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The incidence of breast cancer in the city of Palermo and its Province was investigated. The cancer rate was higher in the city of Palermo (100.8/100,000/year), a great southern urban area, than in the 81 municipalities of the Province (79.2/100,000/year). Rates were also compared with those in other geographic areas of Italy, showing a smaller than expected negative north-south gradient in incidence, especially in the young age group, as shown by the cumulative risk observed in the 0-54-year-old group. These findings confirm the role of recent life style changes in the cancer risk distribution.

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Soluble and nuclear estrogen receptor (ER) content was measured by ligand binding assay, and estrogen and progesterone receptors by immunohistochemical assays (ER-ICA and PR-ICA) in 214 patients with breast cancer recruited at the "M. Ascoli" Cancer Hospital Centre in Palermo, Sicily, to assess the discriminant and predictive value of these parameters. On follow-up, data from both ER-ICA and PR-ICA showed a statistically significant difference, PR-positive patients having longer disease-free (DSF) and overall (OS) survival than PR-negative ones. Conversely, ER status did not correlate significantly with both DFS (P = 0.6) and OS (P = 0.2). In particular, PR-positive patients had 59 +/- 18 months DFS and 67 +/- 12 months OS, compared to 51 +/- 22 months DFS and 57 +/- 17 months OS of PR-negative cases. The present evidence implies that a PR-negative status identifies breast cancer patients with early relapse, as also suggested by previous studies. It also agrees with the results of ligand binding assay of ER, where ER status is a good discriminant and predictor of response to endocrine treatment, but is unable to anticipate early relapse in breast cancer patients. Evidence that PR status is a statistically significant prognostic indicator deserves further study to ascertain whether or not PR should be regarded as an ER-dependent parameter or be related to other biological variables such as growth factor (e.g., EGF), oncogene (e.g., Her2/Neu), or tumor suppressor gene (e.g., p53) products.

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Preliminary evidence from a case control study of healthy postmenopausal women living in Palermo, Sicily, is presented to investigate the potential impact of a traditional Mediterranean diet on the risk of developing breast cancer. Of the 230 women who fulfilled specific eligibility criteria, 115 were enrolled in the study based on serum testosterone values equal to or greater than the median population value (0.14 microg/ml). Women were then individually randomized into a diet intervention (n = 58) and a control (n = 55) group. Women in the intervention group attended a weekly "cooking course" for 1 year, being trained by professional chefs in the correct use of the natural ingredients of the traditional Mediterranean diet, including whole cereals, legumes, seeds, fish, cruciferous vegetables, and many others. The intervention group was subsequently instructed to follow the learned diet at home, while the control group was only advised to increase the consumption of fruits and vegetables, as recommended by WHO. The following measures were taken at the beginning, middle, and end of the study: (a) fasting blood and 12-hour urine samples to assay defined hormonal endpoints; (b) height, weight, and circumference of the waist and hip; and (c) a food frequency and computerized 24-hour dietary recall questionnaire. After 1 year, both the control and the intervention groups showed satisfactory compliance rates (81 and 85%, respectively). In addition, preliminary results so far obtained reveal an unequivocal trend towards weight loss, a strong reduction in cholesterol levels, and a psychophysical feeling of well-being by women adopting the Mediterranean diet. The study is currently ongoing to verify the association of changes in serum and urine hormone levels and breast cancer risk in the intervention group.

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Data on androgen receptor (AR) status of nontumoral and malignant human colorectal tissues are compared using ligand binding assay in 22 patients who underwent surgery for colorectal cancer at the "M. Ascoli" Cancer Hospital Centre in Palermo, Sicily. In nontumoral tissues, ARs were predominantly (67%) positive, with 25% of cases having a 0/+ status. Conversely, malignant tissues showed only 32% of cases with a positive (+/+) AR status, with a proportional increase of 0/+ cases (from 25% to 55%); the extent of AR-negative (0/0) cases remained fairly constant (8-9%). Overall, our evidence indicates that nontumoral colorectal tissues have a predominantly positive (+/+) AR status and that this condition shifts towards a significant decrease of AR-positive cases in cancer tissues. Studies on the relation between status of sex steroid receptors and specific biomolecular markers in human colorectal tumors are currently being carried out in our laboratories.

