Prognostic role of clusterin in resected adenocarcinomas of the lung.

RATIONALE: Clusterin expression may change in various human malignancies, including lung cancer. Patients with resectable non-small cell lung cancer (NSCLC), including adenocarcinoma, have a poor prognosis, with a relapse rate of 30-50% within 5 years. Nuclear factor kB (Nf-kB) is an intracellular protein involved in the initiation and progression of several human cancers, including the lung. OBJECTIVES: We investigate the role of clusterin and Nf-kB expression in predicting the prognosis of patients with early-stage surgically resected adenocarcinoma of the lung. FINDINGS: The level of clusterin gradually decreased from well-differentiated to poorly differentiated adenocarcinomas. Clusterin expression was significantly higher in patients with low-grade adenocarcinoma, in early-stage disease and in women. Clusterin expression was inversely related to relapse and survival in both univariate and multivariate analyses. Finally, we observed an inverse correlation between Nf-kB and clusterin. CONCLUSIONS: Clusterin expression represents an independent prognostic factor in surgically resected lung adenocarcinoma and was proven to be a useful biomarker for fewer relapses and longer survival in patients in the early stage of disease. The inverse correlation between Nf-kB and clusterin expression confirm the previously reported role of clusterin as potent down regulator of Nf-kB.

Chronic administration of green tea extract to TRAMP mice induces the collapse of Golgi apparatus in prostate secretory cells and results in alterations of protein post-translational processing.

Considering its long latency, prostate cancer (PCa) represents an ideal target for chemoprevention strategies. Green tea extract (GTE) has been proved to be one of the most promising natural substances capable of inhibiting PCa progression in animal models (transgenic adenocarcinoma of mouse prostate), as well as in humans. However, the cellular targets of the GTE action are mostly unknown. The main objective of this work was to investigate whether the endoplasmic reticulum (ER) and the Golgi apparatus (GA), known to be actively involved in sensing stress stimuli and initiating and propagating cell death signalling, may represent the subcellular targets of GTE action. To this end, 42 TRAMP mice were divided into four experimental groups: groups II and IV, received GTE in tap water (0.3 g/100 ml solution) starting at 8 weeks of age and up to the time of sacrifice. Groups I and III were respective age-matched water-fed controls. The animals were sacrificed after 4 weeks (groups I and II) or 40 weeks of treatment (groups II and IV). We also treated TRAMP-C2 cells with GTE (20 µg/ml for 7 days) to check the expression profile of clusterin (CLU), a protein involved in prostate tumourigenesis, extensively processed through ER-GA before being secreted through the plasma membrane. In vivo we found that chronic administration of GTE in TRAMP mice results in collapse of ER and GA in prostate epithelial cells. Consistently, in vitro we found that the mature, fully processed form of CLU, sCLU, is strongly reduced by GTE treatment in TRAMP-C2 cells. Taking into account the sCLU biogenesis dependence on the ER-GA integrity and the proposed anti-apoptotic role of sCLU, the possibility for GTE to counteract PCa progression by interfering with sCLU biogenesis is suggested.

Hyper-homocysteinemia alters amyloid peptide-clusterin interactions and neuroglial network morphology and function in the caudate after intrastriatal injection of amyloid peptides.

Amyloid peptides (Abeta) are fragments of the Amyloid Precursor Protein (APP), an integral membrane protein. Abeta peptides are continuously generated by neurons and non-neuronal cells via sequential cleavage of APP by secretases. In particular, Abeta1-42 is the main component of the senile plaques associated with Alzheimer's disease (AD). Glial cells participate in the uptake of soluble extra-cellular Abeta and in the clearance of this material at localized sites where the Abeta are concentrated. It has been shown that clusterin (Apo J) and apolipoprotein E (ApoE) exert important additive effects in reducing Abeta deposition. In agreement with the fact that homocysteine (Hcy) potentiates Abeta peptide neurotoxicity, and Hcy brain levels increase with age, it has been demonstrated that high plasma levels of Hcy are a risk factor for AD. In the present paper, we used animals subjected to chronic intake of methionine (1 g/kg/day) in the drinking water, since this treatment can increase plasma Hcy levels by 30%. By means of this animal model, interactions between the Abeta beta-sheet rich fibrils and clusterin, have been evaluated in striata of animals after Abeta injection. Furthermore, it has been demonstrated that Abeta peptides are not only signals capable of activating astrocytes but also capable of reducing tyrosine-hydroxylase immunoreactivity in the basal ganglia probably leading to a reduction of volume transmission. These alterations in the neuroglial network morphology and function can, at least in part, explain the enhanced pain threshold observed in the Abeta intra-striatally injected animals.

Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke.

RATIONALE: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. OBJECTIVES: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. METHODS: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. CONCLUSIONS: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.

Molecular classification of green tea catechin-sensitive and green tea catechin-resistant prostate cancer in the TRAMP mice model by quantitative real-time PCR gene profiling.

