Prevention of azoxymethane/dextran sodium sulfate-induced mouse colon carcinogenesis by processed Aloe vera gel.

The preventive effect of a processed Aloe vera gel (PAG) on colon carcinogenesis was examined using an azoxymethane (AOM)-initiated and dextran sodium sulfate (DSS)-promoted mouse colon carcinogenesis model. Oral administration of PAG (200, or 400mg/kg/day) significantly reduced the multiplicity of colonic adenomas and adenocarcinomas compared with the AOM/DSS only-treated mice. In the mice treated with 400mg/kg of PAG, adenoma and adenocarcinoma development was reduced to 80% and 60%, respectively, compared to 100% in the PAG-untreated AOM/DSS-treated mice. Western blot analysis using colon extracts showed that PAG reduced the activation of nuclear factor kappa B (NF-κB), resulting in the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression. PAG appeared to inhibit the NF-κB activation through the activation of peroxisome proliferator-activated receptor gamma. PAG also inhibited the expression and phosphorylation of signal transducer and activator of transcription 3, which is known to connect inflammation and cancer. In addition, PAG inhibited cell cycle progression-inducing cellular factors, such as extracellular signal-regulated kinases 1/2, cyclin-dependent kinase 4, and cyclin D1. On the other hand, PAG increased the expression of Caudal-related homeobox transcription factor 2, which is known to be a tumor suppressor in colorectal cancer. These findings show that PAG suppresses colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon.

From the Cover: Ethylmercury-Induced Oxidative and Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death: Involvement of Autophagosome-Lysosome Fusion Arrest.

Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Ca2+ levels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

Induction of mast cell degranulation by triterpenoidal saponins obtained from Cimicifugae rhizoma.

Cimicifugae rhizoma has been widely used as a traditional herbal medicine to treat inflammation and menopausal symptoms. In this study, we found that some of the triterpenoidal saponins purified from the ethanol extract of Cimicifugae rhizoma dramatically induced histamine release. The structure-related induction of mast cell degranulation by them and the mechanism of action were determined. ß-Hexosaminidase release in HMC-1 cells was increased in a concentration-dependent manner, with maximal 6.5- and 8.5-fold increases, by 200 µg/mL 24-epi-7,8-didehydrocimigenol-3-O-xyloside (comp 1) and cimigenol 3-O-beta-d-xyloside (comp 4) compared with those treated with phorbol 12-myristate 13-acetate and A23187 (PMACI), respectively. However, ß-hexosaminidase release was not changed by 7,8-dihydrocimigenol (comp 3), or 23-OAc-shengmanol-3-O-xyloside (comp 7). These triterpenoidal saponins changed neither the intracellular Ca(2+ )level nor the activation of PKC, both of which play essential roles in histamine release. However, cromolyn and ketotifen, membrane stabilizers, effectively inhibited the ß-hexosaminidase release induced by comp 1 or comp 4 by 39 and 45%, respectively. Collectively, xylose on the cimigenol-related backbone among triterpene glycosides isolated from Cimicifugae rhizoma may play an important role in activating mast cells and induction of degranulation partly via membrane destabilization of mast cells.

Diagnosis and treatment of patients with thyroid cancer.

BACKGROUND: Thyroid cancer is the most common malignancy of the endocrine system, representing 3.8% of all new cancer cases in the United States and is the ninth most common cancer overall. The American Cancer Society estimates that 62,450 people in the United States will be diagnosed with thyroid cancer in 2015, and 1950 deaths will result from the disease. OBJECTIVE: To review the current approach to the diagnosis and treatment of patients with thyroid cancer. DISCUSSION: Over the past 3 decades, there has been a dramatic increase in the number of people diagnosed with thyroid cancer, which may be attributable to the wide use of imaging studies, including ultrasounds, computed tomography, magnetic resonance imaging, and positron emission tomography scans that incidentally detect thyroid nodules. Thyroid cancer is divided into several main types, with papillary thyroid cancer being the most common. The treatment options for patients with thyroid cancer include the surgical removal of the entire thyroid gland (total thyroidectomy), radioactive iodine therapy, and molecular-targeted therapies with tyrosine kinase inhibitors. This article summarizes the diagnosis and treatment of thyroid cancer, with recommendations from the American Thyroid Association regarding thyroid nodules and differentiated thyroid cancer. Recently approved drugs and treatment trends are also explored. CONCLUSION: The prognosis and treatment of thyroid cancer depend on the tumor type and its stage at the time of diagnosis. Many thyroid cancers remain stable, microscopic, and indolent. The increasing treatment options for patients with thyroid cancer, including therapies that were recently approved by the US Food and Drug Administration, have kept the mortality rate from this malignancy low, despite the increase in its incidence. Early diagnosis and appropriate treatment can improve prognosis and reduce mortality.

