.

Core fucosylation and alpha2-3 sialylation in serum N-glycome is significantly increased in prostate cancer comparing to benign prostate hyperplasia.

One of the most urgent requirements in prostate cancer diagnosis is the development of a blood-based test which would be able to distinguish prostate cancer from benign prostate hyperplasia (BPH). Previously published results found a significant difference between specific glycan levels in patients with advanced prostate cancer and healthy controls. N-Glycans from the whole serum glycoproteins were measured using our fully quantitative high-throughput N-glycan analysis in combination with exoglycosidase digestions in sera from 13 BPH and 34 prostate cancer samples (17 Gleason score 5 and 17 Gleason score 7). The levels of core-fucosylated biantennary glycans and α2-3-linked sialic acids were significantly increased in prostate cancer patients compared with patients with BPH. Triantennary trigalactosylated glycans and tetraantennary tetrasialylated glycans with outer arm fucose were significantly decreased, and tetraantennary tetrasialylated glycans increased in Gleason 7 compared with Gleason 5. All these glycans can distinguish prostate cancer patients from BPH or Gleason 7 from Gleason 5 prostate cancer patients better than the current clinical test, prostate-specific antigen; therefore, their measurement may provide a new noninvasive approach to diagnose prostate cancer. However, additional validation studies would need to be carried out to further support this finding. Decreases in triantennary trigalactosylated glycans and/or bisected core-fucosylated biantennary monosialylated glycans and increases in tetraantennary tetrasialylated glycans correlate with perineural invasion, which could further help to diagnose tumor spread and predict patients' survival.

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines.

BACKGROUND: There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. RESULTS: The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. CONCLUSION: This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Generation of an epigenetic signature by chronic hypoxia in prostate cells.

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Effects of titanocene dichloride derivatives on prostate cancer cells, specifically DNA damage-induced apoptosis.

BACKGROUND: While locally advanced prostate cancer is initially treatable with androgen ablation, eventually cells develop a castrate-resistant phenotype. Currently, there are no effective treatments for this form of the disease with Docetaxel only providing a small survival advantage. In this study, the effects of novel derivatives of titanocene dichloride on prostate cancer cell lines has been investigated. METHODS: Cellular effects were assessed using the crystal violet assay and the clonogenic survival assay. Cell cycle and apoptosis were assessed by propidium iodide staining. DNA damage was analyzed by comet assay and Western analysis. DNA damage response inhibition was achieved by pre-incubation with an ATM/ATR inhibitor; CGK733 and DNA-PK inhibitor; DMNB. RESULTS: These analogs caused a reduction in cell number. In particular titanocene Y and C had significant effects in all cell lines. A reduction in clonogenic survival was found in response to titanocene Y in three cell lines while the PC-3 cells exhibited increased resistance.Further analysis showed no effect on cell cycle however, the analogs were found to induce apoptosis in a dose-dependent manner in all cell lines. These analogs associate with DNA, induce DNA damage and a differential damage response. Inhibition of key regulators of this DNA damage response sensitized the PC-3 cell line to titanocene-induced apoptosis and significantly reduced the clonogenic capacity of the cells. CONCLUSION: These results demonstrate the mechanism of action of these novel titanocene dichloride analogs and their potential use in castrate-independent advanced prostate cancer.

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.

digeR: a graphical user interface R package for analyzing 2D-DIGE data.

UNLABELLED: 2D Difference In-Gel Electrophoresis (2D-DIGE) or 2D gel technology is being used as a routine proteomics technique for biomarker discovery. Analyzing such high-dimensional data requires multivariate analysis techniques to be applied. In addition, protein post-translational modification (PTM) information from the 2D gel data is usually overlooked. We report on an R package, digeR, with an easy to use graphical user interface for analyzing 2D-DIGE (2D gel) data. It provides a tool for visually looking for potential PTM changes from different biological states and support biomarker discovery through multivariate analysis techniques. AVAILABILITY: digeR package is freely available from the CRAN: http://cran.r-project.org/web/packages/digeR/index.html.

Prostate cancer grading: the effect of stratification of needle biopsy Gleason Score 4 + 3 as high or intermediate grade.

OBJECTIVES: To assess the discrepancy between needle biopsy (NB) and radical prostatectomy (RP) Gleason score (GS) in Irish men, specifically the influence of the stratification of GS 4 + 3 on overall levels of agreement, levels of discrepancy and kappa coefficients, as the GS assigned to prostate cancer NBs affects clinical decision-making and influences future therapeutic strategies. PATIENTS AND METHODS: We reviewed retrospectively a database of the discrepancies between NB and RP Gleason grades (GG) from 2003 to 2008. All patients had clinically localized prostate cancer, and none had had neoadjuvant therapy. Grading of 206 NB specimens was compared with their corresponding RP specimens. The discrepancy rate between NB and RP GS was assessed for each combination of GG. Intermediate- (GS 7, defined as GS 3 + 4 alone vs GS 7) and high-grade (GS 4 + 3 and GS 8-10 vs GS 8-10) classifications were compared. The level of agreement and the kappa coefficient for each system was assessed. RESULTS: In NB, GS 6 was most frequently diagnosed (53%); after RP, GS 3 + 4 was most frequent (36%). In 42% of cases the exact GG remained unchanged after RP, increasing to 48% for GS 6 and GS 3 + 4. Overall 42% of cases showed an increase in their GG. In GS 6 NBs, the rate of increase in the primary GG or increase in the GS was 52%. Biopsy GS 6 and 3 + 4 showed the highest levels of agreement between NB and RP. Low-grade prostate cancer on NB was upgraded in 52% of cases; high-grade prostatic adenocarcinoma was downgraded in 27-77% of cases depending on the grading system used. CONCLUSIONS: Classification of high-grade prostate cancer as GS 4 + 3 and GS 8-10 results in higher levels of agreement between NB and RP GS. Reliable identification of well differentiated prostatic adenocarcinoma in NB specimens represents an ongoing diagnostic challenge, necessitating careful preoperative consideration of the definitive grade of a patient's disease.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Hypoxia increases normal prostate epithelial cell resistance to receptor-mediated apoptosis via AKT activation.

