Clusterin (CLU) protein is widely distributed in animal tissues and is involved in many different processes, including apoptosis and neoplastic transformation. Green tea catechins (GTC) are known to exert chemopreventive effects in many cancer models, including transgenic adenocarcinoma mouse prostate (TRAMP) mice that spontaneously develop prostate cancer (CaP). We report here that growth of SV40-immortalized human prostate epithelial cells (PNT1A) as well as tumorigenic, poorly differentiated prostate cancer cells (PC-3) was potently inhibited by EGCG, the major green tea catechin, while normal human prostate epithelial cells were not significantly affected. IC(50) doses of EGCG for 24 h caused caspase cascade activation and CLU protein accumulation in both cells lines but not in normal cells, in which CLU remained undetectable. While 100% of TRAMP mice developed CaP, only 20% of those receiving 0.3% GTC in drinking water developed the neoplasm. In TRAMP mice, the CLU gene was dramatically down-regulated during onset and progression of CaP. In GTC-treated TRAMP mice in which tumor progression was chemoprevented, CLU mRNA and protein progressively accumulated in the prostate gland. CLU dropped again to undetectable levels in animals in which GTC chemoprevention failed and CaP developed. Up-regulation of histone H3 and down-regulation of growth arrest-specific gene 1 (Gas1) mRNAs in CaP-developing TRAMP mice demonstrated a high proliferation rate in tumors, while the opposite occurred in the glands of GTC chemoprevented animals. Failure of GTC chemoprevention caused induction of both histone H3 and Gas1 and down-regulation of CLU. Immunohistochemistry experiments confirmed CLU down-regulation during CaP onset and progression, and CLU sustained expression in chemoprevented TRAMP mice. A possible role for CLU as a novel tumor-suppressor gene in the prostate is thus suggested.

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Rationale: Studies in transgenic mice showed the key role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1) in the control of cardiomyocyte function and viability.Objective: We investigated whether Pim-1 represents a novel mechanistic target for the cure of diabetic cardiomyopathy, a steadily increasing cause of nonischemic heart failure. Methods and Results:In streptozotocin-induced type 1 diabetic mice, Pim-1 protein levels declined during progression of cardiomyopathy, along with upregulation of Pim-1 inhibitors, protein phosphatase 2A, and microRNA-1. Moreover, diabetic hearts showed low levels of antiapoptotic B-cell lymphoma-2 (Bcl-2) protein and increased proapoptotic caspase-3 activity. Studies on adult rat cardiomyocytes and murine cardiac progenitor cells challenged with high glucose confirmed the in vivo expressional changes. In rescue studies, anti-microRNA-1 boosted Pim-1 and Bcl-2 expression and promoted cardiomyocyte and cardiac progenitor cell survival under high glucose conditions. Similarly, transfection with Pim-1 plasmid prevented high glucoseinduced cardiomyocyte and cardiac progenitor cell apoptosis. Finally, a single intravenous injection of human PIM-1 via cardiotropic serotype-9 adeno-associated virus (1؋10 10 or 5؋10 10 genome copies per animal) at 4 weeks after diabetes induction led to sustained cardiac overexpression of Pim-1 and improved diastolic function and prevented left ventricular dilation and failure. Histological examination showed reduced cardiomyocyte apoptosis and fibrosis in association with increased c-kit ؉ cells and cardiomyocyte proliferation, whereas molecular analysis confirmed activation of the prosurvival pathway and conservation of sarcoendoplasmic reticulum Ca 2؉-ATPase and ␣-myosin heavy chain in Pim-1-treated hearts. Key Words: diabetic cardiomyopathies Ⅲ diastolic dysfunction Ⅲ Pim-1 kinase Ⅲ gene therapy Ⅲ cardiac stem cells A common form of cardiomyopathy directly related to diabetes mellitus (DM), ie, diabetic cardiomyopathy, typically progresses from diastolic dysfunction to heart failure in the absence of coronary artery disease or hypertension. [1][2][3] Studies in animal models illustrate the complexity of the underpinning pathogenic mechanisms (reviewed in Bugger and Abel 4 ). Hence, a deeper understanding of targets for early therapeutic interventions is critically needed. Conclusions:Recent work from Muraski and colleagues 5 uncovered the pivotal role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1), a member of the serine/threonine protein kinase family, in the cardiac cell response to stressors. Promotion of cardiomyocyte survival by Pim-1 is mediated by activation of B-cell lymphoma-2 (Bcl-2), phosphorylation/ inhibition of Bcl-2-associated death promoter (Bad), and maintenance of mitochondrial integrity. 6,7 By inducing c-Myc, nucleostemin, and cyclin E expression and p21 phosphorylation, Pim-1 increases the proliferative activity of cardiac progenitor cells (CPCs). 8,9 Furthermore, Pim-1 acts...

