Role of insulin-like growth factor binding protein-3 in 1, 25-dihydroxyvitamin-d 3 -induced breast cancer cell apoptosis.

Insulin-like growth factor I (IGF-I) is implicated in breast cancer development and 1, 25-dihydroxyvitamin D3 (1, 25-D3) has been shown to attenuate prosurvival effects of IGF-I on breast cancer cells. In this study the role of IGF binding protein-3 (IGFBP-3) in 1, 25-D3-induced apoptosis was investigated using parental MCF-7 breast cancer cells and MCF-7/VD(R) cells, which are resistant to the growth inhibitory effects of 1, 25-D3. Treatment with 1, 25-D3 increased IGFBP-3 mRNA expression in both cell lines but increases in intracellular IGFBP-3 protein and its secretion were observed only in MCF-7. 1, 25-D3-induced apoptosis was not associated with activation of any caspase but PARP-1 cleavage was detected in parental cells. IGFBP-3 treatment alone produced cleavage of caspases 7, 8, and 9 and PARP-1 in MCF-7 cells. IGFBP-3 failed to activate caspases in MCF-7/VD(R) cells; however PARP-1 cleavage was detected. 1, 25-D3 treatment inhibited IGF-I/Akt survival signalling in MCF-7 but not in MCF-7/VD(R) cells. In contrast, IGFBP-3 treatment was effective in inhibiting IGF-I/Akt pathways in both breast cancer lines. These results suggest a role for IGFBP-3 in 1, 25-D3 apoptotic signalling and that impaired secretion of IGFBP-3 may be involved in acquired resistance to vitamin D in breast cancer

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

This article is available open access through the publisher’s website at the link below. Copyright @ 2009 Landes Bioscience.The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaig

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB