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Targeting clusterin in prostate cancer

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The biology of prostate cancer is still poorly understood. Allelic loss studies indicate that there likely exist multiple sites harbouring candidate tumour suppressor genes (TSG), some of which may have an important role in primary tumours, and some in late stages of prostate cancer. The recent studies on the localization of potential TSG in neoplastic transformation of prostate comprise chromosome regions 7q, 8p, l0p/q, 16q, 17q, and 18q. In connection with accumulation of genetic changes affecting functioning of critical TSG, the multistep cancer progression hypothesis is a useful starting point in efforts to understand the biology of the neoplastic lesions of prostate.
Prostate cancer (CaP) is a fast-growing health and social problem already representing the second leading cause of cancer-related death among men in Western countries. Lifestyle-related factors and diet are major contributors for CaP promotion. Because of unfavourable prognosis of extra-prostatic CaP, prevention is considered the best approach to fight it at present time. Green Tea Catechins (GTCs) were proven effective at inhibiting cancer growth in several laboratory studies. We recently performed a pilot clinical trial in HG-PIN subjects showing that only 1/30 tumour was diagnosed in subjects treated for 1 year with 600 mg/die GTCs, while 9/30 cancers were found in placebo-treated men. CaP is an elusive disease, whose biological behaviour is difficult to predict. We have recently described and validated a RT-qPCR method based on a 8-genes signature that significantly discriminated benign tissue from CaP in both humans and TRAMP mice spontaneously developing CaP. In the animal model, also GTCs-resistant CaP was significantly discriminated from GTCs-sensitive CaP, i.e. responding to GTCs administration. Preliminary experiments in our laboratory have shown that this method can be successfully applied to a single tissue needle biopsy specimen in humans. The combination of these results may be of particular significance on the field. In fact, GTCs treatment for men at high risk of CaP as first line prevention therapy in combination with the 8-genes signature profiling in tissue needle biopsies for real time monitoring of patient's response might importantly change, in the near future, the clinical managing of this highly diffuse malignancy.
Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63, by acting through IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras cycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 cells were (1) IGF-I stimulated and lysates were immunoprecipitated with polyclonal IRS-1 antibody or (2) metabolically labeled with [32P]orthophosphoric acid and then cells were treated with IGF-I. Cell lysates were immunoprecipitated with p21Ras antibody(Y13-259) and bound nucleotides were analysed by thin layer chromatography. We demonstrated tyrosine phosphorylation of IRS-1/2 immunoprecipitated from MG-63 cells stimulated with IGF-I. We also showed an increased level of GTP in p21Ras immunoprecipitates from IGF-I treated cells. Nuclear extracts prepared from 32P-labeled cells before and after addition of IGF-I were immunoprecipitated with c-Jun antibody. After electrophoresis and autoradiography, phosphorylation of the c-Jun band was seen to be IGF-I independent. Phosphoamino acid analysis of the c-Jun band showed that phosphoserine was the major species.
 Although the degradome, which comprises proteolytic fragments of blood proteins, presents a potential source of diagnostic biomarkers, studies on cancer peptide biomarkers have provided inconsistent conclusions. In the present study, we reevaluated the usefulness of serum degradome analyses for searching peptide cancer biomarker candidates. Particular attention was paid to pre-analytical factors influencing the variability of determined peptide levels, including clotting time and control group selection. Studies were conducted on 44 and 86 serum samples collected from cancer patients and healthy individuals, respectively, using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS)-based analyses. We identified 1373 unique peptides, nearly 40% of which originated from five blood proteins: fibrinogen alpha chain, apolipoprotein A-IV (APOA4), complement C3, apolipoprotein A-I, and alpha-1-antitrypsin. A set of 118 and 88 peptides exhibited highly significant differences (adjusted p-value ≤ 0.01 and fold change ≥ 2) in pair-wise comparisons of control vs. prostate cancer and control vs. colorectal cancer, respectively, with 37 peptides displaying a consistent direction of change for these pair-wise comparisons. The levels of 67 peptides differed significantly in serum samples collected from healthy individuals immediately prior to colonoscopy and those who underwent colonoscopic examination at least four weeks earlier. Of them, 49 peptides originated from APOA4. Whereas earlier studies, including ours, have utilized fragments of fibrinopeptide A (FPA) to distinguish cancer from healthy cases, here we show that their absolute abundance is a sensitive indicator of clotting time. These observations may have implications for future serum peptidome studies since these issues have not previously been recognized.
