Inhibition of the estradiol-induced growth of cultured human breast cancer cells by the anti-estrogens tamoxifen, desmethyl-tamoxifen, 4-hydroxy-tamoxifen and enclomiphene

The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor. Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth. In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor. In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected.     

Effect of a carotene concentrate on the growth of human breast cancer cells and pS2 gene expression

Breast cancer is the most common cancer in women worldwide. The growth of breast cancer cells is either hormone-dependent or hormone-independent. Both types are represented in vitro by the estrogen-receptor positive (ER+) MCF-7 and the estrogen-receptor negative (ER-) MDA-MB-231 cell lines, respectively. The pS2 gene is an estrogen-regulated gene and serves as a marker for the ER+ tumours. Carotenoids are pigments with anti-cancer properties besides having pro-vitamin A, antioxidant and free-radical quenching effects. This study was designed firstly, to compare the effect of palm oil carotene concentrate with retinoic acid on the growth of the ER+ MCF-7 and the ER- MDA-MB-231 cells; and secondly to evaluate the effect of the palm oil carotene concentrate on the regulation of pS2 mRNA. The growth experiments were performed with monolayer cells seeded in phenol red free RPMI 1640 culture media and subsequently treated with varying concentrations of either retinoic acid or palm oil carotenoids. The cell numbers were determined at the start of each experiment and then at successive time intervals. The results showed that the palm oil carotene concentrate caused dose-dependent inhibition of estradiol-stimulated growth of MCF-7 cells but did not affect the proliferation of MDA-MB-231 cells. Retinoic acid caused similar, albeit more potent effects, as significant inhibition was observed at lower concentrations than the palm oil carotenoids. In the pS2 gene expression experiment, cell monolayers were treated with the carotene concentrate (10(-6) M), either with or without supplemented estradiol (10(-8) M), and subsequently the RNA was extracted. Northern blotting was performed and the regulation of pS2 mRNA determined using a 32P-labelled pS2 cDNA probe. The results showed that the palm oil carotene concentrate did not affect the expression of pS2 mRNA and are therefore independent of the estrogen-regulated pathway.     

Modifications of benzylphenoxy ethanamine antiestrogen molecules: influence affinity for antiestrogen binding site (AEBS) and cell cytotoxicity

The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.     

Antiestrogen binding site and estrogen receptor mediate uptake and distribution of 4-hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells

The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-TAM, was found to possess superior cell growth inhibition characteristics relative to clinical doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines, three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of 4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding site and estrogen receptor.     

Degradation of bisphenol A by white rot fungi, Stereum hirsutum and Heterobasidium insulare, and reduction of its estrogenic activity

Two lignin-degrading basidiomycetes, Stereum hirsutum and Heterobasidium insulare, were used to degrade bisphenol A (BPA) in culture, and the estrogenic activity of the degradation products was examined using MCF-7 cell proliferation assays (E-screen) and analysis of pS2 mRNA expression in MCF 7 cells. Both S. hirsutum and H. insulare showed high resistance to BPA 100 ppm, and their mycelial growth was fully completed within 8 d of incubation at 30 degrees C. It took 7 to 14 d to achieve complete degradation (ca. 99%) of BPA by both fungi. MCF-7 cells proliferated actively at a BPA concentration of 10(-5) M. However, cell line proliferation was significantly inhibited when the cells were incubated in BPA culture media containing S. hirsutum and H. insulare. Similar results were obtained regarding pS2 mRNA expression. The pS2 mRNA expression levels decreased by 1.5-fold in supernatant from BPA treated with S. hirsutum and H. insulare compared with those treated with BPA alone.     

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.     

Estrogenic and antiproliferative properties of soy sapogenols in human breast cancer cells in vitro

Two soy sapogenols, soyasapogenol A (SA) and soyasapogenol B (SB) were tested for their estrogenic activities in estrogen responsive MCF-7 or estrogen-insensitive MDA-MB-231 (MDA) human breast cancer cells. SB and SA had differential actives on cell proliferation with 10 μ SB being growth inhibitory to MDA cells with no significant effect at any concentration on MCF-7 cells. SA also inhibited MDA cell proliferation at 10 μ , but at this same dose stimulated a 2.5-fold increase in MCF-7 proliferation. SA (0.1–10 μ ) induced pS2 mRNA levels and the induction was blocked by co-treatment of cells with the anti-estrogen ICI 182,780. SA also induced the formation of an ER–ERE DNA complex measured by electrophoretic mobility shift assay. In summary, these results show that soyasapogenol A is estrogenic, whereas soyasapogenol B is growth inhibitory.     

Brominated phenols: characterization of estrogen-like activity in the human breast cancer cell-line MCF-7

A large number of halogenated phenols are detected in the blood of humans, fish and wild-animals. We have characterized the estrogen-like activity of phenol, 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,4,6-tribromophenol (2,4,6-TBP) and 4-tert-butylphenol (tert-BP) using the estrogen-dependent human breast cancer cell line MCF-7. 4-BP, 2,4-DBP and 4-tert-BP all bind to the estrogen receptor (ER) with approximately 10,000-fold less affinity than 17 beta-estradiol (17 beta-E). 2,4,6-TBP was only able to displace 43% of radiolabelled estrogen when tested at concentrations up to 1 microM, whereas phenol had no affinity for the ER. 4-tert-BP stimulated cell growth and induced estrogen-regulated proteins such as the progesterone receptor (PgR) and pS2. The brominated phenols, however, although binding to the ER, did not stimulate cell growth or increase the levels of the PgR or pS2, or reduce the level of 17 beta-E induced pS2. On the contrary, 4-BP, 2,4-DBP and partly 4-tert-BP reduced 17 beta-E-stimulated cell growth apparently by an ER independent mechanism.     

Identification of a novel, orally bioavailable estrogen receptor downregulator

Tamoxifen has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer, but its partial agonist activity is considered to limit the efficacy, and cause tumor flare and endometrial cancer. Fulvestrant, on the other hand, binds and degrades ER, thereby acting as a pure anti-estrogen without partial agonist activity. However, due to its low oral bioavailability, fulvestrant has to be intramuscularly administered to patients, which limits the convenience of the drug, and causes pain and inflammation at the site of injection. In search of a patient- friendly pure anti-estrogen, we screened and identified an ER antagonist, CH4893237, which bound to ER with an IC50 value of 1.4 muM and, by oral administration, inhibited estrogen-stimulated uterine growth in ovariectomized mice. CH4893237 reduced the amount of ER at the protein level and impaired the nuclear accumulation of ER, indicating an orally active pure anti-estrogen. Furthermore, CH4893237 inhibited the estrogen-stimulated proliferation of MCF-7, ZR-75-1 and BT-474 cells, and caused a marked growth inhibition of the MCF-7 xenograft in vivo. Thus, CH4893237 will provide an additional option for second-line hormone treatment of breast cancer.     

Subcellular and extracellular localization of specific binding sites for triphenylethylene antiestrogens in human breast cancer

