Abstract: Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Abstract: Xanthohumol (XN), a natural polyphenol present in beer, is known to exert anti-cancer effects. However, its precise mechanisms are not yet clearly defined. The aim of this study was to investigate the effect of oral administration of XN in breast cancer xenografts in nude mice. Proliferation and apoptosis were first examined in MCF7 cell cultures after incubation with XN by trypan blue exclusion assay, [3H]-thymidine incorporation, KI67 immunostaining and TUNEL. Morphological and histological characteristics of tumours from XN-treated or control (vehicle-treated) mice were compared. Immunohistochemistry for proliferative, inflammatory and endothelial cell markers was performed and activation of nuclear factor kappa B (NFkappaB) pathway was assessed by ELISA. In vitro MCF7 cell proliferation decreased in a dose-dependent manner. Oral administration of XN to nude mice inoculated with MCF7 cells resulted in central necrosis within tumours, reduced inflammatory cell number, focal proliferation areas, increased percentage of apoptotic cells and decreased microvessel density. Anti-angiogenic effects of XN were further confirmed by immunoblotting for factor VIII expression in XN-treated tumours as compared to controls. Decreased immunostaining for NFkappaB, phosphorylated-inhibitor of kappa B and interleukin-1beta were also observed as well as a significant decrease in NFkappaB activity to 60% of control values. These novel findings indicate that XN is able to target both breast cancer and host cells, namely inflammatory and endothelial cells, suggesting its potential use as a double-edge anti-cancer agent.

Abstract: Several studies indicate that progesterone exerts relevant effects in breast tissue. However, the exact role of this steroid in breast cancer development and progression has not been elucidated. Here, we show that platelet-derived growth factor (PDGF)-A is one of the progesterone target genes on breast cancer MCF7 and T47D cells. A paracrine role for PDGF-A was investigated, since its receptor expression was down-regulated from breast cancer cells. Progesterone increased PDGF-A protein release as evaluated by Western blotting and ELISA. Medium from Progesterone-treated MCF7 cells resulted in phosphorylation of smooth muscle cells PDGF receptor alpha. This effect was not observed after treatment with PDGF inhibitor. MCF7 cells-secreted PDGF-A was able to increase smooth muscle cell viability and proliferation and decrease apoptosis, effects that were prevented by the use of a PDGF-A neutralizing antibody. Notably, cell invasion was not influenced by PDGF-A secreted by MCF7 cells. Our results elucidated for the first time the cross talk between progesterone and PDGF signaling pathway. The fact that MCF7-secreted PDGF elicited crucial roles in vascular wall smooth muscle cells, suggested a paracrine pathway for progesterone. Targeting these progesterone-induced processes may provide novel therapeutic strategies for hormone-dependent human breast cancer.

Abstract: OBJECTIVES: To examine the effect of 17beta-estradiol and xanthohumol in alkaline phosphatase (ALP) expression and activity in breast cancer MCF-7 cells. DESIGN AND METHODS: ALP isoenzymes expression was evaluated by RT-PCR and Western blotting. ALP activity was measured by spectrophotometry. Cell proliferation and apoptosis were examined by MTT and immunostaining for KI67 and TUNEL, respectively. RESULTS: ALP isoenzymes expression and activity were decreased by 1 nM 17beta-estradiol. Pure estrogenic antagonist (ICI 182,780) reversed 17beta-estradiol-inhibiting effect in TNS-ALP expression. RNA and protein expression of IALP, but not TNS-ALP, was also decreased by incubation with 10 microM xanthohumol (IC(50)) and was accompanied by a significant reduction in ALP activity. Treatment with 17beta-estradiol enhanced cell proliferation and decreased apoptosis. Conversely, xanthohumol incubation inhibited cell viability and apoptosis. CONCLUSION: Estrogens and xanthohumol differently modulate ALP isoenzymes. ALP loss associated with increased cell proliferation. Modulation of this enzyme by 17beta-estradiol and xanthohumol might provide therapeutic strategies against hormone-dependent breast cancer.