안티에스트로젠이 MCF-7 사람 유방암 세포에서 분비하는 단백질 합성에 미치는 영향

신윤용,Sheen, Yhun-Yhong 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.8

Tamoxifen과 같은 안티에스트로젠은 에스트로젠 수용체를 다량 함유하는 MCF-7 사람 유방암 세포에 작용하여 선택적으로 분자량 37,000 단백질을 세포로부터 합성하여 분비시키고, 에스트로젠 수용체를 함유하지 않는 MDA-MB-231 사람 유방암 세포에서는 그 효과가 없다. 이러한 안티에스트로젠 작용은 에스트로젠을 병용 투여하면 감소한다. 새롭게 합성되는 단백질은 [$^{35}S$]methionine과 [$^{35}S$]cysteine 존재하에서 세포를 배양한 후 배지로 분비된 단백질을 전기 영동하여 자가방사기록법으로 조사한 결과 안티에스트로젠 6시간 투여로 분자량 37,000 단백질의 증가를 관찰할 수 있고 1~2일 투여로 그 효과가 최고 수준에 도달한다. 이 단백질은 $10^{-8}$M trans-hydroxytamoxifen, LY117018, $10^{-6}$M trans-tamoxifen에 의하여 최대로 합성되고, $10^{-6}$M cis-tamoxifen에 의해서는 합성되지 않으므로 안티에스트로젠 특이적인 작용에 의하여 합성됨을 알 수 있다. 대조군 세포에서 분자량 37,000 단백질의 양은 약한 에스트로젠인 phenol red에 의하여 감소되어 있고, phenol red 제외 배지에서 배양된 세포는 이 단백질이 다량 검출된다. 당단백질 합성 억제제인 tunicamycin과 endoglycosidase H를 처리하면 이 단백질의 합성이 저해되는 것으로부터 이 단백질은 당단백질임을 알 수 있다. Antiestrogen, such as tamoxifen, selectively increased the production of secreted protein of 37,000 molecular weight (Mr) in estrogen receptor positive human breast cancer MCF-7 cells but not in estrogen receptor negative MDA-MB-231 cells, and the production of this protein by antiestrogen was inhibited by concomitant estradiol treatment. Proteins were detected by [$^{35}S$]methionine and [$^{35}S$]cysteine labeling of the cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and autoradiography. Enhanced production of 37,000 Mr protein was observed within 6 h of antiestrogen treatment, with maximal synthesis seen at 1~2 days when this protein represents about 6% of the total radiolabeled secreted proteins. This protein was stimulated maximally by $10^{-8}$ M trans-hydroxytamoxifen or LY 117018 or $10^{-6}$ M tamoxifen, and its antiestrogen specificity was seen by the fact that transtamoxifen increased this protein, whereas cis-tamoxifen, an estrogen, does not. Interestingly, the basal level of synthesis of the 37,000 Mr protein was high in the absence of estrogen and was then stimulated only minimally by the addition of antiestrogen, suggesting that this protein was dearly produced as an estrogen antagonistic protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicated that this protein was glycoprotein. This protein might serve as an useful marker for anti estrogen action in MCF-7 human breast cancer cells.

