Metabolic profiles of androgens in malignant human liver cell lines.

In this study we have investigated androgen (testosterone and androstenedione) metabolism in malignant HepG2, Huh-7, and HA22T human liver cell lines. Following 72-h incubation with testosterone or androstenedione, estrogen formation through aromatase activity was consistently higher in HepG2 cells (being nearly 100%) and moderate in Huh7 cells (34%), while it was undetectable in HA22T cells. The produced estrogens are completely conjugated by estrogen sulpho-transferase (EST) in HepG2 cells, while nearly 25% remains in the free form in Huh-7 cells. The HA22T and Huh-7 cells show a markedly different balance of 5alpha- versus 5beta-reduced androgens (65.7% vs. 2.5% and 2.6% vs. 22.2%, respectively), while no detectable 5alpha/5beta-reduced androgen is formed in HepG2 cells. These divergent metabolic profiles, coupling aromatase to EST, and to 5alpha/5beta-reductase, hint at a differential regulation of androgen metabolic pathways that may ultimately lead to a distinct impact of biologically active metabolites on growth and function of human liver cancer cells.

Local estrogen formation by nontumoral, cirrhotic, and malignant human liver tissues and cells.

We have investigated the activity and expression of aromatase enzyme in nontumoral, cirrhotic, and malignant human liver tissues and cells using both chromatographic and reverse transcription (RT)-PCR analyses. After 24- and 72-h incubation of tissue minces or hepatic cell lines with either testosterone or androstenedione as androgen precursor, human hepatocellular carcinoma (HCC) tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates being 20 and >95%, respectively, as opposed to nontumoral hepatic tissues and nonmalignant Chang liver (CL) cells, where no aromatase activity could be detected. Cirrhotic samples exhibited intermediate enzyme activity. Notably, exposure of HepG2 cells to the aromatase inhibitor Letrozole resulted in a striking decrease of estrogen formation, which became virtually absent at a Letrozole dose of 0.4 nM. RT-PCR analysis revealed markedly lower aromatase mRNA in both CL cells and nontumoral liver tissues, as compared with HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels, in turn comparable with those observed in nontumoral or HCC tissues. Exon-specific RT-PCR showed prominent expression of exon I.3A-containing message and exon I.4-containing message in CL and HepG2 cells, as in nontumoral and HCC tissues, respectively. The present evidence implies that locally elevated estrogen formation in malignant human liver tissues and cells may have a role in the development and/or maintenance of human HCC, eventually leading to develop alternative strategies for treatment of HCC patients using antiaromatase agents.

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 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.

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.

Dietary enterolactone affects androgen and estrogen levels in healthy postmenopausal women.

In this randomized dietary intervention study (DI) we analyzed levels of androgens, phytoestrogens, and estrogens in 12-h urine samples of 69 healthy postmenopausal women, 37 of whom followed a traditional Mediterranean diet for 6 months (intervention group) as compared to 32 women who followed their regular diet (control group). Circulating levels of both insulin and testosterone (T) were also assayed. Overall, enterolactone (ENL) was the most prominent phytoestrogen in urines of both control and intervention women, and its levels increased by a 20% after DI. At the baseline the ENL levels were seen to be significantly associated with both the total androgens (TOT-A) (r= 0.371, P= 0.002) and the TOT-A/total estrogen (TOT-E) ratio (r= 0.351, P= 0.005) in all 69 postmenopausal women. Furthermore, the DI resulted in a more pronounced negative association of both ENL with insulin (r=-0.321, P= 0.05) and insulin with TOT-A (r=-0.421, P= 0.01). Regarding urinary androgens, ENL associated with both 3alpha-androsterone (5alpha-androgen, r= 0.363, P= 0.002) and 3alpha-etiocholanolone (5beta-androgen, r= 0.295, P= 0.01) at baseline, while after DI, circulating insulin and T exhibited a significant negative association with the 5beta-androgen metabolite etiocholanolone (r=-0.487, P= 0.002; and r=-0.336, P= 0.042, respectively). We conclude that lignan components of the Mediterranean diet, notably ENL, are associated with urinary levels of products of androgen metabolism, including both 5alpha- and 5beta-reductase enzymes, in healthy postmenopausal women. Further studies are necessary to better understand the interplay of sex hormones with dietary phytoestrogens.

16alpha-hydroxyestrone inhibits estrogen sulfotransferase activity in human liver cancer cells.

In this study we investigated the impact of estrogen antagonists and of 16alpha-OHE1 (an estrogen derivative that binds to and induces transactivation of estrogen receptors) on estrogen metabolism in malignant HepG2 human liver cells featured by high estrogen sulfotransferase (EST); our aim was to clarify the potential correlation of EST and ER. As expected, the HepG2 cells exhibited a very high EST activity, with the majority of estrogen metabolites (over 86%) being detected as sulfates by 24 h. The coincubation of E2 and the antiestrogen tamoxifen induced a weak inhibition of EST activity (from 85.4% to 81.5%), while the coincubation with the pure antagonist ICI-182 and with 16alpha-OHE1 produced a 50% and 90% decrease of EST, respectively. Interestingly, both selective estrogen receptor modulators (SERMs) TAM and ICI-182, along with the same 16alpha-OHE1, gave rise respectively to a 2.8%, 3.2%, and 4.6% of de novo 16alpha-OHE1 formation. The inhibition of EST and the increase of 16alpha-OHE1 formation were both time- and dose-dependent. Our results suggest that EST activity is tightly associated with ER transactivation and can be regulated by selective estrogen receptor modulators (SERMs), including antiestrogens and 16alpha-OHE1. In this framework, 16alpha-OHE1 may have a potential role in human liver carcinogenesis, also through the inhibition of EST and the production of unconjugated, bioavailable estrogens.

Profiling cancer stem cells in androgen-responsive and refractory human prostate tumor cell lines.

In this study, we investigated androgen metabolism in two different human prostate cancer cell lines, the androgen-responsive LNCaP cells and the nonresponsive PC3 cells. Following 24-h and 72-h incubation with either testosterone (T) or androstenedione (Ad) used as precursor, divergent patterns and rates of androgen metabolism were observed. Given the recent interest in the multiple uses of embryonic and adult stem cells for basic and applied research, we compared the expression of three presumptive stem cell markers (Oct-4, SUZ-12, and Cripto-1), along with connexin 43 (Cx43), Cx32, and androgen receptor (AR), used as cell differentiation gene markers. In anchorage-independent cell growth conditions, the expression levels of candidate markers of cancer stem cells initially increased (days 2-4) but drastically fell thereafter (day 6) in both cell lines. Results of immunocytochemical assay (ICA) largely confirmed those obtained by RT-PCR. Interestingly, both symmetrical and asymmetrical cell divisions were revealed in PC3 cells using Oct-4 immunostaining. Our data suggest that both androgen-responsive and androgen-nonresponsive prostate tumor cell lines contain a presumptive cancer stem cell population that can be identified using a panel of selected gene markers, including Oct-4, SUZ-12, and Cripto-1.

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.

Comparison of female breast cancer registration in the city and province of palermo with other italian cancer registries.

The aim of this study was to assess the incidence of breast cancer in women from the city and province of Palermo (Sicily) in 5 yr, 1999-2003, using a population based cancer registry approach. In the last years, a sharp increase of breast cancer incidence has been observed worldwide. Overall, direct age-standardized incidence rates were 89.3 per 100,000 person-yr, being markedly higher in Palermo City (101.0) than in Palermo Province (75.0). Results show a highly significant difference in breast cancer incidence in different areas of Sicily, particularly in the older (>50 yr) age groups and a profound difference between the metropolitan area of Palermo and the surrounding areas. The evidence of the different rates of breast cancer incidence in Palermo City and in the other small municipalities of the Palermo Province suggests a different pattern of breast cancer risk as a consequence of different lifestyle and diet modifications in the urban population of Palermo City.