Valentina Martineti

Azidothymidine Induces Apoptosis and Inhibits Cell Growth and Telomerase Activity of Human Parathyroid Cancer Cells in Culture

Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase‐positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation.

Estrogen synthesis in human colon cancer epithelial cells.

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [(3)H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis-Menten kinetic with apparent Km values of approximately 20 nM and V(max) values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 microM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 microM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients.

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.

Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus

The effects of phytoestrogens have been studied in the hypothalamic‐pituitary‐gonadal axis and in various non‐gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor β, the predominantly expressed estrogen receptor subtype in colon mucosa.

Aromatase expression and activity in the human leukaemic cell line FLG 29.1

The recent observation that estrogen synthesis occurs in osteoblast-like cells has suggested the aromatase activity as a possible local modulator of bone remodeling in post-menopausal women. To provide further insights into the androstenedione conversion to estrogen in bone-derived cells, we examined the human leukaemic cell line FLG 29.1, which is induced to differentiate toward the osteoclastic phenotype by TPA and TGF-beta1. Southern blot of RT-PCR products with a 32P-labeled cDNA probe for the human aromatase demonstrated that FLG 29.1 cells express aromatase mRNA. The enzyme activity, determined by measuring [3H]H2O release from [3H]androstenedione, obeyed Michaelis-Menten kinetic with apparent Km and Vmax values ranging from 5 to 10 nM and from 200 to 400 fmol/mg protein/6 h. Gene expression, enzyme activity and protein immunoreactivity, evaluated by immunocytochemistry, were stimulated in a time-dependent fashion by 5% charcoal-stripped FCS and by either 1-100 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 h exposure. After 24 h incubation of FLG 29.1 cells in the absence of these stimuli the aromatase mRNA and the protein were barely detectable. These findings demonstrate that cells of the osteoclastic lineage synthesize estrogen in vitro and that local cytokines, such as TGF-beta1, are able to induce androstenedione conversion.

In vitro effects of oestrogens, antioestrogens and SERMs on pancreatic solid pseudopapillary neoplasm-derived primary cell culture

Background: Solid-pseudopapillary neoplasms of the pancreas (SPNs) are uncommon tumours usually frequent in young women. Although the pathogenesis of SPNs is uncertain a potential influence of the sex hormone milieu on the biology of these tumours has been suggested. The controversial expression of oestrogen receptors (ERs) in SPNs, provide a rationale for studying the effects of oestrogenic molecules on SPN development. Methods: The expression of a large series of hormonal ligands and receptors was evaluated in tissue specimens and in a primary cell culture (SPNC), obtained from a SPN in young female patient. The effects of 17β-oestradiol (17βE2), ICI 182,780 and tamoxifen (Tam) on cell replication and growth were examined. Results: We have established SPNC primary line. Immunocytochemical analysis was positive for vimentin, cyclin D1 and β-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERα, ERβ and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17βE2 and Tam respectively were proved in SPNC. Conclusions: In conclusion, we provide the first direct evidence that oestrogenic molecules can influence proliferation of SPNC, offering future strategies in the control of this neoplasia via selective ER modulators.

Genetics and pharmacogenetics of estrogen response

Estrogens are a steroid hormone group distributed widely in animals and human beings. Estrogens diffuse across cell phospholipidic membranes and interact with estrogen receptors. Their highest concentration is found in target tissues with reproductive function (breast, ovary, vagina and uterus). High estrogen levels are usually associated with tumor onset and progression, while loss of estrogen or its receptor(s) contributes to development and/or progression of various diseases (osteoporosis, neurodegenerative disease and cardiovascular disease). Despite the numerous efforts to highlight estrogen’s mechanism of action, recent discoveries showed an unexpected degree of complexity of estrogenic response.

Control of colon cancer development and progression by selected estrogen receptor modulators

Estrogens behave as protective agents on the development of colorectal cancer, and hormonal-replacement therapy is associated with an increased survival rate in women with this disease, indicating that estrogenic therapy correlates with a better prognosis. The protective effect of estrogens on Fcolorectal cancer development and progression is presumably related to the expression of estrogen receptors in colon mucosa, with the estrogen receptor-β isoform being the predominant one. This observation suggests that estrogen receptor-β could have an inhibitory effect on colorectal cancer cell proliferation and a regulatory effect on colonic mucosa cell growth, opening the discussion on a pharmacologic approach to colorectal cancer prevention and therapy based on estrogenic compounds.