Malignant prostate tissues have markedly reduced zinc (Zn2+) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn2+ level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn2+ (10 μg/ml) over 5 weeks. The intracellular Zn2+ level increased in the Zn2+-treated cells, and there was a marked increase in the presence of zincosomes, a Zn2+-specific intracellular organelle. The proliferation rate of the Zn2+-treated cells was markedly reduced. There was also a significant increase (36.6% ± 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn2+, reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn2+-treated LNCaP cell proliferation to a rate comparable to that of the non Zn2+-treated cells. These results highlight the importance of a high intracellular Zn2+ content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.
Apigenin (4′,5,7,-trihydroxyflavone) is a flavonoid abundant in the common fruits, herbs and vegetables constituting the bulk of the human diet. This study was aimed at quantifying the effects of apigenin on the basic cellular traits determining cancer development, i.e. cell proliferation, gap junctional coupling, and motility, using the Dunning rat prostate MAT-LyLu cell model. We demonstrated that apigenin considerably inhibits MAT-LyLu cell proliferation and significantly enhances the intensity of connexin43-mediated gap junctional coupling. This effect correlates with an increased abundance of C×43-positive plaques at the cell-to-cell borders seen in apigenin-treated variants. Moreover, we observed an inhibitory effect of apigenin on the motility of MAT-LyLu cells. The basic parameters characterising MAT-LyLu cell motility, especially the rate of cell displacement, considerably decreased upon apigenin administration. This in vitro data indicates that apigenin may affect cancer development in general, and prostate carcinogenesis in particular, via its influence on cellular activities decisive for both cancer promotion and progression, including cell proliferation, gap junctional coupling and cell motility and invasiveness.
The results of several epidemiological studies have suggested that a soybean-based diet is associated with a lower risk of prostate cancer. We investigated the effect of the soy isoflavone genistein on the proliferation and contact-stimulated migration of rat prostatic carcinoma MAT-LyLu and AT-2 cell lines. Genistein almost completely inhibited the growth of both MAT-LyLu and AT-2 cells in the concentration range from 25 to 100 μM, but the addition of 1 μM genistein to the medium significantly stimulated the proliferation of both cell lines. Additionally, at concentrations above 25 μM, genistein showed a potent cytotoxic effect. However, the central finding of this study is that at physiologically relevant concentrations (1 μM and 10 μM), genistein inhibits the motility of prostate cancer cells stimulated by homo-and heterotypic contacts. These results show that at physiological concentrations, genistein exerts an inhibitory effect on the migration of prostate cancer cells and suggest that it may be one of the factors responsible for the anti-metastatic activity of plant isoflavonoids.
Id1 (inhibitor of differentiation 1) is a member of the bHLH protein family. Consistent with its role in promoting proliferation and inhibiting differentiation, Id1 expression is low or negligible in normal prostate epithelial cells but is high in prostate cancer. Ectopic expression of Id1 in normal prostate epithelial cells could therefore provide a model for understanding early events involved in initiation of prostate cancer. Over-expression of Id1 immortalized but did not transform ventral prostate epithelial cells (Id1-RPE). Immortalization was associated with decreased Cdkn2a, Cdkn1a, androgen receptor and increased Tert expression. Gene expression profiling over successive doublings was used to identify transcriptomic changes involved during immortalization (Tieg, Jun, alpha actin, Klf10, Id2) and in maintaining the immortalized phenotype (Igfbp3, Igfbp5, Mmp2, Tgfb3). Network analysis indicated that Id1 promotes cancer/tumor morphology, cell cycle and epithelial to mesenchymal transition by influencing AP1, tnf, tgfβ, PdgfBB and estradiol pathways. During immortalization, the expression of majority of differentially expressed genes reduced over progressive doublings suggesting a decline in transcriptional regulatory mechanisms. The associated molecular/gene expression profile of Id1-RPE cells provides an opportunity to understand the molecular pathways associated with prostate epithelial cell survival and proliferation.
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