Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERβ and ERβ by immunohistochemistry and in situ hybridization. ERα, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERα-positive cells increased signiWcantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17- estradiol (E2), but not E 2 + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a signiWcant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy

Telomerase repeat amplification protocol (TRAP): A new molecular marker for parathyroid carcinoma

Telomerase results to be active in human germ, stem cells, several malignant cell tumors and in immortalized cell lines. In order to investigate if molecular mechanisms other than Rb gene inactivation can be helpful to diagnose malignancy of parathyroid tumors, we decided to investigate the presence of active telomerase in homogenates from different pathological parathyroid tissues (hyperplastic, adenomatous, carcinomatous, and normal) and primary cell cultures. The TRAP assay was performed to detect this activity in histologically characterized normal, hyperplastic, adenomatous, and carcinomatous human parathyroid tissues, primary cell lines, and one metastatic tissue from parathyroid carcinoma. Only malignant parathyroid glands and the metastatic tissue were TRAP positive. Our findings suggest that telomerase expression could represent an important molecular mechanism underlying the acquisition and progression of an aggressive phenotype of epithelial parathyroid cells and it may help to predict their malignant potential. The TRAP assay is easy to perform and it could become an additional tool to be included in the harmamentarium for the molecular diagnosis of parathyroid carcinoma

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 ER beta and not expressing ER alpha; 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. Copyright (C) 2010 John Wiley & Sons, Ltd

AZIDOTHYMIDINE INDUCES APOPTOSIS AND INHIBITS CELL GROWTH AND TELOMERASE ACTIVITY OF HUMAN PARATHYROID CANCER IN CELL 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. 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

Tumoral calcinosis: Identification of a novel recessive mutation in fibroblast growth factor-23 (FGF-23)

Tumoral calcinosis (TC) is a rare genetic disorder characterized by periarticular cystic and solid tumorals calcifications. It is characterized by hyperphospatemia and an elevated serum of calcitriol concentration in every patients. The hyperphosphatemia results form an increase in capacity of renal tubular phosphate reabsorption. The identification of phosphotonin family hormones suggest that mutations of these molecules could be involved in the pathogenesis of TC. One of these molecules is represented by FGF-23. The TC phenotype is similar to that described in the FGF-23 knockout mice. In the present study we described a new FGF-23 mutation in a subject affected by TC.A Caucasian women (years 67) was examined for a history ofectopic calcification. Biochemical exams showed and hyperphosphathemia and hyperphosphaturia with normal value ofPTH and inappropriately normal level of 1–25 (OH)2 D3. The patient presented a big shoulder calcification and also a calcification of femoral artery. We expanded the family tree through detailed family histories, which importantly revealed that parents were consanguineous. Hystologically the mass was characterized by calcium deposition and granulomatous reaction around the mass. Genomic DNA was extracted from blood collected from the patient, her daughter and her grandchild by standard procedure. DNA was not available from her parents. All three FGF-23coding exons, as well as conserved splice sites, were amplified by standard PCR procedure. Nucleotide sequences were determined by direct sequencing with a DNA kit and an automated DNA sequencer (ABI PRISM 3100 - Perkiln-Elmer Corp). We discovered a new homozygous codon 41, His/Gln (CAC-CAA) substitution in exon 1 of FGF-23 gene in the affected patient. A heterozygous substitution was present in the daughter. No mutation were found in the two children. FGF-23 gene mutation was not found in the SNP database (www.ncbi.nih.gov/snp). In summary, a recessive mutation in FGF 23 causes TC. Understanding the functional significance and molecular physiology of this novel mutation will reveal critical information regarding the role of FGF-23 in states of normal and of disorder of phosphate homeostasis