Purpose: Wedelia chinensis is a common ingredient of anti-inflammatory herbal medicines in Taiwan and southern China. Inflammation is involved in promoting tumor growth, invasion, and metastasis. This study aims to test the biological effects in vivo of W. chinensis extract on prostate cancer. Experimental Design: The in vivo efficacy and mechanisms of action of oral administration of a standardized extract of W. chinensis were analyzed in animals bearing a subcutaneous or orthotopic prostate cancer xenograft. Results: Exposure of prostate cancer cells to W. chinensis extract induced apoptosis selectively in androgen receptor (AR)-positive prostate cancer cells and shifted the proportion in each phase of cell cycle toward G 2 -M phase in AR-negative prostate cancer cells. Oral herbal extract (4 or 40 mg/kg/d for 24-28 days) attenuated the growth of prostate tumors in nude mice implanted at both subcutaneous (31% and 44%, respectively) and orthotopic (49% and 49%, respectively) sites. The tumor suppression effects were associated with increased apoptosis and lower proliferation in tumor cells as well as reduced tumor angiogenesis. The antitumor effect of W. chinensis extract was correlated with accumulation of the principle active compounds wedelolactone, luteolin, and apigenin in vivo. Conclusion: Anticancer action of W. chinensis extract was due to three active compounds that inhibit the AR signaling pathway. Oral administration of W. chinensis extract impeded prostate cancer tumorigenesis. Future studies of W. chinensis for chemoprevention or complementary medicine against prostate cancer in humans are thus warranted. (Clin Cancer Res 2009;15(17):5435-44) Carcinoma of the prostate gland is the most common malignancy in males in the western world (1). Despite the low incidence of prostate cancer in oriental countries, statistics from Taiwan reveal prostate cancer deaths have continued to increase in the past two decades.5 Androgen ablation therapy remains the most effective means of treating metastatic prostate cancer tumors (2, 3). This therapy often induces apoptosis in the majority of prostate cancer cells by blocking testosterone signaling at the androgen receptor (AR) and lowering the expression of AR-regulated genes including prostate-specific antigen (PSA), a serologic biomarker up-regulated by androgens (4, 5). The CWR22 tumor model, based on implantation of a human prostate cancer in an athymic nude mouse, progresses in the same androgen-dependent manner as observed in clinical prostate cancer tumors. Further development from castrationrelapsed CWR22 tumors created subline tumors and a cell line, 22Rv1, which retain expression of AR and show androgen stimulation of neoplasia (6, 7). Earlier, we devised the androgeninduced PSA-luciferase activity in prostate cancer 22Rv1 cells and a derived stable clone, 103E, as a cell-based assay to detect any AR modulator effect of herbal extract or compounds (8-10). To determine whether herbal remedies or compounds can inhibit prostate cancer growth in o...

dCell-cell fusion and cell invasion are essential for placental development. Human cytotrophoblasts in the chorionic villi may undergo cell-cell fusion to form syncytiotrophoblasts to facilitate nutrient-gas exchange or differentiate into extravillous trophoblasts (EVTs) to facilitate maternal-fetal circulation. The placental transcription factor glial cells missing 1 (GCM1) regulates syncytin-1 and -2 expression to mediate trophoblast fusion. Interestingly, GCM1 and syncytin-1 are also expressed in EVTs with unknown physiological functions. In this study, we performed chromatin immunoprecipitation-on-chip (ChIP-chip) analysis and identified the gene for high-temperature requirement protein A4 (HtrA4) as a GCM1 target gene, which encodes a serine protease facilitating cleavage of fibronectin and invasion of placental cells. Importantly, HtrA4 is immunolocalized in EVTs at the maternal-fetal interface, and its expression is decreased by hypoxia and in preeclampsia, a pregnancy complication associated with placental hypoxia and shallow trophoblast invasion. We further demonstrate that HtrA4 interacts with syncytin-1 and suppresses cell-cell fusion. Therefore, HtrA4 may be crucial for EVT differentiation by playing a dual role in prevention of cellcell fusion of EVTs and promotion of their invasion into the uterus. Our study reveals a novel function of GCM1 and HtrA4 in regulation of trophoblast invasion and that abnormal HrtA4 expression may contribute to shallow trophoblast invasion in preeclampsia.H uman placentation proceeds fast after embryo implantation, and different classes of specialized trophoblast cells have evolved to establish blood circulation for nutrient, gas, and waste exchange between mother and fetus. In brief, the mononuclear cytotrophoblasts in chorionic villi proliferate and differentiate through cell-cell fusion into a multinucleated syncytiotrophoblast layer, which is in direct contact with maternal blood to mediate the above-mentioned exchanges and produce hormones and growth factors for pregnancy maintenance. On the other hand, cytotrophoblasts in the chorionic villi that are anchored to uterine decidua proliferate into cell columns from which some cytotrophoblasts migrate and invade deeper layers of decidua. The migratory and invasive cytotrophoblasts, termed interstitial extravillous trophoblasts (EVTs), may further invade the uterine myometrium and replace the endothelial cells of spiral arteries. This phenomenon, called spiral artery remodeling, is essential for sufficient blood flow into intervillous spaces of the placenta, as remodeled arteries become dilated and nonvasoactive. Indeed, insufficient spiral artery remodeling due to shallow trophoblast invasion may result in placental hypoxia and pregnancy complications such as preeclampsia and intrauterine growth retardation with clinical features of gestational hypertension, proteinuria, and failure of optimal fetal growth (6).Glial cells missing 1 (GCM1), also known as GCMa, is a transcription factor critical for placental developmen...

