Orazia M. Granata

A Traditional Mediterranean Diet Decreases Endogenous Estrogens in Healthy Postmenopausal Women

Abstract: Breast cancer incidence and mortality rates are markedly lower in the south than in the north of Europe. This has been ascribed to differences in lifestyle and, notably, dietary habits across European countries. However, little information exists on the influence of different dietary regimens on estrogens and, hence, on breast cancer risk. Here we report results of our MeDiet Project, a randomized, dietary intervention study aimed to assess the effect of a Mediterranean diet on the profiles of endogenous estrogens in healthy postmenopausal women. Out of the 230 women who initially volunteered to participate in the study, 115 were found to be eligible and were enrolled. Women were then randomly assigned into an intervention (n = 58) and a control (n = 57) group. Women in the intervention group adhered to a traditional, restricted Mediterranean diet for 6 mo, whereas women in the control group continued to follow their regular diet. Women in the intervention group changed their dietary regimen substantially, and this eventually led to a shift from a prevalent intake of animal fat and proteins to a prevalent intake of vegetable fat and proteins. Regarding urinary estrogens, no significant difference was observed between the intervention and control groups at baseline. After 6 mo, however, control women did not show any major change but women in the intervention group exhibited a significant decrease (over 40%) of total estrogen levels (P < 0.02). The largest part of this modification was based on a marked decrease of specific estrogen metabolites, including hydroxyand keto-derivatives of estradiol or estrone. To our knowledge, this is the first report to show that a traditional Mediterranean diet significantly reduces endogenous estrogen. This may eventually lead to identify selected dietary components that more effectively decrease estrogens levels and, hence, provide a basis to develop dietary preventive measures for breast cancer.

Endocrine End‐Points in Rheumatoid Arthritisa

ABSTRACT: 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.

Expression of Wild-Type and Variant Estrogen Receptor Alpha in Liver Carcinogenesis and Tumor Progression

Although estrogen receptors (ERs) are expressed in human hepatocellular carcinoma (HCC), several clinical trials have failed to demonstrate the efficacy of antiestrogen treatment in HCC patients. Recently, the identification of several ER splicing variants has enlightened the complex nature of estrogen signaling in peripheral tissues; this may help understanding estrogen role in either nontumoral or malignant nonclassical target organs, including liver. In this work we have investigated mRNA expression of wild-type and splice variants of ERα in nontumoral, cirrhotic, and malignant human liver, as well as in HCC cell lines, using an exon-specific reverse transcription polymerase chain reaction (RT-PCR). In particular, ERα66 was detected in nontumoral and, to a lesser extent, in cirrhotic liver tissues, whereas its expression decreased or became undetectable in HCC tissues and cell lines. The ERα46 splicing variant was detected ubiquitously in all samples; interestingly, however, the ERα36 variant was inversely expressed with respect to ERα66, being highest in HepG2 cells, intermediate in Huh7 cells, and lowest in HA22T cells. It is noteworthy that aromatase was correspondingly expressed with ERα36 and inversely related to ERα66. This observation suggests that a switch from ERα66 to a predominant expression of ERα36 may be associated with development and/or progression of human HCC.

Simple approach to measure metabolic pathways of steroids in living cells

A simple, rapid approach to the study of conversion rates and metabolic patterns of the steroids testosterone and estradiol is presented. It includes an optimized isocratic high-performance liquid chromatographic procedure in the reversed-phase mode and radioactive on-line detection. The purpose was to estimate the activity of key enzymes of steroid pathways, such as 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase, in in vivo conditions. Using this system, we obtained good efficiency and linearity of radio detection, under continuous flow conditions. Sensitivity limits were of the order of 50 and 70 cpm for [3H]estradiol and [14C]estrone, respectively, even though the efficiency was quite dissimilar (17.3% versus 56.2%). The applicability of this approach to studies of steroid metabolic pathways in growing cancer cells in culture is illustrated with examples of the conversion rates of both testosterone and estradiol. The high reproducibility (coefficients of variation of 2.7 and 5.1% for 3H and 14C, respectively) and good extraction efficiency (ranging from 86 to 94%) indicate the feasibility and reliability of this approach.

Sex Steroids, Carcinogenesis, and Cancer Progression

Abstract: 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 intratissue 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.

A Role for Sex Steroids in Autoimmune Diseases

Abstract: In recent years there has been a continuingly increasing interest in novel research subjects, as yet poorly explored, either because they relate to aspects previously thought to be marginal with respect to classical fields of investigation, or because they require both specialized competence and intense cross‐talk by researchers from disparate areas. The potential interaction between immunity and cancer has generated a remarkable number of studies, including those related to the newly explored immune‐neuro‐endocrine system. In this paper, we review a few autoimmune diseases as examples of a mutual relationship between immune diseases and malignancies. We also review our previous studies on patients with rheumatoid arthritis (RA). In particular, aiming to define the hormone‐responsive or ‐sensitive status of synovial tissues and cells, we have inspected different endocrine end‐points, including (1) high‐ and low‐affinity sites of androgen and estrogen binding; (2) the activity of key enzymes of steroid metabolism; and (3) the hormonal profile of synovial fluids as an indication of local endocrine milieu. Overall, our data provide convincing evidence for synovial macrophage‐like cells and a subset of T lymphocytes to be considered as target cells for gonadal steroids. This provides a basis for developing new strategies for alternative treatments of RA and possibly unveils novel perspectives in both research and the clinic for other autoimmune diseases as well. In addition, the association of autoimmunity and cancer may disclose promising new avenues of research linking steroid hormones, the immune system, and malignant transformation.

