Paracrine Regulation of Human Prostate Cancer Cell Growth and Function

This thesis mainly deals with steroid-growth factor interaction in the regulation of growth and steroid metabolism of LNCaP, DU145 and PC3 human prostate cancer cell lines. Growth response to both androgens and oestrogens and to TGFalpha and TGFbeta1 were investigated; in addition, content and status of steroid (AR and ER) and growth factor (EGFR and TGP?R) receptors were assessed using multiple approaches, including ligand binding assay, immunocytochemistry and reverse transcriptase-PCR (RT-PCR). Furthermore, immunofluorescent staining was used to evaluate expression of EGF, TGFalpha and TGFbeta1 and of the 27 kDa heat shock protein (hsp27) as a marker of oestrogen sensitivity. On the other hand, patterns of both testosterone (T) and oestradiol (E2) metabolism were studied using incubation of cultured cells with labelled steroid precursor and reverse phase-HPLC (RP-HPLC) analysis of precursor degradation and formation of metabolic products. Possible influence of both TGFalpha and TGFbeta1 on rates and direction of metabolism of both steroids was also determined. Growth of LNCaP cells was significantly stimulated by physiological concentrations of the two major androgens (T and dihydrotestosterone: 30.2% and 33.8% respectively, P<0.03) and of E2 (65.8%, P=0.009), using stringent culture conditions. Interestingly, increasing concentrations of E2 (0.01-100 nM) induced a significant inhibition of the proliferative activity of PC3 cells (55.2% at 100 nM E2, P<10-6), while neither androgen SUMMARY significantly affected growth of this cell line. In contrast, DU145 cells proved insensitive to the addition of either androgens or oestrogens. Presence of androgen binding sites in prostate tumour cell lines was ascertained using radioligand binding assay and RT-PCR approaches. High affinity AR were detected in both soluble and pellet fractions of LNCaP and DU145 cells, whilst PC3 cells showed only nuclear AR. These results were also supported by PCR system, where ampifiable AR mRNA was found in all three cell lines. Multiple evidence for high affinity sites of oestrogen binding in LNCaP cells was obtained: i) biochemical assay allowed the detection of high affinity, low capacity binding sites in both soluble and nuclear cell fractions; ii) immunocytochemical and immunofluorescent assays showed a consistently intensive staining for both ER and PgR, as well as hsp27; iii) the RT-PCR system documented the presence of normal or a variant ER mRNA in PC3 cells; the latter, which lacks the entire exon 4, has been recently characterised in our laboratories in human mammary carcinoma cells. Presence of ER in PCS cells was also documented by biochemical and cytochemical assays as well as, indirectly, by hsp27 staining. However, the relative estimate of ER expression displayed levels significantly and consistently lower than those found in LNCaP cells. This finding was confirmed using the RT-PCR approach, where transcript levels for both normal and variant ER mRNA were proportionally lower than in LNCaP cells. Conversely, DU145 cells were found to be consistently ER-negative using biochemical and cytochemical assays, hsp27 staining and RT-PCR system. The evidence that the E2-induced growth was completely reversed in LNCaP cells by the addition of the pure antioestrogen ICI-182,780, clearly suggests that E2 acts via its own receptor. The possibility that the inhibitory effect exerted by E2 on growth of PC3 cells could be mediated via an increase of TGFalpha production was also supported by the fact that use of a neutralising antibody raised against TGFbeta1 produced a three-fold increase of cell growth; this effect was almost completely abolished after addition of 100 nM E2. However, Northern blot analysis did not reveal any increase of TGFbeta1 mRNA following E2 administration in this cell line. Growth of PC3 cells was significantly stimulated by TGFalpha (36% at 50 ng/ml, P<0.003) and inhibited by TGFbeta1 (55% at 5 ng/ml, P<10-6) after 48 hours exposure in routine medium. Proliferative activity of DU145 cells was minimally affected by TGFalpha, but significantly inhibited by TGFbeta1 (28% at 5 ng/ml, P<0.009). In contrast, LNCaP cells proved to be poorly sensitive, at least in the short-term, to either growth factor. Radioreceptor assay showed presence of high affinity binding sites for EGF in all three cell lines and for TGFalpha in DU145 and PC3 cells, whilst no detectable site of TGFalpha binding was found in LNCaP cells. DU145 cells displayed the highest EGFR content, LNCaP the lowest; TGFalphaR were expressed in greater amounts in PC3 than in DU145 cells. EGFR binding data were also confirmed using Western blot analysis, the DU145 cells having EGFR expression levels higher than PC3 and LNCaP cells. In addition, immunofluorescent staining revealed high amounts of both EGFR and TGFalpha, and fairly high EGF in DU145 and, to a lesser extent, in LNCaP cells; by contrast, PC3 cells exhibited low levels of both receptor (EGFR) and ligands (EGF, TGFalpha), but appreciable levels of endogenous TGFbeta1. Overall, these results suggest a differential sensitivity to TGFalpha and TGFbeta1 by prostate cancer cells; TGFalpha response seems to be not proportional to the EGF-R content of individual cell lines, while TGFbeta1 response appears to be inversely related to the androgen-sensitivity of cells. (Abstract shortened by ProQuest.)

P/CAF-mediated spermidine acetylation regulates histone acetyltransferase activity

Histones and polyamines are important determinants of the chromatin structure. Histones form the core of nucleosome particles and their modification by acetylation of N-terminal tails is involved in chromatin structural changes and transcriptional regulation. Polyamines, including spermidine, are also targets of both cytoplasmic and nuclear acetylation, which in turn alters their affinity for DNA and nucleosomes. Previous studies report the interplay between polyamines metabolism and levels of histone acetylation, but the molecular basis of this effect is still unclear. In this work, we have analyzed the in vitro effect of spermidine on histone H3 acetylation catalyzed by P/CAF, a highly conserved histone acetyltransferase (HAT) (E.C. 2.3.1.48). We have observed that spermidine at very low concentrations activates P/CAF, while it has an inhibitory effect at concentrations higher than 4 μM. In addition, the in vitro bimodal effect of spermidine on histone H3 acetylation was also distinctly observed in vivo on polytene chromosomes of Drosophila melanogaster. We also performed kinetic studies indicating that the activating effect of low spermidine concentrations on P/CAF-HAT activity is based on its involvement as a substrate for P/CAF to produce

Nutrigerontology: A key for achieving successful ageing and longevity

During the last two centuries the average lifespan has increased at a rate of approximately 3 months/year in both sexes, hence oldest old people are becoming the population with the fastest growth in Western World. Although the average life expectancy is increasing dramatically, the healthy lifespan is not going at the same pace. This underscores the importance of studies on the prevention of age-related diseases, in order to satisfactorily decrease the medical, economic and social problems associated to advancing age, related to an increased number of individuals not autonomous and affected by invalidating pathologies. In particular, data from experimental studies in model organisms have consistently shown that nutrient signalling pathways are involved in longevity, affecting the prevalence of age-related loss of function, including age-related diseases. Accordingly, nutrigerontology is defined as the scientific discipline that studies the impact of nutrients, foods, macronutrient ratios, and diets on lifespan, ageing process, and age-related diseases. To discuss the potential relevance of this new science in the attainment of successful ageing and longevity, three original studies performed in Sicily with local foods and two reviews have been assembled in this series. Data clearly demonstrate the positive effects of nutraceuticals, functional foods and Mediterranean Diet on several biological parameters. In fact, they could represent a prevention for many age-related diseases, and, although not a solution for this social plague, at least a remedy to alleviate it. Thus, the possibility to create a dietary pattern, based on the combined strategy of the use of both nutraceuticals and functional foods should permit to create a new therapeutic strategy, based not only on a specific bioactive molecule or on a specific food but on a integrated approach that, starting from the local dietary habits, can be led to a "nutrafunctional diet" applicable worldwide

Sex steroids, carcinogenesis, and cancer progression

The relationship between sex steroids and cancer has been studied for more than a century. Using an original intact cell analysis, we investigated sex steroid metabolism in a panel of human cancer cell lines, either hormone responsive or unresponsive, originating from human breast, endometrium, and prostate. We found that highly divergent patterns of steroid metabolism exist and that the catalytic preference (predominantly reductive or oxidative) is strictly associated with the steroid receptor status of cells. We explored intra-tissue concentrations and profiles of estrogens in a set of human breast tumors as compared to normal mammary tissues, also in relation to their estrogen receptor status. In particular, we showed that, with hydroxyestrogens representing the majority of all tissue estrogens, concentrations of individual metabolites, as well as their ratios, significantly differ when comparing normal tissue with cancer tissues or when they are related to the overall survival of cancer patients. © 2004 New York Academy of Sciences