Estrogenic endocrine-disrupting chemicals modulate the production of inflammatory mediators and cell viability of lipopolysaccharide-stimulated macrophages.

Estrogenic endocrine-disrupting chemicals (EDCs) are exogenous substances that act as competitive inhibitors of estrogen in the endocrine system. By disrupting the endocrine system, EDCs can cause severe disabilities and diseases, including cancers and altered sexual development. Although the influence of these molecules in the endocrine system is evident, the effects of EDCs on the immune system as well as their cytotoxicity have been poorly examined. Therefore, we selected 21 EDCs that are commonly found in Korean ecosystems, such as surface waters and effluents, and studied their immunologic effects by comparing nitric oxide (NO) production and cytotoxicity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (RAW cells), a macrophage cell line. Among the EDCs tested, fenitrothion (FTH) inhibited the messenger RNA (mRNA) expression of inducible NO synthase (iNOS), resulting in reduced NO production, while treatment with andostenedione (AD), diethyl phthalate, di-n-butyl phthalate (DBP), estriol, or molinate decreased production of NO in an iNOS-independent fashion. In contrast, benzo(a)pyrene (B(a)P) increased the production of NO in RAW cells. In addition, AD, DBP, or FTH inhibited the mRNA expression of tumor necrosis factor alpha or interleukin-1 beta. Treatment with 17-α-ethynylestradiol, 17-ß-estradiol, 4-n-butyl phenol, or alachlor induced apoptosis of RAW cells, while dicyclohexyl phthalate and B(a)P caused cell death in an apoptosis-independent manner. These data suggest that EDCs can influence the immune response to pathogens by modulating the functions of macrophages.

Processed Aloe vera gel ameliorates cyclophosphamide-induced immunotoxicity.

The effects of processed Aloe vera gel (PAG) on cyclophosphamide (CP)-induced immunotoxicity were examined in mice. Intraperitoneal injection of CP significantly reduced the total number of lymphocytes and erythrocytes in the blood. Oral administration of PAG quickly restored CP-induced lymphopenia and erythropenia in a dose-dependent manner. The reversal of CP-induced hematotoxicity by PAG was mediated by the functional preservation of Peyer's patch cells. Peyer's patch cells isolated from CP-treated mice, which were administered PAG, produced higher levels of T helper 1 cytokines and colony-stimulating factors (CSF) in response to concanavalin A stimulation as compared with those isolated from CP-treated control mice. PAG-derived polysaccharides directly activated Peyer's patch cells isolated from normal mice to produce cytokines including interleukin (IL)-6, IL-12, interferon-γ, granulocyte-CSF, and granulocyte-macrophage-CSF. The cytokines produced by polysaccharide-stimulated Peyer's patch cells had potent proliferation-inducing activity on mouse bone marrow cells. In addition, oral administration of PAG restored IgA secretion in the intestine after CP treatment. These results indicated that PAG could be an effective immunomodulator and that it could prevent CP-induced immunotoxic side effects.

Effects of a new sustained-release microsphere formulation of exenatide, DA-3091, on obese and non-alcoholic fatty liver disease mice.

The aim of this study was to examine the effects of a new sustained-release (SR) microsphere formulation of exenatide, DA-3091, on body weight gain and hepatic injury in high fat diet (HFD)-induced obese mice and high sucrose diet (HSD)-induced non-alcoholic fatty liver disease (NAFLD) mice. Then, we determined whether DA-3091 has the potency as a drug for the treatment of metabolic disease. In obese mice, after 8-week treatment, the body weight gain was significantly more suppressed by both 1 mg/kg and 2 mg/kg of DA-3091, monthly subcutaneous administered, than by 10 mg/kg/day of sibutramin, a drug against obesity. In NAFLD mice, a significant reduction in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, representative markers of hepatic injury, was observed after biweekly subcutaneous administration of 1 mg/kg and 2 mg/kg of DA-3091 for 8 weeks. A significant reduction in hepatic lipid accumulation was observed in DA-3091 treated groups as well. Based on these results, it is demonstrated that DA-3091 has the potency as a drug for the treatment of metabolic disease.

2,3,6-Trisubstituted quinoxaline derivative, a small molecule inhibitor of the Wnt/beta-catenin signaling pathway, suppresses cell proliferation and enhances radiosensitivity in A549/Wnt2 cells.

GDK-100017, a 2,3,6-trisubstituted quinoxaline derivative, reduced ß-catenin-T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity and inhibited cell proliferation in a dose-dependent manner with an IC50 value of about 10 µM in A549/Wnt2 cells. GDK-100017 down-regulated the expression of Wnt/ß-catenin pathway target genes such as cyclin D1 and Dkk1 but not c-myc or survivin. GDK-100017 inhibited cell proliferation by arresting the cell cycle in the G1 phase not only in A549/wnt2 cells but also in SW480 colon cancer cells. In addition to its wnt signaling inhibitory properties, GDK-100017 also enhanced the radiosensitivity of the A549 human NSCLC line. These results suggest that GDK-100017 possesses potential anti-cancer activity by inhibiting the Wnt/ß-catenin signal pathway, blocking the ß-catenin-TCF/LEF interaction, and enhancing radiosensitivity.

A tobacco CBL-interacting protein kinase homolog is involved in phosphorylation of the N-terminal domain of the cucumber mosaic virus polymerase 2a protein.

The replication and transcription of cucumber mosaic virus (CMV) are catalyzed by multi-protein complex RNA-dependent RNA polymerase (RdRp), which is composed of the viral-encoded 1a and 2a proteins with host factors. We have reported that the N-terminal region of the polymerase 2a protein, composed of 126 amino acids, is required for interaction with the helicase 1a protein, and that the phosphorylation of the region abrogated interaction with the 1a protein, suggesting a mechanism of resistance in host plants against viral infection. Here, we found that three protein 2a kinases, of 60, 55, and 38 kDa, co-purified with the tobacco membrane fraction in an in-gel kinase assay. By yeast two-hybrid library screening using the N-terminal 126 amino acids of 2a as a bait, we identified CBL-interacting protein kinase 12 (NtCIPK12) corresponding to 55 kDa protein 2a kinase. The bacterially expressed protein kinase showed protein 2a kinase (t2aK) activity in vitro. We found that NtCIPK12 stabilized upon CMV infection at the post-translational level, and accumulated more heavily to the membrane than in the cytosol.

Estrogenic/antiestrogenic activities of a Epimedium koreanum extract and its major components: in vitro and in vivo studies.

The estrogenic and antiestrogenic activities of Epimedii Herba, which is a traditional medicinal herb used in Korea and China were investigated in this study. The in vitro estrogen receptor (ER) mediated estrogenic/antiestrogenic activities of an Epimedii Herba extract (Epi ext) and its major components were determined using an estrogen responsive element driven reporter gene assay in MCF-7/ERE and HEK293T cells. The Epi ext exhibited ERα- and ERß-mediated estrogenic activity with an EC(50) of 5.0 and 17.8 µM in HEK293T cells, respectively. Prenylflavonoid glycosides such as icariin (ICA), epimedin A, B, and C did not show any in vitro estrogenic or antiestrogenic activities. Icaritin (ICT) and quercetin exhibited in vitro ER mediated estrogenic activity with a more potent interaction with ERß. In vivo estrogenic activities of the Epi ext, ICA and ICT were compared using an uterotrophic assay. Although the potency of in vitro estrogenic activity was in the order of ICT>Epi ext>ICA, ICA had the strongest estrogenic activity and next ICT in ovariectomized rats. These results collectively suggest that phytoestrogens possess both estrogenic and antiestrogenic activity, and that the differential expression of these two compounds with opposing activities is dependent on the physiological environment in terms of estrogen level, which may be the case in humans.

Anti-inflammatory activity of 6-hydroxy-2,7-dimethoxy-1,4-henanthraquinone from tuberous roots of yam (Dioscorea batatas) through inhibition of prostaglandin D2 and leukotriene C4 production in mouse bone marrow-derived mast cells.

6-Hydroxy-2,7-dimethoxy-1,4-phenanthraquinone (PAQ) isolated from the tuberous roots of Yam (Dioscorea batatas) inhibited cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) dependent prostaglandin D(2) (PGD(2)) generation in mouse bone marrow-derived mast cells in a concentration-dependent manner with IC(50) values of 0.08 µM and 0.27 µM, respectively. In the Western blotting with specific anti-COX-2 antibodies, the decrease of the quantity of PGD(2) was accompanied by a decrease in the COX-2 protein level. But PAQ did not affect COX-1 protein level. In addition, this compound inhibited 5-lipoxygenase (5-LOX) dependent production of leukotriene C(4) in a dose-dependent manner, with an IC(50) of 0.032 µM. These results demonstrate that PAQ has a dual COX-2/5-LOX inhibitory activity. This compound also inhibited the degranulation reaction in a dose-dependent manner with an IC(50) of 2.7 µM. Thus, these results suggest that PAQ may be useful in regulating mast cell-mediated inflammatory diseases.

Neuroprotective effects of chlorogenic acid on scopolamine-induced amnesia via anti-acetylcholinesterase and anti-oxidative activities in mice.

Chlorogenic acid is a major polyphenolic component of many plants and beverages, and is particularly abundant in coffee. We evaluated the neuroprotective effects of chlorogenic acid on learning and memory impairment induced by scopolamine (0.5 mg/kg, i.p.), a muscarinic antagonist, using the Y-maze, passive avoidance, and Morris water maze tests. The chlorogenic acid significantly improved the impairment of short-term or working memory induced by scopolamine in the Y-maze test, and significantly reversed cognitive impairments in mice as measured by the passive avoidance test. In addition, chlorogenic acid decreased escape latencies in the Morris water maze test. In a probe trial session, chlorogenic acid increased the latency time in the target quadrant in a dose-dependent manner. Ex vivo, chlorogenic acid inhibited acetylcholinesterase activity in the hippocampus and frontal cortex. Chlorogenic acid also decreased malondialdehyde levels in the hippocampus and frontal cortex. In vitro, chlorogenic acid was found to inhibit acetylcholinesterase activity (IC50=98.17 µg/ml) and free radical scavenging activity (IC50=3.09 µg/ml) in a dose-dependent manner. These results indicate that chlorogenic acid may exert anti-amnesic activity via inhibition of acetylcholinesterase and malondialdehyde in the hippocampus and frontal cortex.

Anti-inflammatory activity of bark of Dioscorea batatas DECNE through the inhibition of iNOS and COX-2 expressions in RAW264.7 cells via NF-κB and ERK1/2 inactivation.

We identified a bioactive herbal medicine with anti-inflammatory activity from an ethanol extract derived from the bark of Dioscorea batatas DECNE (BDB) in RAW264.7 cells. We examined the effects of BDB on nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in LPS-induced RAW264.7 cells. BDB consistently inhibited both NO and PGE(2) production in a dose-dependent manner, with an IC(50) of 87-71 µg/ml, respectively. The reduction of NO and PGE(2) production were accompanied by a reduction in iNOS and COX-2 protein expression, as evaluated by Western blotting. To evaluate the action mode of BDB and its ability to inhibit iNOS and COX-2 protein expression, we assessed the effects of BDB on nuclear factor-κB (NF-κB) DNA-binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation and degradation, and p65 nuclear translocation. BDB suppressed DNA-binding activity and reporter gene activity as well as translocation of the NF-κB p65 subunit. BDB also down-regulated IκB kinase (IKK), thus inhibiting LPS-induced both phosphorylation and the degradation of IκBα. In addition, BDB also inhibited the LPS-induced activation of ERK1/2.

Identification of 2,3,6-trisubstituted quinoxaline derivatives as a Wnt2/ß-catenin pathway inhibitor in non-small-cell lung cancer cell lines.

We screened 1434 small heterocyclic molecules and identified thirteen 2,3,6-trisubstituted quinoxaline derivatives that were able to inhibit the Wnt/ß-catenin signal pathway and cell proliferation. In the screen, some of the hit compounds such as the ethylene group-coupled quinoxaline derivatives were shown to hold promise for use as potential small-molecule inhibitors of the Wnt/ß-catenin signal pathway in non-small-cell lung cancer cell lines.

Effects of CYP2C19 and MDR1 genotype on the eradication rate of Helicobacter pylori infection by triple therapy with pantoprazole, amoxycillin and clarithromycin.

BACKGROUND: CYP2C19 polymorphism plays an important role in the metabolism of proton pump inhibitors. The multidrug resistance (MDR)1 genotype is associated with the successful eradication of Helicobacter pylori. The aim of the present study was to investigate the effects of CYP2C19 and MDR1 genotypes on the eradication rate of H. pylori using a pantoprazole-based triple therapy. METHODS: A total of 210 patients infected with H. pylori were treated with 40 mg pantoprazole, 500 mg clarithromycin and 1000 mg amoxicillin twice daily for 7 days. The CYP2C19 genotype was determined with polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. The MDR1 C3435T polymorphism was identified by PCR-based allele-specific amplification (PCR-ASA). RESULTS: Of the 210 patients who completed the study, 174 (82.9%, 95.0% confidence interval [CI], 77.8-88.0%) achieved successful eradication after the first cycle of therapy. The eradication rates for H. pylori were 86.7%, 81.1% and 82.1% in the homozygous extensive, heterozygous extensive and poor metabolizer groups, respectively (P = 0.65). Moreover, the cure rates in the CC, CT, and TT groups were 82.7%, 84.4% and 76.9%, respectively (P = 0.66). Multiple logistic regression analysis revealed that endoscopic diagnosis was a significant independent risk factor for treatment failure. CONCLUSION: The eradication rates of H. pylori by pantoprazole, amoxicillin and clarithromycin were not significantly different among the CYP2C19 and MDR1 genotypes. Hence, the cure rate of H. pylori in the Korean population was no different for the CYP2C19 and MDR1 genotypes.

Antitumor activity of the novel human cytokine AIMP1 in an in vivo tumor model.

Although AIMP1 (previously known as p43) is one of three auxiliary proteins bound to a macromolecular aminoacyl tRNA complex, it is also secreted as a cytokine controlling both angiogenesis and immune responses. Here we show that systemically administered purified recombinant human AIMP1 had anti-tumor activity in mouse xenograft models. In Meth A-bearing Balb/c mice, tumor volume increased about 28 fold in the vehicle treatment group, while an increase of about 16.7 fold was observed in the AIMP1-treated group. We also evaluated the anti-tumor activity of AIMP1 in combination with a sub-clinical dose of the cytotoxic anti-tumor drug, paclitaxel. The growth of NUGC-3 human stomach cancer cells was suppressed by 84% and 94% by the combinations of 5 mg/kg paclitaxel + 25 mg/kg AIMP1 (p = 0.03), and 5 mg/kg paclitaxel + 50 mg/kg AIMP1 (p = 0.02), respectively, while 5 mg/kg paclitaxel alone suppressed growth by only 54% (p = 0.02). A similar cooperative effect of AIMP1 and paclitaxel was observed in a lung cancer xenograft model. These results suggest that AIMP1 may be useful as a novel anti-tumor agent.

Comparison of prostate cancer cell lines for androgen receptor-mediated reporter gene assays.

In order to select a better prostate cancer cell model for androgen receptor (AR)-mediated reporter gene assays, we assessed the androgen response characteristics of three cell lines, LNCaP, PC3/AR(+) and 22Rv1, in this study. Both the mRNA and the proteins of AR and glucocorticoid receptor (GR) were expressed in all three cell lines. Among the three cell lines, only in LNCaP cells, DHT concentration-dependently stimulated proliferation. DHT induced the luciferase activity in three cell lines which were transiently transfected with pMMTV-Luc, in a concentration-dependent manner. The maximum induction was 24.0-fold and 13.4-fold in 22Rv1 and in the LNCaP respectively. PC3/AR(+) were more sensitive to respond to DHT at a minimal concentration of 10(-12)M by 14.0-fold induction. The transcriptional activity induced with 10(-8)M DHT was inhibited about 50-75% in the PC3/AR(+) and 22Rv1, and 98% in the LNCaP, by vinclozolin. Dexamethasone concentration-dependently induced the luciferase activity in PC3 and 22Rv1, but not in the LNCaP. However, the response to dexamethasone in 22Rv1 was very weak compared to DHT. The (anti)androgencity of seven pyrethroids was assessed via an AR-mediated luciferase reporter assay. None of them showed the androgenic action in all three cell lines. Permethrin inhibited the DHT induced luciferase activity about 22%, 35.8% and 75.5% in 22Rv1, PC3/AR(+) and LNCaP, respectively. Based on results from in this study and cell line character, 22Rv1 cells seemed to be an appropriate model for the screening of androgenic endocrine disruptors, although it needs further studies with other steroid receptor and thyroid receptor.

In vitro and in planta interaction evidence between Nicotiana tabacum thaumatin-like protein 1 (TLP1) and cucumber mosaic virus proteins.

Using a yeast two-hybrid system, we identified a plant cellular factor that interacts with the proteins of the Cucumber mosaic virus (CMV). Initially 14 candidate genes were isolated from Nicotiana tabacum, using a full-length CMV 1a gene as bait. Among the candidate genes, two were encoding thaumatin-like proteins (TLP), and were designated as Nicotiana tabacum thaumatin-like protein 1 (NtTLP1). Consistent with this observation, recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein and coat protein, in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation.

Effects of hydroxyl group numbers on the B-ring of 5,7-dihydroxyflavones on the differential inhibition of human CYP 1A and CYP1B1 enzymes.

Flavonoids are polyphenols composed of two aromatic rings (A, B) and a heterocyclic ring (C). In order to determine the effects of the number of hydroxyl groups in the B-ring of the flavonoids on human cytochrome P450 (CYP) 1 family enzymes, we evaluated the inhibition of CYP1A-dependent 7-ethoxyresorufin omicron-deethylation activity by chrysin, apigenin and luteolin, using bacterial membranes that co-express human CYP1A1, CYP1A2, or CYP1B1 with human NADPH-cytochrome P450 reductase. Chrysin, which possesses no hydroxyl groups in its B-ring, exhibited the most pronounced inhibitory effects on CYP1A2-dependent EROD activity, followed by apigenin and luteolin. On the contrary, CYP1A1-mediated EROD activity was most potently inhibited by luteolin, which is characterized by two hydroxyl groups in its B-ring, followed by apigenin and chrysin. However, all of the 5,7-dihydroxyflavones were determined to similarly inhibit CYP1B1 activity. Chrysin, apigenin, and luteolin exhibited a mixed-type mode of inhibition with regard to CYP1A2, CYP1B1, and CYP1A1, with apparent Ki values of 2.4, 0.5, and 2.0 microM, respectively. These findings suggested that the number of hydroxyl groups in the B-ring of 5,7-dihydroxyflavone might have some influence on the degree to which CYP1A enzymes were inhibited, but not on the degree to which CYP1B1 enzymes were inhibited.

Effects of 2,4-D and DCP on the DHT-induced androgenic action in human prostate cancer cells.

2,4-Dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (DCP) are used extensively in agriculture as herbicides, and are suspected of potential endocrine disruptor activity. In a previous study, we showed that these compounds exhibited synergistic androgenic effects by co-treatment with testosterone in the Hershberger assay. To elucidate the mechanisms of the synergistic effects of these compounds on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. In in vitro assay systems, while 2,4-D or DCP alone did not show androgenic activity, 2,4-D or DCP with 5alpha-dihydroxytestosterone (DHT) exhibited synergistic androgenic activities. Co-treatment of 10 nM 2,4-D or DCP with 10 nM DHT was shown to stimulate the cell proliferation by 1.6-fold, compared to 10 nM DHT alone. In addition, in transient transfection assays, androgen-induced transactivation was also increased to a maximum of 32-fold or 1.28-fold by co-treatment of 2,4-D or DCP with DHT, respectively. However, 2,4-D and DCP exerted no effects on either mRNA or protein levels of AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to AR, up to 50% at concentrations of approximately 0.5 microM for both compounds. The nuclear translocation of green fluorescent protein-AR fusion protein in the presence of DHT was promoted as the result of the addition of 2,4-D and DCP. Collectively, these results that 2,4-D and DCP enhanced DHT-induced AR transcriptional activity might be attributable, at least in part, to the promotion of AR nuclear translocation.