납(Lead)이 취외분비 기능에 미치는 영향

신윤용(Yhun Yhong Sheen),김원준(Won Joon Kim) 대한약리학회 1981 대한약리학잡지 Vol.17 No.1

No evidence has accumulated that lead compound is an essential component for biological function in animals. Lead is absorbed primarily through the epithelial mucosal cells in duodenum and the absorption can be enhanced by the substances which bind lead and increase its solubility. Iron, zinc and calcium ions, however, decrease the absorption of lead without affecting its solubility, probably by competing for shared absorptive receptors in the intestinal mucosa. Therefore, the absorption of lead is increased in iron deficient animals. Lead shows a strong affinity for ligands such as phosphate, cysteinyl and histidyl side chains of proteins, pterins and porphyrins. Hence lead can act on various active sites of enzymes, inhibiting the enzymes which has functional sulfhydryl groups. lead inhibits the activity of δ-aminolevulinic acid dehydratase for the biosynthesis of hemoproteins and cytochrome, which catalyzed the synthesis of monopyrrole prophobilinogen from δ-aminolevulinic acid. Accordingly lead decrease hepatic cytochrome p-450 content, resulting an inhibition of the activity of demethylase and hydroxylase in liver. Little informations are available on the effect of lead on digestive system although the catastrophic effects of lead intoxication are well documented. The present study was, therefore, attempted to investigate the effect of lead on pancreaticobiliary secretion in rats. Albino rats of both sexes weighing 170 ~ 230g were used for this study. The animals were divided into one control and three treated groups, i.e., control (physiologic saline 1.5ml/kg i.p.), lead acetate (l0μmole/kg/day i.p.), Pb(Ac)<Sub>2</sub> and EDTA(each 10μmole/kg/day i.p.), Pb(Ac)<Sub>2</sub> and FeSO<Sub>4</sub>(each l0μmole/kg/day hp). The pancreatico-biliary juice was collected under urethane anesthesia, and activities of amylase and lipase were determined by employing Sumner s and Cherry and Crandall s methods. The summarized results are follows. 1) In the experiment for acute toxicity of lead acetate, 20% of mortality was observed in rat treated with lead acetate as well as inhibition of the activity of amylase in the juice at the 3 rd day of the treatment. 2) No increases in body weight were observed in rats treated with lead acetate, while in control group the significant increases were observed. However, the body weights of animals were increased in the group lead acetate plus EDTA or FeSO<Sub>4</sub>. 3) Lead acetate decreased significantly the volume of pancreatico-biliary juice whereas additional treatment of EDTA and FeSO<Sub>4</sub> prevented it. 4) Total activity of amylase was markedly reduced due to lead acetate treatment, but no change was showed following additional treatment with EDTA and FeSO<Sub>4</sub>. 5) No changes in the cholate and lipase output were observed in rats treated with lead acetate as compared with that of control rats. 6) Increase in bilirubin output in rats treated with lead acetate was shown on the 2nd and 3rd weeks treatment. 7) In the case of in vitro experiment, lead acetate also markedly inhibited release of amylase from pancreatic fragment. 8) Histologic finding indicated that acini vacuolation was induced in the pancreatic tissue of rat treated with lead acete. From the above results, it might be concluded that lead acetate decreases the volume of pancreatico-biliary secretion and inhibits the amylase activity, by acting directly on pancreatic cells.

안티에스트로젠이 MCF - 7 사람 유방암 세포에서 분비하는 단백질 합성에 미치는 영향

신윤용 ( Yhun Yhong Sheen ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

Antiestrogen, such as tamoxifen, selectively increased the production of secreted protein of 37,000 molecular weight (Mr) in estrogen receptor positive human breast cancer MCF7 cells but not in estrogen receptor negative MDA-MB-231 cells, and the production of this protein by antiestrogen was inhibited by concomitant estradiol treatment. Proteins were detected by [^(35)S]methionine and [^(35)S]cysteine labeling of the cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and autoradiography. Enhanced production of 37,000 Mr protein was observed within 6 h of antiestrogen treatment, with maximal synthesis seen at 1∼2 days when this protein represents about 6% of the total radiolabeled secreted proteins. This protein was stimulated maximally by 10^(-8) M traps-hydroxytamoxifen or LY 117018 or 10^(-6) M tamoxifen, and its antiestrogen specificity was seen by the fact that transtamoxifen increased this protein, whereas cis-tamoxifen, an estrogen, does not. Interestingly, the basal level of synthesis of the 37,000 Mr protein was high in the absence of estrogen and was then stimulated only minimally by the addition of antiestrogen, suggesting that this protein was clearly produced as an estrogen antagonistic protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicated that this protein was glycoprotein. This protein might serve as an useful marker for antiestrogen action in MCF-7 human breast cancer cells.

에스트로젠이 MCF - 7 사람 유방암 세포에서 분비하는 단백질 합성에 미치는 영향

신윤용 ( Yhun Yhong Sheen ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

Estrogen selectively increased the production of sercreted proteins of 32,000 Mr, 52,000 Mr, 160,000 Mr in estrogen receptor positive human breast cancer MCF-7 cells but not in estrogen receptor negative MDA-MB-231 cells, and the production of these proteins by estrogen was inhibited by concomitant antiestrogen treatment. Proteins were detected by [^(35)S]methionine and [^(35)S]cysteine labeling of the cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and autoradiography. Enhanced production of 32,000 Mr, 52,000 Mr, 160,000 Mr proteins were observed within 6 hours of estrogen treatment, with maximal synthesis seen at 2 days. When cells were grown in estrogen free condition, i.e. in charcoaldextran treated serum in medium lacking the estrogen phenol red, the basal level of the 32,000 Mr protein was extremely low, and estrogen stimulation results in 10-fold increase in the production of this protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicates that this protein is glycoprotein. This protein might serve as an useful marker for estrogen action and may be involved in estrogen modulation of cell proliferation and/or cell function.

Radiation enhanced TGF-β signaling and the epithelial-to-mesenchymal trnasition (EMT) in syngeneic breast mouse models

Yhun Yhong Sheen(신윤용),So Yeon Park(박소연),Sin Ri Kim(김신리),Hyun Joo Nam(남현주),Jeong Hwa Jeon(전정화),Hee Won Jeong(정희원) 환경독성보건학회 2016 한국독성학회 심포지움 및 학술발표회 Vol.2016 No.6

에스트로젠의 작용 메카니즘

신윤용 ( Yhun Yhong Sheen ) 생화학분자생물학회 1992 생화학총설집 Vol.4 No.-

Daidzein이 benzo(k)fluoranthene에 의한 사람 유방암 세포 MCF-7의 CYP1A1 유전자 발현 조절에 미치는 영향

양소연,신윤용,Yang, So-Yeon,Sheen, Yhun-Yhong 한국환경성돌연변이발암원학회 2004 한국환경성돌연변이·발암원학회지 Vol.24 No.4

CYP1A1 is known to be inducible by xenobiotic compouds such as polyciclic aromatic hydrocarbons(PAHs) and 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). These chemicals have been identified worldwide and can have a significant impact on the human health and well being of human and wildlife. Given these issues, the detection and quantification of these chemicals in biological, environmental and food samples is important. We investigated the effect of dietaty flavonoid, such as CYP1A1 promoter activity, 7-ethoxyresorufin-O-deethylase(EROD) activity and CYP1A1 mRNA expression induced by benzo(k)fluoranthene(B(k)F) in MCF-7 cells. Based on the three criteria of frequency of occurrence in the environment, toxicity and potential exposure to humans, B(k)F is one of the top-listed PAHs. We found that B(k)F significantly up-regulates the level of CYP1A1 promoter activity, EROD and CYP1A1 mRNA. when cells were treated with daidzein inhibited the B(k)-induced CYP1A1 prompter activity and mRNA level at high concentration. But daidzein exhibited stimulatory effects B(k)F-induced CYP1A1 promoter activity and mRNA level at low concentration. Overall, results from these studies demonstrate flavonoids might interfere the action of B(k) with AhR system to stimulate CYP1A1 gene expression.

Genistein이 Benzo(k)fluoranthene에 의한 CYP1B1 유전자조절 작용에 미치는 영향

서미정,신윤용,Seo, Mi-Jung,Sheen, Yhun-Yhong 한국환경성돌연변이발암원학회 2004 한국환경성돌연변이·발암원학회지 Vol.24 No.4

CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydroxyestradiol that is considered as carcinogenic metabolite. Luciferase activity was induced about 20 folds over that control by 1 nM TCDD (2,3,7,8-tetrchlorodibenzo-p-dioxin) and these inductions were dose-dependent. Recent industrialized society, human hasbeen widely been exposed to widespread environmental contaminants such as PAHs (polycyclic aromatic hydrocarbon) that are originated from the incomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR (aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarket for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounda such as policyclic aromatic hydrocarbon (PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. We examined effects of PAHs on the CYP1B1-lucifrease reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. flvonoids such as genistein decreased B(k)F induced luciferase activity at low concentration. it exhibited stimulatory effect at high concentration.

PAH가 송어 RTH-149세포에서 CYP1A1 유전자 발현에 미치는 영향

김지선,신윤용,Kim, Ji-Sun,Sheen, Yhun-Yhong 한국환경성돌연변이발암원학회 2004 한국환경성돌연변이·발암원학회지 Vol.24 No.4

In mammalian, cytochrome P4501A1 (CYP1A1) is very important for metabolism of xenobiotics such as PAHs(Polycyclic aromatic hydrocarbon) and heterocyclic amine, and it is induced by environmental contaminants such as PAHs, TCDD(2,3,7,8-tetrchlorodibenzo-p-dioxin) and 3-MC (3-methylcholanthrene). In fish, like mammalian, when it is exposed to environmental contaminants, they cause specific and sensitive induction of CYP1A. Therefore, induction of CYP1A in fish and mammalian is widely used as a biomarker for exposure of environmental contaminants. In this study, to compare the function of Cyp1a1 in fish with it in mammalian, we have used rainbow trout(Oncorhynchys mykiss) hepatoma cells (RTH-149) and mouse hepatocyte (Hepa-I). in order to examine induction of Cyp1a1 by TCDD, we have used the bioassay system. We examined effects of TCDD on the Cyp1a1-luciferase reporter gene activity, 7-ethoxyresorufin O-deethylase(EROD) activity and Cypa mRNA level.

Daisdzein이 Benzo(k)fluoranthene에 의한 CYP1B1 유전자조절 작용에 미치는 영향

서미정,김여운,신윤용,Seo, Mi-Jeong,Kim, Yeo-Woon,Sheen, Yhun-Yhong 한국환경성돌연변이발암원학회 2004 한국환경성돌연변이·발암원학회지 Vol.24 No.4

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Recent industrialized industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA. Some flavonoids such as genistein, daidzein, chrysin, naringenin and morin were also investigeted. These flavonoids decreased B(k)F infuced luciferase activity at low concentration. But, these flavonoids exhibited stimulatory effect at high concentration.