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 17beta-oestradiol (17betaE2), 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 beta-catenin and negative for cytokeratin, CD10 and neuroendocrine markers, in line with the immunostaining features of the tumoral tissue. Expression of ERalpha, ERbeta and progesterone mRNAs was demonstrated in SPNC and tumor tissue. A proliferative and antiproliferative action of 17betaE2 and Tam respectively were proved in SPNC. CONCLUSION: 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.

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 beta, the predominantly expressed estrogen receptor subtype in colon mucosa.To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor beta, beside its native cell line, expressing very low levels of ERbeta and not expressing ERalpha; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor beta overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G(0)/G(1) cell fraction and in estrogen receptor beta overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor beta.Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications.

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-ß isoform being the predominant one. This observation suggests that estrogen receptor-ß 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.

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.

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.

ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components.

Several strands of evidence indicate that oestrogens exert a protective role against the development of colon cancer through indirect and direct effects on colonic epithelium. Oestrogen receptor beta (ERbeta), the predominant ER subtype in human colon, is significantly decreased in colonic tumours compared with normal mucosa suggesting a potential role in the regulation of colon tumour growth. To investigate this hypothesis we engineered human colon cancer ERalpha-negative HCT8 cells in order to obtain ERbeta protein over-expression. Stably transfected cells were cloned and ERbeta expression and functionality were monitored by RT-PCR, Western blotting and transactivation in an assay using oestrogen-responsive reporter constructs. Over-expression of ERbeta inhibited cell proliferation and increased cell adhesion in a ligand-independent manner. Its constitutive activation is possibly due to cross-talk with intracellular signalling pathways, as epidermal growth factor and IGF-I were able to induce ERbeta transactivation. A possible mechanism by which ERbeta over-expression inhibits proliferation in HCT8 cells is by modulation of some key regulators of the cell cycle; there is a decrease in cyclin E and an increase in the cdk inhibitor p21CIP1. In fact, flow cytometry analysis provided evidence for blocking of the G1-S phase progression induced by ERbeta over-expression. The magnitude of this effect was affected by the level of ERbeta expression. These results provide the first direct evidence that ERbeta plays an important role in colon cancer as a regulator of cell proliferation through the control of key cell cycle modulators and arrest in G1-S phase transition. These findings are compatible with the hypothesis that the loss of ERbeta expression could be one of the events involved in the development or progression of colon cancer.

Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture.

UNLABELLED: 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. INTRODUCTION: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions. MATERIALS AND METHODS: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol-positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3'-azido-3'deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes. RESULTS: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT-monophosphate (AZT-MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture. CONCLUSIONS: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.

Growth response of colon cancer cell lines to selective estrogen receptor modulators.

BACKGROUND: The purpose of this study was to compare the "in vitro" effects of the selective estrogen receptor modulators, tamoxifen and raloxifene, on two human colon cancer cell lines. MATERIALS AND METHODS: Serial concentrations (0.1, 1, 5 and 10 microM) of tamoxifen and raloxifene were used and evaluated for cell proliferation, viability and apoptosis in HCT8 and HCT116 cells. RESULTS: Micromolar doses of raloxifene significantly reduced HCT116 and HCT8 cell proliferation. Tamoxifen (5 microM) strongly reduced HCT8 cell growth with minor effects on HCT116 cells. Raloxifene (10 microM) was lethal on both cell lines, while 10 microM tamoxifen caused lethality only in HCT8 cells. Five microM raloxifene reduced cell viability in HCT8 and HCT116 cells, while 5 microM tamoxifen halved only HCT8 cell viability. Raloxifene and tamoxifen did not induce apoptosis in the two cell lines. CONCLUSION: Tamoxifen, and even more raloxifene, were effective in reducing HCT8 and HCT116 cell proliferation and viability, suggesting their potential application in the prevention and therapy of colorectal cancer.