Novel insights into the expression of CGB1 & 2 genes by epithelial cancer cell lines secreting ectopic free hCGβ

BACKGROUND Ectopic secretion of human chorionic gonadotrophin free beta (hCGβ) by epithelial cancer is associated with aggressive tumors which more readily metastasize, possibly by acting as an autocrine anti-apoptotic agent. hCGβ is encoded by six homologous CGB genes, with poorly-understood variable transcriptionally active expression profiles; CGB1 and CGB2 have always been considered pseudogenes. However, transcripts from CGB1 and -2 can be detected in placental, testicular and pituitary tissues. The expression and function of these genes in cancer is less well-known. MATERIALS AND METHODS Expression profiles of CGB genes in epithelial cancer cells by quantitative polymerase chain reaction (qPCR) were explored, along with the consequence of specific siRNA silencing of CGB1 and 2. Immunohistochemical and immunoassay techniques were used to detect the translation and secretion of hCGβ in these cells. RESULTS CGB1 and -2 gene transcripts were only detected in cells which secreted hCGβ. siRNA-mediated silencing of CGB1 and -2 transcripts significantly reduced secreted protein in concordance with a reduction in cell survival to a greater degree than that of other CGB genes. CONCLUSION CGB genes 1 and 2, previously considered as pseudogenes, are notably expressed by epithelial cancer cell lines. The transcription of these genes, but not other CGB genes, correlates with a functionally expressed protein and propensity for cancer growth

GLI1 confers profound phenotypic changes upon LNCaP prostate cancer cells that include the acquisition of a hormone independent state

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active NGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, beta1-integrin, Np63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function

Pulse wave velocity and neutrophil gelatinase-associated lipocalin as predictors of acute kidney injury following aortic valve replacement

Background Accurate prediction, early detection and treatment of acute kidney injury (AKI) are essential for improving post-operative outcomes. This study aimed to examine the role of aortic stiffness and neutrophil gelatinase-associated lipocalin (NGAL) as predictors of AKI or need for early medical renal intervention following aortic valve replacement (AVR). Methods Aortic pulse wave velocity and plasma NGAL were measured pre-operatively in recruited patients undergoing AVR for aortic stenosis (AS). Plasma NGAL was also measured at 3 and 18–24 hours after cardiopulmonary bypass (CPB). AKI was defined using RIFLE criteria. Early medical renal intervention included diuretics or dopamine infusion exclusively for renal causes. Results Fifty-three patients aged 71 ± 9 years were included. Sixteen (30%) developed AKI (AKI-Yes) and 24 patients (45%) received early medical intervention (Intervention-Yes). There was no significant difference in the demographic, clinical or operative characteristics between the two groups for either outcome. PWV did not significantly correlate with AKI (r = 0.12, P = 0.13) or early intervention (r = 0.18, P = 0.18). At 3 h post-CPB, plasma NGAL was a much stronger predictor of both AKI and the need for early medical intervention than conventional markers such as creatinine (AKI: AUC 83%, 95% CI 0.70–0.95 vs. AUC 65%, 95% CI 0.47- 0.82; Medical intervention: AUC 84%, 95% CI 0.72–0.96 vs. AUC 56%, 95% CI 0.38–0.73). Post-CPB (3 hr) plasma NGAL was also significantly associated with AKI (r = 0.68, P < 0.001) at levels above 150 ng/ml; and significantly associated with early intervention (r = 0.64, P < 0.001) above 136 ng/ml. Simple linear regression showed no relationship between PWV and NGAL levels. Conclusion Aortic PWV does not correlate significantly with post-operative AKI or plasma NGAL levels in surgical AS patients. Post-operative NGAL is however an early and powerful predictive biomarker of both post-operative AKI and the need for early medical renal intervention and should consequently be considered in prediction models for AKI after cardiac surgery

Aortic stiffness as a marker of cardiac function and myocardial strain in patients undergoing aortic valve replacement

Background: Cardiac function and myocardial strain are affected by cardiac afterload, which is in part due to the stiffness of the aortic wall. In this study, we hypothesize that aortic pulse wave velocity (PWV) as a marker of aortic stiffness correlates with conventional clinical and biochemical markers of cardiac function and perioperative myocardial strain in aortic valve replacement (AVR). Methods: Patients undergoing AVR for aortic stenosis between June 2010 and August 2012 were recruited for inclusion in this study. PWV, NYHA class and left ventricular (LV) function were assessed pre-operatively. PWV was analysed both as a continuous and dichotomous variable according to age-standardized reference values. B-type natriuretic peptide (BNP) was measured pre-operatively, and at 3 h and 18-24 h after cardiopulmonary bypass (CPB). NYHA class, leg edema, and LV function were recorded at follow-up (409 ± 159 days). Results: Fifty-six patients (16 females) with a mean age of 71 ± 8.4 years were included, with 50 (89%) patients completing follow-up. The NYHA class of PWV-norm patients was significantly lower than PWV-high patients both pre- and post-operatively. Multiple logistic regression also highlighted PWV-cut off as an independent predictor of NYHA class pre- and post-operatively (OR 8.3, 95%CI [2.27,33.33] and OR 14.44, 95%CI [1.49,139.31] respectively). No significant relationship was observed between PWV and either LV function or plasma BNP. Conclusion: In patients undergoing AVR for aortic stenosis, PWV is independently related to pre- and post-operative NYHA class but not to LV function or BNP. These findings provisionally support the use of perioperative PWV as a non-invasive marker of clinical functional status, which when used in conjunction with biomarkers of myocardial strain such as BNP, may provide a holistic functional assessment of patients undergoing aortic valve surgery. However, in order for PWV assessment to be translated into clinical practice and utilised as more than simply a research tool, further validation is required in the form of larger prospective studies specifically designed to assess the relationship between PWV and these functional clinical outcomes

GLI1 Confers Profound Phenotypic Changes upon LNCaP Prostate Cancer Cells That Include the Acquisition of a Hormone Independent State

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active ΔNGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, β1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function