• Biomedical Science Letters
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• v.11 no.2
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• pp.175-183
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• 2005

The two major isoflavones in soy, genistein and daidzein, are well known to prevent hormone-dependent cancers by their anti estrogenic activity. The exact molecular mechanisms for the protective action are, however, not provided yet. It has been reported that genistein and daidzein have a potential anticancer activity through their antiproliferative effect in many hormone-dependent cancer cell lines. Transforming growth $factor-\beta1(TGF-\beta1)$ has also been found to have cell growth inhibitory effect, especially in mammary epithelial cells. This knowledge led to a hypothetical mechanism that the soy isoflavones-induced growth inhibitory effect can be derived from the regulation of $TGF-\beta1$ and $TGF-\beta$ receptors. In order to test this hypothesis, the effects of the soy isoflavones at various concentrations and periods on the expression of $TGF-\beta1$and $TGF-\beta$ receptors were investigated by using Northern blot analysis in human breast carcinoma epithelial cell lines, an estrogen receptor positive cell line (MCF-7) and an estrogen receptor negative cell line (MDA-MB-231). As a result, only genistein has shown a profound dose-dependent effect on $TGF-\beta1$ expression in the $ER^+$ cell line within the range of doses tested, and the expression levels are correspondent to their inhibitory activities of cell growth. Moreover, daidzein showed down-regulated $TGF-\beta1$ expression at a low dose, the cell growth proliferation was promoted at the same condition. Therefore, antiproliferative activity of the soy isoflavones can be mediated by $TGF-\beta1$ expression, and the effects are mainly, if not all, occurred by ER dependent pathway. The expression of $TGF-\beta$ receptors was induced at a lower dose than the one for $TGF-{\beta}1$ induction regardless of the presence of ER, and the expression patterns are similar to those of the cell growth inhibition. These results indicated that the regulation of $TGF-\beta$ receptor expression as well, prior to $TGF-\beta1$ expression, may be involved in the antiproliferative activity of soy isoflavones. Little or no expression of $TGF-\beta$ receptors was found in the MCF-7 and MDA-MB-231 cells, suggesting refractory properties of the cells to growth inhibitory effect of the $TGF-\beta$. The soy isoflavones can seemingly restore the sensitivity of growth inhibitory responses to $TGF-\beta1$ by re-inducing $TGF-\beta$ receptors expression. In conclusions, our findings presented in this study show that the antitumorigenic activity of the soy isoflavones could be mediated by not only $TGF-\beta1$induction but $TGF-\beta$ receptor restoration. Thus, soy isoflavones could be good model molecules to develop new nonsteroidal antiestrogenic chemopreventive agents, associated with, regulation of $TGF-\beta$ and its receptors.

• Journal of Life Science
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• v.29 no.2
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• pp.256-264
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• 2019

Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. In contrast, MLCK and ROCK play critical roles in the regulation of stress fiber (SF) formation in cells. To determine whether $LT{\beta}R$ stimulation in fibroblastic reticular cells (FRCs) is involved in these signaling pathways, myosin light chain kinase inhibitor-7 (ML-7) was used to inhibit them. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic anti-$LT{\beta}R$ antibody-treated cells. The inhibition of ROCK activity with Y27632-induced changes in actin cytoskeleton organization and cell morphology in FRCs. Actin bundles rearranged into SFs, and phospho-myosin light chain (p-MLC) co-localized in FRCs. We checked the level of Rho-guanosine diphosphate (RhoGDP)/guanosine triphosphate (GTP) exchange activity using FRC lysate. When $LT{\beta}R$ was stimulated with agonistic anti-$LT{\beta}R$ antibodies, Rho-GDP/GTP exchange activity was markedly reduced. Regarding $LT{\beta}R$ signaling with a focus on MLCK inhibition, we showed that the phosphorylation of MLCs was reduced by $LT{\beta}R$ stimulation in FRCs. Cytoskeleton components, such as tubulin, b-actin, and phospho-ezrin proteins acting as membrane-cytoskeleton linkers, were produced in de-phosphorylation, and they reduced expression in agonistic anti-$LT{\beta}R$ antibody-treated FRCs. Collectively, the results suggested that MLCK and ROCK were simultaneously responsible for SF regulation triggered by $LT{\beta}R$ signaling in FRCs.

• International Journal of Oral Biology
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• v.37 no.1
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• pp.1-7
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• 2012