Clusterin is overexpressed during tissue and cell involution and downregulated in proliferating cells. Its role in cell survival, cell death and neoplastic transformation remains debated. We studied the expression and distribution of clusterin mRNA and protein in human prostate carcinoma (CaP) specimens of different degrees of malignancy. Fresh CaP specimens were obtained from 25 patients subjected to longterm androgen ablation before surgery. Clusterin expression was studied by Northern and Western analysis, in situ hybridization and immunohistochemistry, in comparison with Gas1 and histone H3 mRNA (markers of cell quiescence and S phase of the cell cycle, respectively). Clusterin is downregulated in CaP in comparison with matched benign controls. In low-grade CaP, clusterin colocalized with Gas1 to the stromal compartment, and in some glands, the basal lamina was heavily stained. In high-grade CaP clusterin stained the remnants of stromal matrix while histone H3 localized to cancer cells, which were very rarely clusterin positive. High clusterin expression was found in the branches of a nerve infiltrated by tumor. The periglandular clusterin expression found in lowgrade CaP could result from induction of quiescence and/or apoptosis of prostatic fibroblasts lining those glands in which tumor invasion is at an initial stage, involving basal lamina. In advanced CaP, the staining of the remnants of the extracellular matrix suggests a role for clusterin in the process of dismantling the stromal organization caused by cancer progression.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼22 nucleotides, which have increasingly been recognized as potent post-transcriptional regulators of gene expression. MiRNA targeting is defined by the complementarities between positions 2-8 of miRNA 5'-end with generally the 3'-untranslated region of target mRNAs (messenger RNAs). The capacity of miRNAs to simultaneously inhibit many different mRNAs allows for an amplification of biological responses. Hence, miRNAs are extremely attractive targets for therapeutic regulation in several diseases, including cardiovascular. Novel approaches are emerging to identify the miRNA functions in cardiovascular biology processes and to improve miRNA delivery in the heart and vasculature. In the present study, we provide an overview of current studies of miRNA functions in cardiovascular cells by the use of high-content screening. We also discuss the challenge to achieve a safe and targeted delivery of miRNA therapeutics in cardiovascular cells.

Expression of the castration-induced clusterin protein is incompatible with the survival of human prostate cancer cells in tissues and in cell culture. To investigate the fate of human prostate epithelial cells, when engineered to maintain expression of clusterin protein, we have used an IRES-hyg vector and hygromycin selection. PC-3 prostate tumour cells were substantially more sensitive to clusterin expression than nonmalignant PNT1a cells, showing multiple phenotypic changes including cell cycle arrest and increased apoptosis. The results strengthen the hypothesis that clusterin expression is proapoptotic. Expression of exogenous clusterin in both cell types resulted in its relocation from the cytoplasm and a nuclear accumulation of the protein, as was also seen in the same cells when apoptosis was induced by etoposide treatment. To survive clusterin expression, the PC-3 tumour cells developed apoptosis-inhibitory properties. This could have significance for the resistance of prostate cancers to chemo/radiotherapy, where clusterin overexpression is observed.

Objective: Calcitriol analogues might represent an interesting new therapy for benign prostate hyperplasia (BPH). We here report the preclinical characterization of BXL-628, an analogue selected for an ongoing double-blind, randomized, placebo-controlled phase II trial in BPH. Design: Experiments with BXL-628 were carried out in human BPH cells and in the ventral prostate of intact and castrated rats. Methods: BPH cell and rat prostate growth were evaluated along with morphological and biochemical hallmarks of apoptosis. Results: BXL-628 inhibited human BPH cell proliferation and induced apoptosis even in the presence of androgens or growth factors. It also decreased prostate growth to an extent similar to finasteride, inducing DNA fragmentation and apoptosis, both in intact and in testosterone-supplemented castrated rats. Accordingly, BXL-628, like finasteride, increased the expression of clusterin, a prostatic atrophy marker. However, BXL-628 did not inhibit 5a-reductase 1 and 2, did not bind to the androgen receptor (AR) in BPH homogenates and did not affect AR-coupled luciferase activity. In addition, BXL-628 did not affect rat pituitary and testis activity or calcemia. Conclusions: BXL-628 inhibited in vitro and in vivo prostate cell proliferation, and therefore might represent a novel, interesting option for the treatment of BPH.European Journal of Endocrinology 150 591-603

Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G0/G1-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell-detachment-induced apoptosis, suggesting a possible major role for clusterin as an anti-proliferative gene in human prostate epithelial cells.