Letizia Cocciadiferro

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.

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.

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.

Intercellular Communication and Human Prostate Carcinogenesis

Abstract: Gap‐junction‐mediated intercellular communication (GJIC) is required for completion of embryonic development, tissue homeostasis, and regulation of cell proliferation and death. Although, as emphasized in several reports, defects or disruption of GJIC may be important in carcinogenesis, the potential role of GJIC in the onset and progression of human prostate cancer remains ill‐defined. The gap junction channel‐forming connexins (Cx) comprise a multigene family of highly conserved proteins that are differentially expressed in a tissue‐ and development‐specific manner; changes in connexin expression are also commonly seen during cellular differentiation. However, when multiple connexins are concurrently expressed, gap junction channels may consist of more than one connexin species. This is important, because only certain pairings give rise to functional channels. In our studies, we investigated GJIC in a panel of both nontumorigenic (RWPE‐1) and malignant (RWPE‐2, LNCaP, DU‐145) human prostate epithelial cells, compared to a normal rat liver epithelial F344 (WB‐1) cell line, as it was found to be junctionally proficient. In addition, expression and regulation of Cx43 and Cx32 were also inspected using western blot analysis. The ability of hormones, antihormones, and the antihypertensive drug forskolin to restore GJIC in nontumorigenic and malignant human prostate epithelial cells was examined by the scrape‐loading/dye transfer (SL/DT) or fluorescence recovery after photobleaching (FRAP) methods using an Ultima laser cytometer. Results from both assays showed that neither nontumorigenic nor malignant prostate cells have functional GJIC. However, both estrone (E1) and forskolin (FK) induced a significant increase (4.4‐ and 2.8‐fold, respectively) in cell‐cell communication only in the RWPE‐1 cells. Interestingly, the use of Matrigel, a solubilized basement membrane, as substrate for cell attachment and growth resulted in the rescue of GJIC activity in RWPE‐1 cells, as revealed by the SL/DT method. Furthermore, E1 induced a twofold increase in connexin 43 (Cx43), whereas forskolin caused a 50% reduction in Cx32 expression in RWPE‐1 cells. These data suggest that agents that increase Cx43:Cx32 ratio may restore GJIC in junctionally deficient cells, providing a basis for the development of new strategies for the prevention and treatment of human prostate cancer.

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.

Intercellular Communication and Human Hepatocellular Carcinoma

Abstract: We have previously reported that gap junction‐mediated intercellular communication (GJIC) can be restored in junctionally deficient human prostate epithelial cells, also suggesting that GJIC activity is regulated by estrogen. In the present work, we report studies on sex steroid regulation of GJIC and proliferative activity in both nontumoral (Chang liver, CL) and malignant (HepG2, Huh7) human liver cells. Junctional activity and liver cell growth were measured using the scrape‐loading/dye‐transfer (SL/DT) and the MTS assay, respectively. Using the SL/DT, only Huh7 cells exhibited a moderate degree of junctional activity in basic conditions, while neither CL nor HepG2 cells showed functional GJIC. Under exactly the same experimental approach used for prostate studies, we observed that, once again, both estrogen (either estradiol or estrone) and FK induce a significant increase of GJIC in Huh7 cells, while exposure of HepG2 cells to FK produces only a limited rise of junctional activity in this cell line. However, estrogen induced a significant increase and reduction of the proliferative activity of CL and Huh7 cells, respectively, while growth of HepG2 cells was not affected. While the above evidence suggests that estrogens are primarily implicated in growth regulation and communication of both prostate and liver epithelial cells, it also implies that compounds able to restore GJIC in junctionally deficient cells or prevent its disruption in junctionally proficient cells may be used for development of new strategies in the prevention and/or treatment of several human malignancies, including hepatocellular carcinoma (HCC).

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.

Sildenafil protects epithelial cell through the inhibition of xanthine oxidase and the impairment of ROS production.

Abstract Xanthine oxidase (XO) plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases and chronic heart failure. The XO inhibitors allopurinol and oxypurinol held considerable promise in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil and with zaprinast the authors have investigated the potential effects of these latter compounds on the buttermilk XO and on non-tumourigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthine-induced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5.