MCF-7 human breast cancer cell homogenates and subcellular organelles were submitted to isopycnic centrifugation on Percoll gradients to investigate the subcellular localization of triphenylethylene antiestrogen specific binding sites (AEBS). Electron microscopy revealed that gradient fractions coincident with the migration of [3H]tamoxifen-AEBS complexes were homogeneously represented by rough and smooth endoplasmic reticulum. Eighty percent of AEBS were localized in the endoplasmic reticulum [45,000 +/- 4,000 sites/cell, mean +/- S.D.), while 20% of these sites were also found in the nuclear fraction (12,000 +/- 1,000 sites/cell, mean +/- S.D.). A similar subcellular distribution of AEBS was observed in human breast cancer bioptic specimens. No differences in [3H]tamoxifen binding affinity between microsomal and nuclear AEBS were observed in MCF-7 and bioptic breast cancer. No major differences in microsomal AEBS levels were observed in the limited number of estrogen receptor-positive or -negative breast cancer specimens we have studied, whereas estrogen receptor-negative samples had higher levels of nuclear AEBS with respect to estrogen receptor-positive tumors. The presence of AEBS was also detected in the human serum of healthy and tumor-bearing subjects. The affinity and the binding specificity of serum AEBS were similar to those of intracellular AEBS. No differences in the levels of serum AEBS were observed between healthy and tumor-bearing subjects [19 +/- 4 and 22 +/- 4 pmoles/ml (mean +/- S.D.) respectively. Human serum AEBS did not appear to be associated to lipoproteins, whereas it migrated as a 5.5 S sedimenting molecule.     

Mitogen-activated protein kinase (MAPK) pathway mediates the oestrogen-like activities of ginsenoside Rg1 in human breast cancer (MCF-7) cells

Background and purpose:

The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen-like actions in MCF-7 cells by activating the mitogen-activated protein kinase (MAPK) pathway in a ligand-independent manner.

Experimental approach:

MCF-7 cells were co-incubated with the MAPK inhibitor PD98059 to determine whether the stimulant effects of Rg1 on cell proliferation, the induction of IGF-IR and pS2, the functional transactivation of oestrogen receptor-α (ERα), as well as ERα phosphorylation are dependent on MAPK. The time-dependent responses of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) to Rg1 in MCF-7 cells were studied. The responses of MEK phosphorylation to Rg1 in oestrogen receptor (ER)-negative HEK293 cells were also determined. The effects of Rg1 on cell proliferation and IGF-IR protein expression were studied in the presence of tyrosine kinase inhibitor genistein to elucidate the involvement of tyrosine kinase in mediating these effects.

Key results:

The oestrogenic effects of Rg1 in MCF-7 cells were abolished in the presence of PD98059. Rg1 could induce MEK protein expression and the phosphorylation level of MEK and ERK significantly in a time- and dose-dependent manner. Rg1 activated MEK phosphorylation in ER-negative HEK293 cells in a time- and dose-dependent manner. Rg1 induction of cell proliferation and IGF-IR protein expression was abolished by co-treatment with genistein.

Conclusions and implications:

Taken together, these results show that the MAPK pathway is involved in mediating the oestrogen-like actions of Rg1 in MCF-7 cells and suggest that Rg1 may activate ERα via MEK/ERK in a ligand-independent manner.     

Chemosensitization by fibroblast growth factor-2 is not dependent upon proliferation,S-phase accumulation,or p53 status

Fibroblast growth factor-2 (bFGF/FGF-2) is a pleiotropic growth factor that functions as a survival factor and directs apoptosis during embryogenesis and development. As a survival factor, FGF-2 would be expected to protect cells against drug toxicities. Such protection has been reported in some cells treated with some chemotherapeutic drugs. However, we recently demonstrated that FGF-2 can sensitize NIH 3T3 mouse fibroblasts to the cytotoxic and apoptotic effects of cisplatin. Sensitization requires prolonged incubation of cells with FGF-2 before the addition of cisplatin, and it requires an FGF-2 concentration (5-10 ng/mL) that is higher than that needed for its mitogenic effects (0.5 ng/mL). We now report that FGF-2 can also sensitize MCF7 human breast cancer cells and A2780 human ovarian cancer cells, as well as NIH 3T3 cells, to cisplatin. FGF-2 did not affect the cisplatin sensitivity of SKOV3 ovarian cancer cells or a panel of seven pancreatic cancer cell lines. We have demonstrated that the sensitizing effect is not simply a function of the mitogenic activity of FGF-2 on cells, as we did not observe sensitization with other growth-stimulatory factors (FGF-1 and epidermal growth factor); the sensitizing effect of FGF-2 was observed even with cell lines that were not growth-stimulated by FGF-2; and sensitization was not restricted to cells in S-phase of the cell cycle. These results indicate that cell proliferation is neither necessary nor sufficient for sensitization by FGF-2. Moreover, sensitization to cisplatin appears to be p53-independent, as p53-null 3T3 10-1 cells were equally sensitized by FGF-2. Finally, FGF-2 also sensitized NIH 3T3 and MCF7 cells to carboplatin, and had smaller effects on the sensitivity of these cell lines to doxorubicin and docetaxel. FGF-2 had no effect on sensitivity to etoposide in any cell line tested. Therefore, sensitization by FGF-2 was most effective with the platinum compounds, suggesting that this activity may be specific to particular mechanisms of drug action.     

Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of oligomeric products thought to be responsible for the biological effects of dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided by cell proliferation assay in human MCF-7 cells, resulted in the isolation of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells by approximately 60% at a non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the estradiol (E2)-induced binding of the ER to its cognate DNA responsive element. The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2 gene expression and in cells transiently transfected with an estrogen-responsive reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit the proliferation of both estrogen-dependent and -independent breast tumor cells and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist of Ah receptor function.     

A novel non-H1, non-H2 histamine antagonist protects against cysteamine-induced duodenal ulcers in rats

A newly synthesized para-diphenylmethane derivative, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine-HCl (DPPE), binds with high affinity to the microsomal anti-estrogen binding site (AEBS). Recent data suggest that the DPPE/AEBS binding site is closely related to a novel low-affinity, non-H1, non-H2 histamine site which may be associated with a calcium channel. We previously have shown that DPPE markedly reduces stress-induced and ethanol-induced gastric ulcers and attenuates gastric acid secretion. We now show that DPPE also profoundly reduces cysteamine-induced duodenal ulcers in rats.     

人乳腺癌细胞MCF-7紫杉醇耐药株的建立及其生物学特性研究

目的建立紫杉醇(Taxol)的人乳腺癌耐药细胞株MCF-7/Taxol,并初步研究其生物学特性。方法采用低浓度加量持续诱导法建立MCF-7/Taxol;细胞模型的多药耐药性以SRB法检测;流式细胞术分析细胞周期分布;免疫组化法检测细胞表型变化;高效液相鉴定细胞中药物蓄积;光镜和电子扫描显微镜观察细胞形态。结果SRB法检测,MCF-7/Taxol细胞的紫杉醇半数抑制浓度(IC50)是亲代MCF-7细胞的525倍,撤药3mon后细胞的耐药指数保持在150倍以上,并对羟喜树碱、表柔比星、多柔比星、米托蒽醌、硫酸长春新碱、博来霉素和丝裂霉素等多种化疗药物产生交叉耐药性。MCF-7/Taxol细胞分化程度低于同步传代的MCF-7细胞,倍增时间明显高于亲本细胞,S期细胞明显增加,G1期细胞减少;随撤药时间的延长,细胞的增殖速度加快。MCF-7/Taxol细胞P-gp、LRP和GSTπ的表达水平较亲本细胞有明显增加,ER、PR阳性表达丢失;光镜下MCF-7/Taxol细胞明显变大并且形态不规则;电镜下其表面纤绒毛成小球状隆起和絮状突起;在稳定生长的耐药细胞和撤药培养的细胞中都有Taxol蓄积。结论该MCF-7/Taxol模型具有多药耐药细胞的基本生物学特性,可用于肿瘤多药耐药机制药的研究,推测耐药细胞株中含有天然MCF-7肿瘤干细胞。     

Activation of insulin-like growth factor I receptor-mediated pathway by ginsenoside Rg1

Ginsenoside Rg1, an active ingredient in ginseng, was previously shown to be a novel class of potent phytoestrogen. The present study aims at investigating the molecular mechanisms involved in mediating its actions in human breast cancer (MCF-7) cells. Rg1 (1 pM) stimulates cell proliferation (P<0.01) and estrogen-responsive pS2 mRNA expression (P<0.05) without alteration of estrogen receptor alpha (ERalpha) protein or mRNA expression in MCF-7 cells. In addition, 10(-14)-10(-4) M of Rg1 does not demonstrate specific binding to ERalpha.We hypothesize that Rg1 may exert its actions in MCF-7 cell via the activation of crosstalk between ER- and insulin growth factor I receptor (IGF-IR)-dependent pathways. The results indicate that Rg1 significantly increases IGF-IR expression and IGF-IR promoter activity in MCF-7 cells (P<0.05). Cotreatment of MCF-7 cells with 1 muM of estrogen antagonist ICI 182,780 completely abolishes the effects of Rg1 on IGF-IR expression.Furthermore, Rg1 enhances tyrosine phosphorylation of IRS-1 in MCF-7 cells upon IGF-I stimulation and the activation of IRS-1 phosphorylation is also ER-dependent. Taken together, our results suggest that Rg1 not only increases IGF-IR expression but also enhances IGF-IR-mediated signaling pathways in MCF-7 cells. The stimulation of IGF-IR expression by Rg1 in MCF-7 cells appears to require ER, and its actions might involve ligand-independent activation of ER.     

FGF-21通过上调肝脏LDL受体的表达降低血浆中LDL水平

目的成纤维细胞生长因子-21(FGF-21)可降低实验动物血浆中的低密度脂蛋白(LDL)的浓度,但其作用机制尚不清楚,本实验试图通过研究FGF-21对LDLR的调节作用揭示FGF-21调节血浆LDL浓度的机制。方法向谷氨酸钠(MSG)肥胖大鼠连续注射FGF-21蛋白40d后,检测其血浆中LDL的变化,并用荧光定量PCR的方法结合免疫荧光检测FGF-21对肝脏组织中LDLR mRNA及蛋白的影响。结果 MSG肥胖鼠经FGF-21处理,血浆内的LDL降低18%,其肝脏组织中LDLR mRNA含量提高9倍;HepG2细胞内的LDLR mRNA表达量随FGF-21处理时间增加而提高,且呈剂量依赖性;流式细胞仪检测显示,经FGF-21处理后HepG2细胞表面LDLR蛋白数量增加。结论 FGF-21通过增加肝细胞的LDLR表达量从而降低血浆中LDL浓度。     

Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.     

GPER抑制剂对雌激素长期诱导的MCF-12A乳腺上皮细胞生长和克隆形成的影响

目的：研究GPER（G protein-coupled estrogen receptor,GPER）抑制剂对雌激素（estrogen,E2）长期作用下乳腺上皮细胞生长和克隆形成的影响,探讨阻断GPER受体防治乳腺癌的可能性。方法：分别使用E2、E2+G15和G15持续作用MCF-12A细胞5周（共传11代）建立细胞模型,通过光镜观察细胞形态的变化、台盼蓝计数法分析细胞生长变化,采用Western blot检测雌激素受体α（estrogen receptor α,ERα）及GPER蛋白表达变化,软琼脂糖克隆形成实验分析细胞克隆形成能力。结果：（1）MCF-12A经E2（10,20和40 nmol·L^-1）连续处理5周后,其体外生长能力与E2浓度呈一定剂量依赖性,其中,E2（20 nmol·L^-1）组细胞呈多层重叠样生长,排列紊乱,形态变化最为显著。（2）相比于对照组,E2组中不同代数细胞的ERα蛋白表达明显下调,且呈一定时间依赖性,而GPER蛋白表达未见明显变化。（3）GPER抑制剂G15能抑制E2诱导的细胞生长。（4）G15抑制E2诱导的细胞克隆形成。结论：G15可抑制E2对乳腺上皮细胞MCF-12A的促生长及克隆形成作用,提示GPER抑制剂可能可作为乳腺癌的防治药物。