A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Different inhibitory actions of cyclosporine A and cyclosporine A-acetate on lipopolysaccharide-, interleukin 1-, 1,25 dihydroxyvitamin D3- and parathyroid hormone-stimulated calcium and lysosomal enzyme release from mouse calvaria in vitro.

The effects of cyclosporine A (CsA) and one of its immunologically inactive structural analogues, cyclosporine A acetate (CsA-A) on lipopolysaccharide (LPS)-, interleukin-1 (IL-1)-, 1,25 dihydroxyvitamin D3 (1,25 D3)- and parathyroid hormone (PTH)-stimulated bone resorption were tested in mouse calvaria cultures. The release of calcium and a lysosomal enzyme, N-acetylglycosaminidase (NAG) was determined after 3 days of culture. All bone resorbing agents potently stimulated calcium and NAG release. At therapeutic concentration levels of 0.1 and 1.0 micrograms/ml, the immunologically active CsA was significantly more potent than the inactive CsA-A against LPS- and 1,25 D3-induced calcium and NAG release. The inhibition by both cyclosporines of IL-1 and PTH stimulated calcium release was not significantly different. CsA was however more potent than CsA-A against IL-1 stimulated NAG release. PTH-stimulated NAG release was not inhibited by CsA or CsA-A. These findings suggest that both cyclosporines interfere with more than one mechanism of activation of bone resorption. The specific effect of CsA against LPS and 1,25 D3 may be related to its known inhibition of immune cell derived cytokine expression.

In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate.

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid beta were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples.