Expression of 25 hydroxyvitamin D3-1alpha-hydroxylase in human endometrial tissue.

1,25(OH)(2)D(3) (calcitriol) has been shown to play an important role in cell proliferation, differentiation and immune responsiveness. The enzyme responsible for calcitriol synthesis 25 hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase) has been reported in many human tissues. The aim of this study was to investigate the expression of 1alpha-OHase in gynaecological tissues. Using a highly specific nested touchdown PCR we examined the expression of 1alpha-OHase in normal and malignant endometrial tissue and in human endometrial Ishikawa cells. In addition, we analyzed the protein expression of 1alpha-OHase by Western blot. The expression of 1alpha-OHase in normal and malignant endometrial tissue and Ishikawa cells was detected and splice variants of the enzyme in Ishikawa cells were identified. These data suggest an alternative splicing of 1alpha-OHase in malignant endometrial tissue and cells. We postulate that the expression of 1alpha-OHase gene variants may contribute to the antiproliferative effects of calcitriol. In conclusion, the modulation of the 1alpha-OHase opens up a new target for vitamin D(3) related therapies in endometrial cancer.

Vitamin D--metabolism in the human breast cancer cell line MCF-7.

BACKGROUND: The three main vitamin D metabolizing enzymes, vitamin D3-25-hydroxylase (25-OHase, 25-hydroxylase), 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase, 1alpha-hydroxylase) and 25-hydroxyvitamin D3-24-hydroxylase (24-OHase, 24-hydroxylase), have been described in malignant breast tissue. This in vitro study aimed to obtain more information regarding the regulation of these enzymes in the human breast cancer cell line MCF-7. MATERIALS AND METHODS: Vitamin D receptor (VDR)- positive MCF-7 cells in culture were stimulated with the vitamin D metabolites vitamin D3, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 for 24, 48; 72 and 96 hours in physiological and supraphysiological concentrations. The expressions of 25-hydroxylase, 1alpha-hydroxylase and 24-hydroxylase and their changes after stimulation were assessed by real-time PCR. RESULTS: The expression of 25-hydroxylase was slightly influenced by vitamin D3. The expression of 1alpha-hydroxylase was induced after stimulation with vitamin D3 and 25-hydroxyvitamin D3. Stimulation with 1,25-dihydroxyvitamin D3 markedly increased the expression of 24-hydroxylase time- and dose-dependently. CONCLUSION: Our results demonstrated that MCF-7 cells are able to regulate the expression of 24-hydroxylase. This might be a mechanism for these tumor cells to protect themselves against the antiproliferative and apoptosis-inducing effects of calcitriol.

Analysis of 25-hydroxyvitamin D3-1alpha-hydroxylase in normal and malignant breast tissue.

BACKGROUND: The presence of extra-renal 25-hydroxyvitamin D3 [25(OH)D3]-1alpha-hydroxylase (1alpha-OHase) has been reported in several cell types including prostate and colon cancer cells. Additionally, alterations in the local production of 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D3] have been implicated in the tumorigenesis of these malignancies. The aim of this study was to analyze whether normal breast tissue or breast cancer cells expressed 1alpha-OHase and to evaluate whether breast tissue possessed the capacity to produce 1alpha,25(OH)2D3 from 25(OH)D3. MATERIALS AND METHODS: Total RNA was extracted from normal breast tissue (n = 11), breast carcinomas (n = 12) and cultured MCF-7 breast cancer cells for real-time (LightCycler using specific hybridization probes) and conventional PCR analysis. RESULTS: mRNA for 1alpha-OHase was detected in breast cancer tissue and in MCF-7 breast cancer cells. Interestingly, the mRNA levels for 1alpha-OHase were significantly increased in breast cancer compared to normal breast tissue. When the MCF-7 cells were treated with 1alpha,25(OH)2D3, cell proliferation was inhibited in a dose-dependent manner. Incubation of the MCF-7 cells with [3H]-25(OH)D3 resulted in its conversion to [3H]-1,25(OH)2D3. The 1alpha-OHase activity in the MCF-7 cells was blocked by a specific cytochrome P450 inhibitor, clotrimazole. CONCLUSION: The data suggest that at least breast cancer cells expressed 1alpha-OHase mRNA and, therefore, might have the ability to synthesize 1alpha,25(OH)2D3 within the cells. The local production of 1alpha,25(OH)2D3 might play an important role in regulating the proliferation and differentiation of breast cells. We hypothesize that alterations in the local production of 1alpha,25(OH)2D3 may be involved in the tumorigenesis of breast cancer. Additionally, breast cancer may be a target for treatment with precursors of biologically-active vitamin D analogs.

Vascular endothelial growth factor response to exogenous chorionic gonadotropic hormone in the luteal phase of women with a history of severe ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a severe complication of ovarian stimulation. No reliable test exists to predict the syndrome. The objective of the present prospective observational study was to examine vascular endothelial growth factor (VEGF) secretion after human chorionic gonadotropin (hCG) administration in the luteal phase of a spontaneous cycle of women with a history of severe OHSS. Five women with a history of severe OHSS were administered 250 mug recombinant hCG intravenously on day 21 of a spontaneous menstrual cycle. Plasma samples were collected at regular intervals from 15 min before hCG to 6 h thereafter and the free VEGF plasma concentrations were determined. Plasma levels of free VEGF remained at the lower detection limit of the assay throughout the observational period. Women with previous severe OHSS do not show a significant short-time response of VEGF secretion upon hCG administration. No evidence was found to support the notion that women inclined to develop a severe form of the syndrome after ovarian stimulation could possibly be identified by the VEGF short-time secretory response to exogenous hCG in the luteal phase of a spontaneous cycle.

Granulomatous mastitis.

INTRODUCTION: Granulomatous mastitis (GM) is a rare disease which predominantly occurs in premenopausal women shortly after their last childbirth. ETIOLOGY: Its etiology is unclear, however, the disease has been shown to be correlated with breast-feeding and the use of oral contraceptives. An autoimmune component has also been discussed. PRESENTATION: It presents with the clinical symptoms of galactorrhea, inflammation, breast mass, tumorous indurations and ulcerations of the skin. In mammography and sonography nodular opacities and hypoechoic nodules are found. Very often clinical and radiological findings mimic breast cancer. HISTOLOGICAL DIAGNOSIS: The diagnosis is made by histopathology. Histological features in GM include signs of a chronic granulomatous inflammation with giant cells, leucocytes, epitheloid cells and macrophages as well as abscesses. TREATMENT: Therapy of GM consists of complete surgical excision combined with oral steroid therapy, eventually in combination with anti-inflammatory drugs or colchicine. Use of methotrexate has also been successful. In case of formation of abscesses antibiotic therapy should be applied before steroid therapy. Immune-suppressive therapy should be performed until complete remission as rates of recurrence can be up to 50%.