Lucia M. Polito

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.

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.

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.

Intra-tumoural variation of oestrogen receptor status in endometrial cancer

Soluble and nuclear oestrogen receptor status was determined in both the central and peripheral portions of tumour for 37 cases of adenocarcinoma of the endometrium. Of these, 29 had functional receptor in the peripheral biopsy, but only 19 retained functional receptor in the centre. Six of the 10 patients whose tumours showed this difference came from the group of 12 patients who were immediately post-menopausal (4.50 +/- 1.45 y post-menopausal age). Receptor status was not related to tumour classification into histological grades I and II. However, receptor-negative central biopsies were significantly more likely (P less than 0.05) to be Grade III. Early relapse was also related to a receptor-negative central biopsy.

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.

Molecular expression of 17βhydroxysteroid dehydrogenase types in relation to their activity in intact human prostate cancer cells

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.

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.

Metabolic profiles of androgens in malignant human liver cell lines.

Abstract:  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 5α‐ versus 5β‐reduced androgens (65.7% vs. 2.5% and 2.6% vs. 22.2%, respectively), while no detectable 5α/5β‐reduced androgen is formed in HepG2 cells. These divergent metabolic profiles, coupling aromatase to EST, and to 5α/5β‐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.

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 3α‐androsterone (5α‐androgen, r= 0.363, P= 0.002) and 3α‐etiocolanolone (5β‐androgen, r= 0.295, P= 0.01) at baseline, while after DI, circulating insulin and T exhibited a significant negative association with the 5β‐androgen metabolite etiocolanolone (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 5α‐ and 5β‐reductase enzymes, in healthy postmenopausal women. Further studies are necessary to better understand the interplay of sex hormones with dietary phytoestrogens.

Comparison of Female Breast Cancer Registration in the City and Province of Palermo With Other Italian Cancer Registries

Abstract: 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.