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In the present work, we have inspected expression of estrogen receptors (ER) and their regulation by the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) in a leukemic cell line, the THP-1 cells, using multiple experimental approaches. Firstly, ligand binding assay (LBA) revealed that control (unstimulated) THP-1 cells express type I (high affinity, limited capacity) ER in the nuclear fraction only, whilst treatment of cells with TPA resulted in the appearance of type I ER in the soluble fraction as well, with the 50 ng/ml dose and the 48 h incubation time being the most effective experimental condition. A concomitant increase of type II ER was also seen in both soluble and nuclear cell fractions. Unstimulated THP-1 cells were found to be ER negative by immunocytochemistry; conversely, cells exposed to 50 ng/ml TPA for 48 h stained positively for ER, with the majority of cells having a strong nuclear staining. Scrutiny of ER mRNA expression using reverse transcriptase-polymerase chain reaction showed the presence of a wild type ER transcript in both control and TPA-treated THP-1 cells, though levels of ER mRNA were found to be comparatively higher in the latter. This combined evidence would imply that the TPA-induced differentiation of THP-1 cells is accompanied by the rise of high affinity (type I) ER, suggesting that estrogens may play a role in the regulation of macrophage activity during the inflammatory and/or the immune response.

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Our previous studies are reviewed and at the same time preliminary experimental observation to the topic of endocrine end-points in autoimmune disease is introduced. To this end, we have used rheumatoid arthritis (RA), including synovial fluids and primary cultures of synovial macrophages, as a model system in order to investigate (a) expression and subcellular localization of high-affinity sites of steroid binding in immune effector cells; (b) steroid metabolic profiles in both male and female RA patients, as compared to healthy subjects; and (c) activities of key steroid enzymes that govern intratissue accumulation of sex hormones. In RA tissues and cells, the concurrent evidence for (1) androgen and/or estrogen receptors, (2) high concentrations of biologically active steroids, (3) key enzymes of steroid metabolism, and (4) significant changes of estrogen to androgen ratio, all strongly suggests that individual immune cells, including synovial macrophages, may behave as steroid-sensitive cells, namely, they may represent a target for sex steroids, supporting the hypothesis of a potential endocrine regulation of the immune response also in RA disease. In this respect, definition of several endocrine end-points may have important implications for the treatment of rheumatic disease and other immunological disorders.

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OBJECTIVE: To investigate gap-junctional intercellular communication (GJIC) in LNCaP and DU145 human prostate cancer cells. STUDY DESIGN: Normal rat liver F344 (WB1) cells were used as positive controls. Functional GJIC was inspected using either the scrape-loading/dye transfer (SL/DT) method or fluorescence recovery after photobleaching (FRAP) analysis. In the former, GJIC activity was expressed as a measure of the extent of diffusion of Lucifer Yellow after cell monolayers were scraped using a surgical blade and exposed to dye for a few minutes at room temperature. In the latter, cells were incubated for 15 minutes at 37 degrees C with 5,6-carboxyfluorescein diacetate dye and the dye transfer visualized by photobleaching individual cells with a 488-nm laser and monitoring the recovery of fluorescence using a laser cytometer. RESULTS: The preliminary results obtained indicate that neither LNCaP nor DU145 cells have functional GJIC, while, as expected, WB1 cells show unimpaired GJIC activity. Equivalent results were consistently obtained using either SL/DT or the FRAP approach. However, using FRAP analysis, DU145 cells only showed weak recovery of fluorescence after a total observation interval of 15 minutes. CONCLUSION: The present data, though preliminary, suggest that disruption of GJIC may play a role in development of malignancy in the human prostate.

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OBJECTIVE: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis. STUDY DESIGN: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system. RESULTS: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse. CONCLUSION: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.

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Targeted overexpression of the c-myc oncogene induces neoplastic transformation in immortalized, non-tumorigenic mouse mammary epithelial cells (MMEC). Experiments in the present study were conducted to examine whether cellular transformation induced by c-myc oncogene is associated with altered metabolism of 17beta-oestradiol (E2). The parental, MMEC and the stable c-myc transfectant (MMEC/myc3) cell lines were compared for major oestrogen metabolic pathways, namely E2 and E1 interconversion, and C2- and C16alpha-hydroxylation by both high-pressure liquid chromatography (HPLC) analysis and the 3H release assay using specifically labelled [C2-3H]E2 or [C16alpha-3H]E2. The reductive conversion of E1 to E2 was about 14-fold and 12-fold higher than the oxidative conversion of E2 to E1 in MMEC and MMEC/myc3 cells respectively. However, in MMEC/myc3 cells, both reductive and oxidative reactions were decreased by about 32% and 12% relative to those seen in the parental MMEC cells (P = 0.0028). The extent of C16alpha-hydroxylation was increased by 164.3% (P < 0.001), with a concomitant 48.4% decrease (P < 0.001) in C2-hydroxylation in MMEC/myc3 cells; this resulted in a fourfold increase in the C16alpha/C2 hydroxylation ratio in this cell line. Thus, a persistent c-myc expression, leading to aberrant hyperproliferation in vitro and tumorigenesis in vivo, is associated with an altered oestrogen metabolism. However, it remains unclear whether this represents a result of oncogene expression/activation or is rather a consequence of phenotypic transformation of the cells.