No abstract

The present study investigated the modulatory role of transforming growth factor beta 1 (TGFbeta1) on the secretion of matrix metalloproteinases (MMPs) and tested whether the altered secretion of MMPs could directly affect the invasive behavior of ovarian cancer cells. To this aim, human ovarian cancer SKOV3 cells were treated once with vehicle or various concentrations of TGFbeta1 for 24 h. Gelatinase activities in conditioned media were analyzed by zymography and densitometry. TGFbeta1 dose-dependently stimulated the secretion of a 68-kDa gelatinase, which was characterized as an MMP because its activity was inhibited by a metalloproteinase inhibitor 1,10-phenanthroline, and by a synthetic MMP inhibitor BB3103. In addition, we used aminophenylmercuric acetate (APMA) to activate latent gelatinases. APMA time-dependently decreased the activity of 68-kDa gelatinase, and increased the activities of 64- and 62-kDa gelatinolytic bands. The 68-kDa gelatinase was further characterized as MMP2 (gelatinase A) by immunoblotting analysis. We then tested TGFbeta1 effect on the invasive potential of SKOV3 cells as assessed by the migration ability through reconstituted basement membrane, and further investigated whether TGFbeta1 may act through modulating the MMP activity to affect ovarian cancer cell invasion. The results show that TGFbeta1 stimulated the invasive behavior of SKOV3 cells, and that MMP inhibitor BB3103 abrogated this effect of TGFbeta1. In conclusion, this study indicates that TGFbeta1 may act partly through stimulating the secretion of MMP in promoting the invasive behavior of human ovarian cancer cells. Furthermore, this work supports the idea that specific MMP inhibitors of the hydroxamate class could be therapeutically useful in controlling cancer cell invasion/metastasis.

Cholesterol is an essential structural component of mammalian cell membrane and a precursor for the synthesis of steroid hormones and bile acids ( 1 ). During ovarian steroid hormone synthesis, cholesterol is fi rst transported into mitochondrial inner membrane facilitated by the steroidogenic acute regulatory protein (StAR), and then converted to the important sex steroid progesterone under sequential actions of the mitochondrial enzyme P450 cholesterol side chain-cleavage enzyme (P450scc) and endoplasmic reticulum enzyme 3 ␤ -hydroxysteroid dehydrogenase (3 ␤ -HSD). Progesterone could be further enzymatically processed into androgens and estrogens ( 2 ). Cellular cholesterol could be derived from the de novo synthesis pathway or from circulating lipoproteins. For steroidogenic cells, lipoproteins are the major source that provides suffi cient cholesterol to meet the demand of steroid hormone synthesis ( 3 ). Aside from the ubiquitous LDL receptor (LDLR)-mediated endocytic uptake of LDL-cholesterol adapted by most cell types, steroidogenic cells additionally utilize HDL-derived cholesterol through scavenger receptor class B member I Abstract Cellular cholesterol is known to be under homeostatic control in nonsteroidogenic cells, and this intrigued us to understand how such control works in ster oidogenic cells that additionally use cholesterol for steroid hormone synthesis. We employed primary culture of rat ovarian granulosa cells to study how steroidogenic cells adapt to acquire suffi cient cholesterol to meet the demand of active steroidogenesis under the stimulation of gonadotropin follicle-stimulating hormone (FSH) and cytokine transforming growth factor (TGF) ␤ 1. We found that TGF ␤ 1 potentiated FSH to upregulate scavenger receptor class B member I (SR-BI) and LDL receptor (LDLR), both functional in uptaking cholesterol as hHDL 3 NSC99-2320-B-010-013-MY3 (to J.-J.H.) and NSC98-2311-B-002-005-MY3 (to F.-C.K.) Abbreviations: 25-OHC, 25-hydroxycholesterol; 3 ␤ -HSD, 3 ␤ -hydroxysteroid dehydrogenase; ACTH, adrenocorticotropic hormone; AMG, aminoglutethimide; CE, cholesteryl ester; ChIP, chromatin immunoprecipitation; FSH, follicle-stimulating hormone; LDLR, LDL receptor; LRH-1, liver receptor homolog-1; P450scc, P450 cholesterol side chain-cleavage enzyme; SCAP, SREBP cleavage-activating protein; SR-BI, scavenger receptor class B member I; SRE, sterol response element; SREBP, sterol regulatory element-binding protein; StAR, steroidogenic acute regulatory protein; TGF ␤ 1, transforming growth factor ␤ 1. This study was supported by National Science Council of Taiwan Grants

No abstract

Purified bovine liver beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32) and wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) were inhibited with freshly dissolved and 24 h aquated tetrahaloaurate (III) compounds. Rate and equilibrium inhibition constants were measured. From this data two acid phosphatases species were observed. Equilibrium inhibition constants ranged from 1 to 12.5 microM for the various gold compounds toward both enzymes. The first order rate constants ranged between 0.005 and 0.04 min.-1 for most reactions with the exception of the fast reacting acid phosphatase which had values as high as 2.6 and 2.8 min.-1. It is observed that the beta-glucuronidase is rapidly inhibited during the equilibrium phase before the more slower reaction covalent bond formation takes place. The acid phosphatases form the covalent bonds more rapidly, especially the faster reacting species suggesting a unique difference in the active site geometry to that of the more slowly reacting species. The tightly bonded gold (III)-enzyme complex is probably the reason for its toxicity and non-anti-inflammatory use as a drug.

Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.