A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer.

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.

Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways.

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)2D3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)2D3 inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)2D3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 +/- 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)2D3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)2D3 survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)2D3-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)2D3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)2D3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)2D3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.

Pamidronate prevents the development of skeletal metastasis in nude mice transplanted with human breast cancer cells by reducing tumor burden within bone.

Pamidronate is used routinely in the treatment of established bone metastasis. However, pamidronate has not yet been assessed in the prevention of osteolytic bone metastasis and its precise mechanism of action in this disorder remains to be established. In the present study, pamidronate or vehicle alone was administered subcutaneously to nude mice either simultaneously or as post intracardiac injection of the human breast cancer MDA-MB-231 cells. Radiographs were used first to assess the presence of osteolytic bone metastases. Kaplan-Meier analysis demonstrated that animals treated with pamidronate early, but not late, showed a slower progression of bone metastases and hind limb paralysis than did vehicle-treated animals. Mann-Whitney analysis showed that only 44.4% of mice treated with pamidronate at the time of tumor cell inoculation developed bone metastases as compared to over 80% (p<0.05) of mice receiving vehicle alone. We then analyzed the number of bone lesions and their volume at time of sacrifice by bone histomorphometry. In contrast to X-ray analysis, morphometric analysis indicates that the number of lesions within bone was similar in pamidronate and vehicle-treated mice but that the lesions were significantly smaller and therefore, often not visible on radiographs. These results demonstrate that pamidronate is effective in reducing tumor burden in breast cancer metastatic to bone and is most effective as a preventative agent when administered closest in time to implantation of tumor cells. Our data also suggest that pamidronate acts mainly by inhibiting the growth of established bone metastatic lesions but has no effect on the metastatic spread itself.

Estrogens (E2) regulate expression and response of 1,25-dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation.

Studies on the effect of estrogens (E(2)) on the expression of vitamin D receptor (VDR) and its bioresponse in bone have demonstrated that E(2) modulate activity and increase the number of VDRs in vitro; however, no in vivo studies have been pursued to assess this interaction. Our study identifies the changes in the number of VDR-expressing cells in bone of C57BL/6J young and old oophorectomized mice (4 and 24 months) with and without 17beta estradiol (E(2)) replacement. A total of 36 mice were sacrificed; both tibiae and femora were isolated and VDR expression was quantified by Northern blot, immunohistochemistry, immunofluorescence, and flow cytometry. Among the intact mice there was a significant difference in the number of VDR-expressing osteoblasts between young (68%) and old (56%) (p<0.04). In young oophorectomized mice the number of VDR-expressing osteoblasts decreased from 68% to 46% after oophorectomy and recovered to 72% after E(2) administration (p<0.02), while in the group of old mice, the number of VDR-expressing osteoblasts decreased from 56% to 48% after oophorectomy (p<0.01) and recovered to 85% after E(2) administration (p<0.001). Our results show that VDR expression in bone decreases with aging and estrogen deprivation but recovers after E(2) supplementation in both young and old mice with a more significant level of response in older bone. To evaluate the level of VDR bioresponse to E(2) we assessed the effect of E(2) supplementation to human osteoblasts (N-976) in vitro. Northern blot showed a significant up-regulation of VDR expression in E(2) treated cells as compared to non-treated cells (p<0.05). We also assessed the previously known anti-apoptotic effect of vitamin D in osteoblasts in vitro after serum deprivation by using either E(2), E(2)+1,25(OH)(2)D(3), or 1,25(OH)(2)D(3) alone. We found a lower number of apoptotic cells and longer cell survival after 48 h of treatment with 1,25(OH)(2)D(3)+E(2) as compared to 1,25(OH)(2)D(3) or E(2) alone (p<0.002). In summary, our results demonstrate that E(2) increases VDR expression in bone in vivo and potentiate the bioresponse of VDR in osteoblasts in vitro.