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The 17beta-hydroxysteroid dehydrogenase (17betaHSD) enzyme system governs important redox reactions at the C17 position of steroid hormones. Different 17betaHSD types (no. 1-4) have been identified to date in peripheral human tissues, such as placenta, testis, and breast. However, there is little information on their expression and activity in either normal or malignant prostate. In the present work, we have inspected pathways of 17beta-oxidation of either androgen or estrogen in human prostate cancer cells (LNCaP, DU145, and PC3) in relation to the expression of messenger RNAs (mRNAs) for 17betaHSD types 1-4. These cell systems feature distinct steroid receptor status and response to hormones. We report here that high expression levels of 17betaHSD4 were consistently observed in all three cell lines, whereas even greater amounts of 17betaHSD2 mRNA were detected solely in PC3 cells. Neither 17betaHSD1 nor 17betaHSD3 mRNAs could be detected in any cell line. From a metabolic standpoint, intact cell analysis showed a much lower extent of 17beta-oxidation of both androgen [testosterone (T)] and estrogen [estradiol (E2)] in LNCaP and DU145 cells compared to PC3 cells, where a greater precursor degradation and higher formation rates of oxidized derivatives (respectively, androstenedione and estrone) were observed. Using subcellular fractionation, we have been able to differentiate among 17betaHSD types 1-4 on the basis of their distinct substrate specificities and subcellular localization. This latter approach gave rise to equivalent results. PC3 cells, in fact, displayed a high level of microsomal activity with a low E2/T activity ratio and approximately equal apparent Km values for E2 and T, suggesting the presence of 17betaHSD2. Dehydrogenase specific activity with both E2 and T was also detected, although at lower levels, in LNCaP and DU145 cells. No evidence for reductase activity could be obtained in either the soluble or microsomal fraction of any cell line. As comparable expression levels of 17betaHSD4 were seen in the three cell lines, 17betaHSD2 is a likely candidate to account for the predominant oxidative activity in PC3 cells, whereas 17betaHSD4 may account for the lower extent of E2 oxidation seen in both LNCaP and DU145 cells. This is the first report on the expression of four different 17betaHSD types in human prostate cancer cells. It ought to be emphasized that for the first time, analysis of different 17betaHSD activities in either intact or fractionated cells harmonizes with the expression of relevant mRNAs species.

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In the present study we have inspected estrogen metabolism in cultured human prostate cancer cells (LNCaP, DU145, PC3), in relation to the expression of mRNAs for different 17 beta hydroxysteroid dehydrogenase (17 beta HSD) enzymes (from 1 to 4). Using an intact cell analysis, we have compared precursor degradation and product formation after incubation of cells with physiological amounts of radioactive E2 or estrone (E1) for 24-72 h and subsequent reverse-phase high performance liquid chromatography analysis. The LNCaP and DU145 cells only partly converted E2 to E1 (26 and 13% at 72 h, respectively), giving rise to an appreciable production of E2 from E1 (nearly 20% in all cases). Conversely, PC3 cells revealed a massive E2 oxidation to E1 (up to 90% by 72 h) and a scant formation of E2 (<2%) from E1. In addition, an appreciable formation of 16 alpha OHE1 was seen in either PC3 (11%) or DU145 (5%) cells. respectively using E2 or E1 as precursor. All three cell lines exhibited marked amounts of 17 beta HSD4 mRNA species, whilst even greater amounts of 17 beta HSD2 transcript were found in PC3 cells only. No mRNA for either 17 beta HSD1 or 17 beta HSD3 could be detected in any cell line. The present evidence indicates that pathways of estrogen metabolism are distinctly governed in prostate cancer cells depending on their endocrine status, being associated with a differential expression of mRNA for different 17 beta HSD enzymes.

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We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 microM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (delta4Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 microM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to delta4Ad. In either cell line, T transformation to delta4Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.