We previously found that human prostate cancer (CaP) progression is accompanied by differential expression of a panel of 8 informative genes, some of which are metabolically related. Gene profiling focused on this 8-gene pack by northern blot analysis in combination with standard clinical information provided reliable prognostic prediction of human CaP. For a better insight into the potential of this 8-gene signature in tumor detection/classification and therapeutic response, we determined, by qPCR, the expression of these informative genes in the TRAMP mice model of CaP progression. The 8-genes signature resulted effective in discriminating, by linear discriminant analysis (LDA), the prostate of wild type mice from transgenic TRAMP mice developing CaP (P < 0.0002). Since it is known that Green Tea Catechin (GTC) administration to TRAMP mice results in a substantial delay of CaP progression in 80% of the animals, while 20% remain unresponsive, we determined the 8-gene signature in the prostates of GTC-sensitive and GTC-resistant mice. LDA discriminated benign tissue from CaP (i.e. wild-type + chemoprevented, GTC-sensitive TRAMP mice, in which CaP progression was delayed, was discriminated from TRAMP mice + GTC-resistant TRAMP mice, in which CaP developed irrespective of GTC administration; P < 0.01). Moreover, GTC-sensitive TRAMP mice bearing CaP were discriminated from GTCs-resistant ones, (P = 0.0001). These results show that qPCR gene profiling, based on the signature of the 8-genes selected by us, could represent an appropriate means for studying the biological behavior of CaP, which may lead to identifying new tools of potential prognostic value, in that a molecular classification for the presence/absence of cancer and for discriminating GTCs-responsive from GTC-resistant CaP is provided.

The chemopreventive action of catechins in the TRAMP mouse model of prostate carcinogenesis is accompanied by clusterin over-expression.

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.

Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin.

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.

Cisplatin-resistance modulates the effect of protein synthesis inhibitors on spermidine/spermine N(1)-acetyltransferase expression.

Cisplatin (DDP)-resistance confers a deficient expression of spermidine/spermine N(1)-acetyltransferase (SSAT) gene in response to the spermine analog N(1),N(12)-bis(ethyl)spermine (BESpm) in the DDP-resistant human ovarian carcinoma cell line (C13*), compared with their parental DDP-sensitive 2008 cells. This SSAT gene deficiency is correlated with a reduced growth sensitivity to spermine analogs. This study was performed to determine whether SSAT gene expression of resistant cells was kept suppressed by labile repressor proteins developed during resistance selection. We show here that inhibitory concentrations of cycloheximide (CHX) and anisomycin (ANISO) differentially affect BESpm-induced SSAT activity in 2008 and in C13* cells in a concentration-dependent manner and allow resistant cells to reach activation levels comparable to those of the sensitive cells. Northern blot analysis revealed that both CHX and ANISO in combination with BESpm caused a synergistic BESpm-mediated accumulation of SSAT mRNA in C13* cells, with respect to each drug alone, while in 2008 cells only a slight increase was observed. The more pronounced effect of inhibitors on the SSAT activity induced by BESpm in the resistant cells was also the result of a more prolonged stabilization of SSAT mRNA and enzyme protein. By contrast, sub-inhibitory concentrations of CHX and ANISO did not significantly stimulate BESpm-induced SSAT transcription and activity. These results suggest that labile repressor proteins, related to DDP-resistance phenotype, play a regulatory role in SSAT gene expression, and further indicate that by overcoming this inhibitory control it is possible to recover BESpm response.

Clusterin (SGP-2, ApoJ) expression is downregulated in low- and high-grade human prostate cancer.

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 long-term 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 low-grade 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.

Successful prediction of prostate cancer recurrence by gene profiling in combination with clinical data: a 5-year follow-up study.

We show here that gene expression profiling, performed with conventional techniques and focused on a selected group of genes, when used in combination with standard clinical information, provides reliable prognostic prediction of prostate cancer (CaP). We showed previously that changes in the expression of metabolically related genes are involved in CaP progression. We then proceeded to search further for correlations between patients' gene profiling and recurrence with a 5-year follow-up study conducted on the same cohort of patients in which the molecular data were obtained. CaP prognosis was first assessed on the basis of gene expression profiling alone; then the result was compared with the prediction obtained using clinical and pathological information (Gleason score, Tumor-Node-Metastasis staging, prostate volume, or prostate-specific antigen levels at the time of diagnosis). The best result was obtained with a selected combination of gene profiling and clinical/pathological parameters, which resulted in prediction of recurrence in 95.7% of patients.

Clusterin (SGP-2) transient overexpression decreases proliferation rate of SV40-immortalized human prostate epithelial cells by slowing down cell cycle progression.

Clusterin is a highly conserved, widely distributed glycoprotein whose biological significance is still debated. Involved in many biological processes and disease states, clusterin is induced by cell injury and tissue regression, but is repressed during cell proliferation. We have previously reported that clusterin mRNA induction is associated with epithelial cell atrophy in the rat prostate and both clusterin transcript and protein accumulated in quiescent normal human skin fibroblasts. Here we show that transient clusterin overexpression, in SV40-immortalized human prostate epithelial cells (PNT2), resulted in increased accumulation of cells in the G(0)/G(1) phases of the cell cycle, accompanied by slowdown of cell cycle progression and decrease of DNA synthesis. The activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and the level of histone H3 mRNA (markers of cell proliferation) concomitantly decreased, while Gas1 mRNA (a marker of cell quiescence) accumulated. Thus it appears that clusterin, by opposing the effect of SV40 on the proliferation rate of PNT2 cells, acts as an anti-oncogene in the prostate, suggesting a role for this gene in controlling proliferation of normal and transformed prostate epithelial cells.