Androgen metabolism and biotransformation in nontumoral and malignant human liver tissues and cells.

There is indirect multiple evidence that hints at a potential role of sex steroids in development and progression of human hepatocellular carcinoma (HCC). In the present study, we have investigated androgen metabolism in a panel of human liver cancer cell lines (HA22T, Huh7, HepG2) and in normal, cirrhotic and malignant human liver tissues aiming to dissect the potential impact of individual enzyme activities and their products in normal and diseased human liver, both in vivo and in vitro. Using our intact cell analysis we were able to assess rates and pathways of androgen metabolism in living conditions. Overall, incubation of cultured cells or tissue minces with either testosterone (T) or androstenedione (Ad) used as precursor resulted in a large extent of 17betaoxidation of T to Ad (cells: 28-77%; tissues: 35-50%). In malignant liver cell lines, both HA22T and Huh7 cells showed consistent amounts of the 5alpha-reductase enzyme products (18% and 15%, respectively), while 5beta-reductase activity was more pronounced in Huh7 cells (18%) than in HA22T cells (1.8%). Interestingly, a significant extent of estrogen formation could be observed in Huh7 cells (5.4-11.5%), while no aromatase activity could be detected in HA22T cells. In HepG2 cells, along with a relatively high proportion of Ad, estrogens represented the most prominent (50-55%) end product of androgen metabolism, regardless of the precursor used. In liver tissues, equivalent results could be obtained, with a consistent proportion of 17betaoxidation of T to Ad (35-50%) being observed in the majority of samples. However, while normal liver tissue samples exhibited a minor proportion of bioactive androgens (3.4%) with no aromatase products, HCC tissues showed a significant extent of aromatase activity (nearly 20%) with estrogen representing the most prominent metabolic product after 24h incubation with either T or Ad. HCV and alcoholic cirrhotic tissues displayed different patterns of androgen metabolism. The former produced limited amounts of bioactive androgens (5.3%) and considerable levels of the intermediate aromatase product 19OH-Ad (up to 28%), the latter exhibited a prevalence of androgen degradation through the 5beta-reductase pathway (9.8%) and a significant extent of aromatase activity (16% as a whole). In conclusion, three major metabolic states could be depicted, depending on prevalent pathways of androgen metabolism and steroid receptor status: estrogenic, androgenic, and mixed. This model supports the idea that local estrogen biosynthesis may be implicated in human HCC and provides a basis for the exploitation of aromatase inhibitors and/or ER antagonists or selective estrogen receptor modulators (SERMs) as a new therapeutic strategy in HCC patients.

Nutrition, aging and cancer: lessons from dietary intervention studies

There is convincing epidemiological and clinical evidence that, independent of aging, lifestyle and, notably, nutrition are associated with development or progression of major human cancers, including breast, prostate, colorectal tumors, and an increasingly large collection of diet-related cancers. Mechanisms underlying this association are mostly related to the distinct epigenetic effects of different dietary patterns. In this context, Mediterranean diet has been reported to significantly reduce mortality rates for various chronic illnesses, including cardiovascular diseases, neurodegenerative diseases and cancer. Although many observational studies have supported this evidence, dietary intervention studies using a Mediterranean dietary pattern or its selected food components are still limited and affected by a rather large variability in characteristics of study subjects, type and length of intervention, selected end-points and statistical analysis. Here we review data of two of our intervention studies, the MeDiet study and the DiMeSa project, aimed at assessing the effects of traditional Mediterranean diet and/or its component(s) on a large panel of both plasma and urine biomarkers. Both published and unpublished results are presented and discussed.

Altered androgen metabolism eventually leads hepatocellular carcinoma to an impaired hormone responsiveness

Sex steroid hormones are thought, among several other risk factors, to play a role in liver malignancies. For example, from epidemiological studies in hepatocellular carcinoma (HCC), a clear disadvantage for male sex is evident. In addition, elevated levels of serum testosterone (T) and increased T to Estradiol (E(2)) ratio have been reported to predict an increased risk of HCC for male cirrhotic patients. On the other hand, palliative treatment of liver cancer patients with anti-hormones has been widely used in the past. However, the molecular mechanism(s) underlying sex steroid action on either normal or transformed liver cells, have not yet been fully clarified, nor endocrine discriminants have been satisfactorily assessed for an adequate characterization of liver cancer tissues. In this paper, we report studies on hormonal status of human liver tissues and cells, especially focusing on androgens, to better define endocrine end-points of interest for HCC. A consistent evidence from ex vivo or in vitro systems strongly suggests that high affinity binding sites of androgens are expressed at sufficient concentrations to induce a biological response in either normal or phenotipically transformed hepatocytes; in the latter, however, high heterogeneity and/or more scattering concentrations were encountered. Further, experimental data seem to suggest that lack of response to androgens may be due to a rapid metabolic conversion of steroids by neoplastic tissues and cells. Cancer hepatocytes privilege in fact 5beta more than 5alpha metabolic pathway of androgens. This may eventually lead biologically active androgens to be transformed into less active derivatives, as it occurs for T which is massively converted (>90% at 6 h) thus hindering the whole mechanism of action of androgens.

The Mediet Project

Abstract: 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 μg/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.