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A constant dose-dependent side-effect in cyclosporin A (CSA)-treated patients is the appearance of hypertrichosis; this occurs in both sexes and suggests an androgenizing activity. To determine the influence of CSA on peripheral androgen metabolism, we evaluated in rheumatoid arthritis (RA) patients treated with low-dose CSA (3.5 mg/kg/day), during a period of 12 months, plasma levels of testosterone (Tes) and of 5alpha-androstane-3alpha, 17beta-diol glucuronide (Adiol-G), an important peripheral Tes metabolite. Clinical and laboratory parameters of RA were also monitored. Furthermore, the metabolism of physiological concentrations of Tes (1 x 10(-8) M) was evaluated in primary cultures of RA synovial macrophages (M phi) in the presence of CSA concentrations close to the pharmacological immunosuppressive doses (100-500 ng/ml). At the final time of observation (12 months), a significant increase in the mean plasma Adiol-G level was observed in patients of both sexes. The increase was evident after 1 month of treatment in male patients (P < 0.01) and after 3 months in female patients (P < 0.05). Almost all the patients experienced the side-effect of a low-degree hypertrichosis after a mean period of 1-2 months. No significant correlations with the laboratory parameters of the disease were observed. Results from in vitro experiments on Tes metabolism by cultured synovial M phi showed at 24 and 48 h, in the presence of CSA, a significantly (P < 0.0001) greater formation of dihydrotestosterone and increased amounts of other Tes metabolites, including androstenedione, androsterone and epiandrosterone, when compared to untreated controls. In conclusion, the appearance of a dose-related hypertrichosis and the increase in plasma androgen metabolites (i.e. Adiol-G) in CSA-treated patients, as well as the hormonal metabolic effects on cultured synovial M phi, should be regarded as possible markers of the influence of CSA on peripheral androgen metabolism at the level of target cells.

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Sex hormones have been proposed to play an important role in promoting liver cancer transformation. The aim of our study was to evaluate changes in circulating levels of estradiol (EII), testosterone (T) and the EII/T relationship (ETR) in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) of viral origin compared with a group of healthy controls (C). The study population included 64 patients (41 M) mean age 62.5 years with HCC; 68 patients (41 M) mean age 61.3 years suffering from LC, while the C included 59 subjects (39 M) mean age 60.0 years recruited from voluntary blood donors. EII and T were assayed using the IEMA method; ultrasonography was performed using a Toshiba SSA 240 A scanner with a convex 3.75 MHz probe. Serum EII levels progressively increased from C to LC and HCC with statistically significant values (H=36.9, p<0.0001). Serum values of T progressively decreased from C to LC and HCC but the difference was not significant (H=3.84, p=ns). ETR values differed in the three groups, with a significant difference between C vs LC and HCC (p<0.0001). There was also a significant difference for EII, with values decreasing as the neoplasm dimension increased (p<0.04), and in particular there were differences between HCC <5 cm vs >5 cm (p<0.05). In contrast, ETR progressively increased as the diameter of neoplasm increased, but differences were significant only between <3 cm vs >5 cm (p<0.05). In conclusion, our data confirm that in LC and HCC there is an increase in serum EII levels, which can be important in the genesis of liver carcinoma. Progressive serum reduction in T may be due to increased androgen uptake and progressive accumulation within the neoplastic mass. Further studies are necessary to determine whether subjects with LC and elevated serum levels of estrogens are at higher risk of developing HCC.

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This paper summarizes our most recent results of steroid enzyme studies on cultured breast and endometrial cancer cells. It deals mainly with estrogen 17 beta-hydroxysteroid oxidoreductase (17 beta HSOR) activity, which presides over estradiol (E2) and estrone (E1) interconversion, a major metabolic pathway of estrogens. Assessment of either the oxidative or reductive component of 17 beta HSOR was carried out on intact cells by means of an original approach based on reverse phase-high performance liquid chromatography and radioactive detection on line. This system allows the continuous monitoring of both precursor degradation and formation of several radiometabolites to assess rates and direction of steroid metabolism. Overall, hormone-responsive, estrogen receptor (ER)-positive cells, regardless of whether they were derived from breast (MCF7) or endometrial (Ishikawa) tumor tissues, showed a prevalence for reductive metabolism (E1-->E2), whilst oxidative pathways (E2-->E1) were largely dominant in non-responsive, ER-poor mammary (MDA-MB231) and endometrial (HEC-1A) cells. The above estimates of 17 beta HSOR activity were at variance with those obtained using the classical enzymology approach, not only in quantitative terms (being markedly lower using intact cell analysis), but also because the prevalent direction of estrogen metabolism was often reversed. Although striking methodological differences may well account for this discrepancy, intact cell analysis is undoubtedly more similar to the in vivo state than the artificial requirements of classical enzymology procedures.

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