A peculiar formula of essential amino acids prevents rosuvastatin myopathy in mice

Aims: Myopathy, characterized by mitochondrial oxidative stress, occurs in ∼10% of statin-treated patients, and a major risk exists with potent statins such as rosuvastatin (Rvs). We sought to determine whether a peculiar branched-chain amino acid-enriched mixture (BCAAem), found to improve mitochondrial function and reduce oxidative stress in muscle of middle-aged mice, was able to prevent Rvs myopathy. Results: Dietary supplementation of BCAAem was able to prevent the structural and functional alterations of muscle induced by Rvs in young mice. Rvs-increased plasma 3-methylhistidine (a marker of muscular protein degradation) was prevented by BCAAem. This was obtained without changes of Rvs ability to reduce cholesterol and triglyceride levels in blood. Rather, BCAAem promotes de novo protein synthesis and reduces proteolysis in cultured myotubes. Morphological alterations of C2C12 cells induced by statin were counteracted by amino acids, as were the Rvs-increased atrogin-1 mRNA and protein levels. Moreover, BCAAem maintained mitochondrial mass and density and citrate synthase activity in skeletal muscle of Rvs-treated mice beside oxygen consumption and ATP levels in C2C12 cells exposed to statin. Notably, BCAAem assisted Rvs to reduce oxidative stress and to increase the anti-reactive oxygen species (ROS) defense system in skeletal muscle. Innovation and Conclusions: The complex interplay between proteostasis and antioxidant properties may underlie the mechanism by which a specific amino acid formula preserves mitochondrial efficiency and muscle health in Rvs-treated mice. Strategies aimed at promoting protein balance and controlling mitochondrial ROS level may be used as therapeutics for the treatment of muscular diseases involving mitochondrial dysfunction, such as statin myopathy

The management of cancer in the elderly: targeted therapies in oncology

Cancer is universally considered a disease of ageing. Today the management of elderly cancer patients poses many specific problems and it should be revisited in the light of the most recent advances in both diagnosis and treatment of human malignancies. In particular, the potential use of novel therapeutic options, based on therapeutic agents raised against molecular targets (the so called targeted therapy), appears to be promising in this clinical settings especially in view of the limited side-effects. The mainstays of cancer treatment during the twentieth century were surgery, radiation and chemotherapy. However, surgery is not curative in metastatic disease, radiation and chemotherapy are limited by side effects because they can't discriminate between healthy and cancerous cells. When key molecular changes responsible for malignant transformation were identified (e.g. growth factors and their receptors), it was hoped that new targeted agents, by inhibiting cancer-specific pathways, would spare normal cells and thereby offer improved safety benefits and a higher therapeutic index over standard chemotherapeutics. The most common targeted therapies used in clinical practice, i.e. monoclonal antibodies and small molecules, are described

Monoaminergic and histaminergic strategies and treatments in brain diseases

The monoaminergic systems are the target of several drugs for the treatment of mood, motor and cognitive disorders as well as neurological conditions. In most cases, advances have occurred through serendipity, except for Parkinson's disease where the pathophysiology led almost immediately to the introduction of dopamine restoring agents. Extensive neuropharmacological studies first showed that the primary target of antipsychotics, antidepressants, and anxiolytic drugs were specific components of the monoaminergic systems. Later, some dramatic side effects associated with older medicines were shown to disappear with new chemical compounds targeting the origin of the therapeutic benefit more specifically. The increased knowledge regarding the function and interaction of the monoaminergic systems in the brain resulting from in vivo neurochemical and neurophysiological studies indicated new monoaminergic targets that could achieve the efficacy of the older medicines with fewer side-effects. Yet, this accumulated knowledge regarding monoamines did not produce valuable strategies for diseases where no monoaminergic drug has been shown to be effective. Here, we emphasize the new therapeutic and monoaminergic-based strategies for the treatment of psychiatric diseases. We will consider three main groups of diseases, based on the evidence of monoamines involvement (schizophrenia, depression, obesity), the identification of monoamines in the diseases processes (Parkinson's disease, addiction) and the prospect of the involvement of monoaminergic mechanisms (epilepsy, Alzheimer's disease, stroke). In most cases, the clinically available monoaminergic drugs induce widespread modifications of amine tone or excitability through neurobiological networks and exemplify the overlap between therapeutic approaches to psychiatric and neurological conditions. More recent developments that have resulted in improved drug specificity and responses will be discussed in this review.peer-reviewe