The aging prostate is associated with changes in its vascular structure, which could lead to changes in oxygen levels. Hypoxia is an important environmental change that leads to the progression of many cancers mediated through a number of cellular changes, which included resistance to apoptosis. The role of hypoxia in initiating tumour development has not been previously investigated. We demonstrate that normal prostate epithelial cells develop a resistance to receptor-mediated apoptosis following 24 hr of 1% hypoxia. This effect is associated with the altered expression of a number of pro- and anti-apoptotic proteins, which leads to inhibition of Cytochrome c release and downstream caspase activation. This is mediated via decreased Bax translocation and upstream Caspase 8 activity. Despite increased expression of cIAP-2, small interfering RNA (siRNA) knockdown does not restore susceptibility to TRAIL-induced apoptosis. Gene expression analysis indicated potential changes in AKT activation, which was confirmed by increased phosphorylation of AKT. Inhibition of this phosphorylation reversed the resistance to TRAIL-induced apoptosis. AKT activation is emerging as a key survival signal in prostate cancer. This study demonstrates that short exposure to low oxygen can increase resistance to immune surveillance mechanisms and might confer a survival advantage onto normal prostate epithelial cells so that they can survive subsequent genomic instability and other carcinogenetic insults leading to the early development of prostate cancer.

Increased E-cadherin expression in the ligated kidney following unilateral ureteric obstruction.

E-cadherin expression in the kidney is used as a surrogate marker of epithelial mesenchymal transition for the testing of various antifibrotic strategies. Here we reexamined E-cadherin expression in the kidneys of rats with unilateral ureteric obstruction, which was previously reported to decrease in parallel with the development of tubulointerstitial disease in this widely used experimental model of renal fibrosis and epithelial mesenchymal transition. E-cadherin mRNA expression was consistently increased both acutely (hours) and chronically (days) in the ligated kidney compared to the cognate non-ligated kidney. Increased E-cadherin protein levels were also found in the ligated kidney particularly in dilated tubular segments. Simulation of early pressure changes in the ligated kidney by mechanical stretch of human renal epithelial cells in culture did not alter E-cadherin expression. Porcine LLCPK-1 cells subjected to hypotonic stretch, however, did have increased E-cadherin mRNA and protein levels, responses that were not prevented by transforming growth factor-beta, a cytokine that promotes epithelial mesenchymal transition. Our findings question the utility of E-cadherin as a marker of epithelial mesenchymal transition in this model of renal fibrosis.

The single insult of hypoxic preconditioning induces an antiapoptotic response in human proximal tubular cells, in vitro, across cold storage.

OBJECTIVE: To examine whether hypoxia (one of the many components of ischaemic preconditioning) can induce a protective response in culture renal tubular cells, and thus determine if non-lethal periods of hypoxia could confer protection against apoptotic injury to human proximal tubular cells during cold storage and subsequent cytotoxic insult, and establish the cellular mechanisms by which this protection is induced. MATERIALS AND METHODS: Human proximal tubular cells (HK-2) were pre-incubated for 24 h in normoxic or hypoxic conditions and then incubated at 4 degrees C for 6 h to mimic cold storage, before being returned to normal conditions and exposed to varying concentrations of cyclosporine A (CSA). Cell viability and apoptosis were measured using propidium iodide staining and flow cytometry. The expression of heat-shock protein (HSP)-70 was determined by Western blotting. RESULTS: Hypoxia had no effect on cell viability or apoptosis. Pre-exposure of cells to hypoxia significantly protected against CSA-induced damage even after a period of cold storage. Western blotting analysis showed that hypoxia up-regulated the anti-apoptotic protein HSP-70. HK-2 cells over-expressing HSP-70 mimicked hypoxia preconditioning, in that they were protected during cold storage and CSA-induced apoptosis. CONCLUSION: Exposure of renal tubular cells to a sequential model of cold storage, reperfusion and incubation with CSA resulted in apoptotic cell death. Preconditioning these cells with hypoxia induced a protective response and up-regulation of the anti-apoptotic protein HSP-70. There was a similar response in non-preconditioned cells over-expressing HSP-70. Further understanding of the cellular changes occurring during this period of preconditioning will allow the development of more targeted, clinically relevant methods of preconditioning in renal transplantation.