Nutritional antioxidants as antidegenerative agents.

In this study, primary cultures of cerebellar granule neurons were prepared from eight-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. All experiments were performed with fully differentiated neurons (eight days). To induce apoptosis, culture medium was replaced with a serum-free medium (containing 5 mM KCl) eight days after plating. In another series of experiments, apoptosis was induced by application of glutamate (50 microM) to the cell cultures. Apoptosis was measured by flow cytometry, the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling) method, and by the classical method of DNA fragmentation. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by both low K(+) concentrations and glutamate, a series of natural antioxidants and a red wine lyophilized extract (which is rich in antioxidant compounds) were tested in our experimental model. It was found that ascorbic acid (30 microM) and a red wine lyophilized extract (5 microgram/ml) were capable of blocking the apoptotic process. Addition of the following natural antioxidants did not have any protective effect on apoptosis induced by low K(+) concentrations: trans- and cis-resveratrol (5-200 microM), alpha-tocopherol (100-200 microM), reduced glutathione (100-400 microM), 3-hydroxytirosol (25-100 microM), epicatechin (25-100 microM), or quercetin (25-50 miroM). It is concluded that only a limited number of natural antioxidants are provided with antiapoptotic activity in cultured cerebellar granule neurons. This effect is probably exerted by reducing ROS formation, and by blocking caspase-3 activity.

Laser scanning analysis of cell-cell communication in cultured human prostate tumor cells.

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.

Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy.

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.

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells.

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.

Sex hormones in patients with liver cirrhosis and hepatocellular carcinoma.

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.

17 beta-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells.

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E2) as precursor resulted in: (1) elevated formation (up to 90%) of E2-sulphate (E2-S), using lower precursor concentrations; (2) very limited conversion to estrone (E1) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E2-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E1 production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E1 (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E2 metabolism could be minor in Ishikawa cells as a consequence of the high rate of E2-S formation encountered is contradicted by the evidence that conversion to E1 also remains limited in the presence of much lower E2-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E2) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17 beta-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.

Androgen metabolism and inhibition of interleukin-1 synthesis in primary cultured human synovial macrophages.

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5alpha-treductase. Furthermore, interleukin-1beta production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10(-8) M). A significant decrease of IL-1beta levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms.