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Raloxifene inhibits estrogen-induced up-regulation of telomerase activity in a human breast cancer cell line.

Kawagoe J, Ohmichi M, Takahashi T, Ohshima C, Mabuchi S, Takahashi K, Igarashi H, Mori-Abe A, Saitoh M, Du B, Ohta T, Kimura A, Kyo S, Inoue M, Kurachi H.

Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, Iidanishi, Yamagata, Japan.

The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17beta-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFkappaB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFkappaB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFkappaB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFkappaB.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12917431&dopt=Abstract raloxifene Evista



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Serum lipids and apolipoproteins in Greek postmenopausal women: association with estrogen, estrogen-progestin, tibolone and raloxifene therapy.

Creatsas G, Christodoulakos G, Lambrinoudaki I, Panoulis C, Chondros C, Patramanis P.

2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, Athens.

The aim of this study was to assess lipid and apolipoprotein levels in postmenopausal women taking various regimens of replacement therapy or no therapy. Seven hundred forty-eight postmenopausal women followed in the Menopause Clinic of the 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital, were studied in a cross-sectional design. Women were either non-users of replacement therapy (no. = 511) or users of one of the following regimens: conjugated equine estrogen 0.625 mg (CEE, no. = 34), CEE 0.625 mg plus medroxyprogesterone acetate 5 mg (CEE/MPA, no. = 60), 17beta-estradiol 2 mg plus norethisterone acetate 1 mg (E2/NETA, no. = 44), tibolone 2.5 mg (no. = 84), raloxifene HCI 60 mg (no. = 51). Total cholesterol (TC), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C), triglycerides (TG), apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) levels were assessed. Women were grouped according to replacement regimen and mean levels of lipid and apolipoproteins were compared between groups. Women in the raloxifene group were older and longer menopaused. After adjustment for age and duration of menopause, TG levels were significantly lower in the tibolone and E2/NETA groups (75 and 89.9 mg/dl, respectively) compared to non-users. TC was lower in all therapy groups, but the difference acquired significance only in the E2/NETA (207.8 mg/dl), compared to non-users (231.5 mg/dl). LDL-C levels were significantly lower in the CEE (133.8 mg/dl), CEE/MPA (130.4 mg/dl) and raloxifene group (129.9 mg/dl) compared to non-users (151.9 mg/dl). There was no difference in HDL-C levels between users and non-users (58.9 mg/dl) except for the tibolone group where HDL-C was significantly lower (48.6 mg/dl). ApoA1 levels were significantly higher in the CEE/MPA group (194.4 mg/dl) and significantly lower in the tibolone group (141.6 mg/dl) compared to non-users (170.4 mg/dl). No difference was detected between groups concerning ApoB levels. In conclusion, tibolone therapy is associated with lower TG levels as well as lower HDL and ApoA1 levels. ERT, continuous combined estrogen-progestin therapy (HRT) and raloxifene are associated with lower LDL-C levels. Among continuous combined HRT users, CEE/MPA is associated with higher ApoA1 levels, while E2/NETA with lower TG levels. Large prospective randomized studies are required to validate these results.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12952369&dopt=Abstract raloxifene Evista



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Estrogen and raloxifene induce apoptosis by activating p38 mitogen-activated protein kinase cascade in synthetic vascular smooth muscle cells.

Mori-Abe A, Tsutsumi S, Takahashi K, Toya M, Yoshida M, Du B, Kawagoe J, Nakahara K, Takahashi T, Ohmichi M, Kurachi H.

Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan.

Proliferation of vascular smooth muscle cells (VSMC) plays a major role as an initiating event of atherosclerosis. Although estrogen directly inhibits the proliferation of VSMC, the mechanism has not been firmly established. In addition, the effect of raloxifene on VSMC remains unknown. 17Beta-estradiol (E(2)) and raloxifene significantly inhibited the growth of VSMC under growth-stimulated conditions. Since mitogen-activated protein (MAP) kinases have been implicated in VSMC proliferation, the role of MAP kinases in both the E(2)- and raloxifene-induced growth inhibition of VSMC was studied. Both E(2) and raloxifene caused rapid, transient phosphorylation and activation of p38 that was not affected by actinomycin D and was blocked by ICI 182,780. In contrast with p38 phosphorylation, extracellular signal-regulated protein kinase (ERK) phosphorylation was significantly inhibited and c-Jun N-terminal kinase (JNK) phosphorylation was not changed by E(2). Because VSMC expressed both estrogen receptor (ER) alpha and ERbeta, it is not known which of them mediates the E(2)-induced phosphorylation of p38. Although E(2) did not affect the p38 phosphorylation in A10 smooth muscle cells, which express ERbeta but not ERalpha, transfection of ERalpha expression vector into A10 cells rendered them susceptible to induction of p38 phosphorylation by E(2). We then examined whether E(2) and raloxifene induce apoptosis through a p38 cascade. Both E(2) and raloxifene induced apoptosis under growth-stimulated conditions. The p38 inhibitor SB 203580 completely blocked the E(2)-induced apoptosis. Our findings suggest that both E(2)- and raloxifene-induced inhibition of VSMC growth is due to induction of apoptosis through a p38 cascade whose activation is mediated by ERalpha via a nongenomic mechanism.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12967334&dopt=Abstract raloxifene Evista



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Raloxifene concurrently stimulates osteoprotegerin and inhibits interleukin-6 production by human trabecular osteoblasts.

Viereck V, Grundker C, Blaschke S, Niederkleine B, Siggelkow H, Frosch KH, Raddatz D, Emons G, Hofbauer LC.

