Maurizio Calabrò

Estrogen regulates cytokine production and apoptosis in PMA-differentiated, macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF‐α) and interleukin‐10 (IL‐10) in phorbol‐myristate‐acetate (PMA)‐differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage‐like PMA‐differentiated U937 cells was also inspected. E2 increased TNF‐α synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI‐182,789 completely abolished the induction of TNF‐α, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF‐α production by U937 cells. Exposure of cells to E2 resulted in a dose‐dependent decrease of IL‐10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage‐like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI‐182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro‐inflammatory action through the modulation of TNF‐α and IL‐10, and regulate the immune effector cells by the induction of programmed cell death. J. Cell. Biochem. 90: 187–196, 2003.

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.

CCR5 proinflammatory allele in prostate cancer risk: a pilot study in patients and centenarians from Sicily.

Prostate cancer (PCa) is the most common malignant neoplasm in older men in Western countries. The number of affected older men is increasing. Therefore, strategies for prevention of prostate cancer are crucial. To this purpose it is essential to know the mechanisms involved in development and progression of this malignancy. Recently, an increasing body of genetic and epidemiological studies proposed new hypotheses for prostate carcinogenesis. It has been suggested that genetic factors as well as exposure to environmental factors such as infectious agents, dietary carcinogens, and hormonal imbalances participate in PCa development. Besides, chronic inflammation plays a key role in PCa. Taking into consideration this complex scenario, in the present study we evaluated whether CCR5Δ32 deletion of CCR5 gene might be associated with PCa susceptibility. For the control group we used centenarians, since they represent a disease‐free human model. These preliminary results suggest that the CCR5Δ32 anti‐inflammatory variant might be a resistance factor for the development of PCa.

HER2/neu expression in relation to clinicopathologic features of breast cancer patients

Abstract:  We have evaluated HER2/neu expression in 1,355 breast cancer patients recruited at the Breast Cancer Registry in Palermo between January 1999 and December 2004. In this retrospective study, HER2/neu expression was related to clinicopathologic features of the disease, including tumor size, nodal and menopausal status, estrogen and progesterone receptors. Statistical analysis on all 1,355 patients showed a significant correlation between HER2/neu and nodal status (P < 0.001), and a significant association between HER2/neu overexpression and estrogen and progesterone receptors status (P < 0.001). In 194 patients without metastasis, with an average follow‐up ≥5 years, only HER2/neu 3+ and histopathologic grading G3 were significantly associated with overall survival.

Expression Levels and Clinical‐Pathological Correlations of HER2/neu in Primary and Metastatic Human Breast Cancer

Abstract: In this retrospective study we assessed the expression of the HER2/neu oncogene product in a series of 574 consecutive breast cancer cases, all recruited at the Maurizio Ascoli Cancer Center of Civico Hospital, in Palermo, between January 1998 and June 2003. The HER2/neu expression was evaluated using immunohistochemistry and scored from 0 to +3 as per FDA recommendations. The HER2/neu expression levels were related to the clinical‐pathological features of the disease, including tumor size, nodal and menopausal status, estrogen and progesterone receptors, and hormonal or chemotherapeutic treatment. In 108 patients with a follow‐up period of 3 years or more, the HER2/neu expression was also related to their survival characteristics. A significant correlation (P= 0.011) between HER2/neu +3 and estrogen receptor‐negative cases was observed in the 487 M0 patients. In addition, HER2/neu +3 cases were associated with a positive nodal status (57.4%), although this association was not quite significant (P= 0.06). More importantly, follow‐up data revealed that, in the 91 M0 patients with an average follow‐up period of 37 months, the percentage of HER2/neu +3 patients who relapsed was remarkably greater (54.8%) than that observed for the HER2/neu +1/0 cases when combined (34.2%). Furthermore, the disease‐free interval (DFI) was 47 months in the HER2/neu +1/0 group, while it dropped to 45 months in c‐HER2/neu +3 cases. Although the limited number of cases does not allow us to draw any definitive conclusions, our data suggest that high expression levels of HER2/neu +3 are associated with an early relapse and a shorter disease‐free interval in M0 breast cancer 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.

Soluble and nuclear oestrogen receptor status of advanced endometrial cancer in relation to subsequent clinical prognosis.

Both soluble and nuclear oestrogen receptors have been measured in at least two separate sections from 72 endometrial cancers and 12 normal endometria. Concentration of oestrogen receptor is shown to be, in our hands, more meaningful when expressed per unit DNA than per unit protein, whether for soluble or nuclear receptor. Endometrial cancer cells from the central part of the tumour are shown to be receptor negative more frequently than those from peripheral tumour. Thus, in large cancers, biopsies from different areas are required before a tumour can be correctly designated as receptor positive, heterogeneous or receptor negative. The intratumoral variation of receptor status may relate to poor prognosis, since patients with homogeneous receptor-positive disease survive significantly longer than those with tumours showing either heterogeneous distribution of receptor or homogeneous absence of receptor. Intratumoral variation in receptor status is found to be more common in the group of patients who are within 7 years of their menopause, than in older patients.

Androgen Receptor Status in Nontumoral and Malignant Human Colorectal Tissues

Abstract: 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.

Aromatase and Amphiregulin Are Correspondingly Expressed in Human Liver Cancer Cells

Human hepatocellular carcinoma (HCC) is associated with high mortality rates, being the third most common cause of cancer death worldwide. Although estrogens have been implicated in HCC, their potential role in development and/or progression of this malignancy remains unclear. In this study we investigated mRNA and protein expression of aromatase (Aro) and amphiregulin (AREG) in relation to estrogen receptors (ERs), in HepG2, Huh7, and HA22T human malignant liver cell lines, using RT‐PCR and Western blot analyses. Aro expression was significantly higher (∼13‐fold, P= 0.003) in HepG2 cells than in Huh7 cells, while no Aro expression could be detected in HA22T cells. Interestingly, the patterns of AREG expression were consistently associated with those of Aro, with ∼3‐fold and ∼8‐fold higher levels being seen in HepG2 cells than in Huh7 cells (P= 0.002) and HA22T cells (P= 0.0014), respectively. Using a specific set of primers, ERα46 is the only ER variant expressed in all cell lines, while the wild‐type ERα66 could not be detected. Western blot analysis revealed a corresponding figure. This evidence suggests that AREG expression may be upregulated by estrogens in human HCC and that locally elevated aromatase activity also may increase malignant cell proliferation through AREG signaling.

Ligand binding and cytochemical analysis of estrogen and progesterone receptors in relation to follow-up in patients with breast cancer.

Abstract: 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.