Department of Obstetrics and Gynecology, Georg-August-University of Goettingen, Goettingen, Germany D-37075. viereck med.uni-goettingen.de

Raloxifene reduces bone loss and prevents vertebral fractures in postmenopausal women. Its skeletal effects are mediated by estrogen receptors (ER) and their modulation of paracrine osteoblastic factors. Receptor activator of nuclear factor-kappa B ligand is essential for osteoclasts and enhances bone resorption, whereas osteoprotegerin (OPG) neutralizes receptor activator of nuclear factor-kappa B ligand. Here, we assessed the effects of raloxifene on OPG production in human osteoblasts (hOB). Raloxifene enhanced gene expression of ER-alpha and progesterone receptor. Moreover, raloxifene increased OPG mRNA levels and protein secretion by hOB in a dose- and time-dependent fashion by 2- to 4-fold with a maximum effect at 10(-7) M and after 72 h (P < 0.001). Treatment with the ER antagonist ICI 182,780 abrogated the effects of raloxifene on OPG production. Moreover, raloxifene enhanced osteoblastic differentiation markers, type 1 collagen secretion, and alkaline phosphatase activity by 3- and 2-fold, respectively (P < 0.001). In addition, raloxifene inhibited expression of the bone-resorbing cytokine IL-6 by 25-45% (P < 0.001). In conclusion, our data suggest that raloxifene stimulates OPG production and inhibits IL-6 production by hOB. Because OPG production increases with osteoblastic maturation, enhancement of OPG production by raloxifene could be related to its stimulatory effects on osteoblastic differentiation.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12970288&dopt=Abstract raloxifene Evista



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Differential effects of estradiol and raloxifene on collagen biosynthesis in cultured human skin fibroblasts.

Surazynski A, Jarzabek K, Haczynski J, Laudanski P, Palka J, Wolczynski S.

Department of Medicinal Chemistry, Medical Academy of Bialystok, 15-230 Bialystok, Poland.

The dose-dependent effect of a 24 h treatment with estradiol (E(2)) (1, 2, 5, 10 nM) and raloxifene (Rx) (1, 5, 10, 20 microM) on ER alpha and ER beta mRNA expression, collagen bio-synthesis, prolidase activity, MMP-2, MMP-9, insulin-like growth factor I receptor expression (IGF-1R) and beta1-integrin expressions in cultured fibroblasts obtained from postmenopausal women were examined. Both ligands increased mRNA expression of ER compared to control. Rx at 5 and 10 microM concentrations had greater stimulative effect on collagen biosynthesis, prolidase activity and IGF-1R expression compared to E(2) at 2 and 5 nM concentration. Both studied ER ligands had no effect on beta1-integrin receptor expressions. MMP-2 expression was not detected in human skin fibroblast culture. In contrast to estradiol raloxifene inhibited the expression of MMP-9. Raloxifene had stronger positive stimulative effects on collagen biosynthesis, through different biochemical mechanisms, than estradiol in human skin fibroblasts and might reverse some of the postmenopausal changes in skin or connective tissue. Increase of collagen synthesis induced by raloxifene may be activated by both estrogen receptor dependent and independent pathways such as up-regulation of estrogen receptors, up-regulation of IGF receptor, transcriptional regulation of collagen genes by estrogen receptor-raloxifene complex, increasing of prolidase activity or finally by inhibition of MMP-9 expression.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14533013&dopt=Abstract raloxifene Evista



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Nitrosation, nitration, and autoxidation of the selective estrogen receptor modulator raloxifene by nitric oxide, peroxynitrite, and reactive nitrogen/oxygen species.

Toader V, Xu X, Nicolescu A, Yu L, Bolton JL, Thatcher GR.

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 Wood Street, Chicago, Illinois 60612-7231, USA.

The regulation of estrogenic and antiestrogenic effects by selective estrogen receptor modulators (SERMs) provides the basis for use in long-term therapy in cancer chemoprevention and postmenopausal osteoporosis. However, the evidence for carcinogenic properties within this class requires study of potential pathways of toxicity. There is strong evidence for the elevation of cellular levels of NO in tissue treated with SERMs, including the benzothiophene derivative, raloxifene, in part via up-regulation of nitric oxide synthases. Therefore, the reactions of 17beta-estradiol (E(2)), raloxifene, and an isomer with NO, peroxynitrite, and reactive nitrogen/oxygen species (RNOS) generated from NO(2)(-)/H(2)O(2) systems were examined. Peroxynitrite from bolus injection or slow release from higher concentrations of 3-morpholinosydnonimine (SIN-1) reacted with the benzothiophenes and E(2) to give aromatic ring nitration, whereas peroxynitrite, produced from the slow decomposition of lower concentrations of SIN-1, was relatively unreactive toward E(2) and yielded oxidation and nitrosation products with raloxifene and its isomer. The oxidation and nitrosation products formed were characterized as a dimer and quinone oxime derivative. Interestingly, the reaction of the benzothiophenes with NO in aerobic solution efficiently generated the same oxidation products. Stable quinone oximes are not unprecedented but have not been previously reported as products of RNOS-mediated metabolism. The reaction of glutathione (GSH) with the quinone oxime gave both GSH adducts from Michael addition and reduction to the corresponding o-aminophenol. The ready autoxidation of raloxifene, observed in the presence of NO, is the first such observation on the reactivity of SERMs and is potentially a general phenomenon of significance to SERM chemical toxicology.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14565768&dopt=Abstract raloxifene Evista