Opioid receptors have been pharmacologically classified as ${\mu}$, ${\delta}$, ${\kappa}$ and ${\varepsilon}$. We have recently reported that the antinociceptive effect of morphine (a ${\mu}$-opioid receptor agonist), but not that of ${\beta}$-endorphin (a novel ${\mu}/{\varepsilon}$-opioid receptor agonist), is attenuated by whole body irradiation (WBI). It is unclear at present whether WBI has differential effects on the antinociceptive effects of ${\mu}-$, ${\delta}-$, ${\kappa}-$ and ${\varepsilon}$-opioid receptor agonists. In our current experiments, male ICR mice were exposed to WBI (5Gy) from a $^{60}Co$ gamma-source and the antinociceptive effects of opioid receptor agonists were assessed two hours later using the hot water ($52^{\circ}C$) tail-immersion test. Morphine and $D-Ala^2$, $N-Me-Phe^4$, Gly-olenkephalin (DAMGO), [$D-Pen^2-D-Pen^5$] enkephalin (DPDPE), trans-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U50,488H), and ${\beta}$-endorphin were tested as agonists for ${\mu}$, ${\delta}$, ${\kappa}$, and ${\varepsilon}$-opioid receptors, respectively. WBI significantly attenuated the antinociceptive effects of morphine and DAMGO, but increased those of ${\beta}$-endorphin. The antinociceptive effects of DPDPE and U50,488H were not affected by WBI. In addition, to more preciously understand the differential effects of WBI on ${\mu}-$ and ${\varepsilon}$-opioid receptor agonists, we assessed pretreatment effects of ${\beta}$-funaltrexamine (${\beta}$-FNA, a ${\mu}$-opioid receptor antagonist) or ${\beta}$-$endorphin_{1-27}$ (${\beta}$-$EP_{1-27}$, an ${\varepsilon}$-opioid receptor antagonist), and found that pretreatment with ${\beta}$-FNA significantly attenuated the antinociceptive effects of morphine and ${\beta}$-endorphin by WBI. ${\beta}$-$EP_{1-27}$ significantly reversed the attenuation of morphine by WBI and significantly attenuated the increased effects of ${\beta}$-endorphin by WBI. The results demonstrate differential sensitivities of opioid receptors to WBI, especially for ${\mu}-$ and ${\varepsilon}$-opioid receptors.

• Biomolecules & Therapeutics
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• v.25 no.1
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• pp.44-56
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• 2017

Insulin resistance is characterized by the reduced ability of insulin to stimulate tissue uptake and disposal of glucose including cardiac muscle. These conditions accelerate the progression of heart failure and increase cardiovascular morbidity and mortality in patients with cardiovascular diseases. It is noteworthy that some conditions of insulin resistance are characterized by up-regulation of the sympathetic nervous system, resulting in enhanced stimulation of ${\beta}$-adrenergic receptor (${\beta}$AR). Overstimulation of ${\beta}$ARs leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, pathological consequences of the cross-talk between the ${\beta}$AR and the insulin sensitivity and the mechanism by which ${\beta}$AR overstimulation promotes insulin resistance remain unclear. This review article examines the hypothesis that ${\beta}$ARs overstimulation leads to induction of insulin resistance in the heart.

• Biomolecules & Therapeutics
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• v.24 no.5
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• pp.517-522
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• 2016

${\beta}$-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of ${\beta}$-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with ${\beta}$-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with ${\beta}$-arrestin2. For this, we employed dopamine $D_2$ and $D_3$ receptors ($D_2R$ and $D_3R$, respectively), since they display distinct agonist-induced interactions with ${\beta}$-arrestins. Our results showed that the second and third intracellular loops of $D_2R$ are involved in the agonist-induced translocation of ${\beta}$-arrestins toward plasma membranes. In contrast, the N- and C-termini of $D_2R$ exerted negative effects on the basal interaction with ${\beta}$-arrestins.

• Molecules and Cells
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• v.38 no.2
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• pp.105-111
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• 2015

The beta2-adrenergic receptor (${\beta}2AR$) belongs to the G protein coupled receptor (GPCR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound ${\beta}2AR$ in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, ${\beta}2AR$ is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of ${\beta}2AR$. In this review, structural features of inactive and active states of ${\beta}2AR$, the interaction of ${\beta}2AR$ with heterotrimeric G protein, and the comparison with ${\beta}1AR$ will be discussed.

• Journal of Yeungnam Medical Science
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• v.28 no.2
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• pp.153-164
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• 2011

Background: It has been reported that estrogen receptor beta ($ER{\beta}$) mRNA expression was down-regulated during carcinogenesis and was inversely related to estrogen receptor alpha ($ER{\alpha}$) expression in breast cancer. The association of $ER{\beta}$ mRNA expression to tamoxifen resistance has also been reported. In this study, the expression of $ER{\alpha}$ and $ER{\beta}$ via immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) was prompted, and an attempt was made to find out the relationship between $ER{\beta}$ expression and recurrence in the hormonal therapy group, and between $ER{\beta}$ expression and known prognostic factors. Methods: Tumor specimens were obtained at surgery from 67 female breast cancer patients during the period of September 1995 to December 2000. All the specimens were frozen in liquid nitrogen and kept at $-70^{\circ}C$ until they were used. The medical records were analyzed retrospectively. The expressions of ER were analyzed using IHC and RT-PCR methods. Results: The median follow-up was at 93.0 months (range: 14-157 months). The percentage of $ER{\alpha}+/ER{\beta}+$, $ER{\alpha}+/ER{\beta}-$, $ER{\alpha}-/ER{\beta}+$, and $ER{\alpha}-/ER{\beta}$ group were 35.9% 9.4%, 47.2%, and 7.5%, respectively, in 53 patients with hormonal therapy. $ER{\beta}$ was positive in 42 (82.3%) of 51 ER-positive patients. In the hormonal therapy group, the recurrence rates of each group was 15.8%, 0%, 40.0%, and 0%, respectively. In this group, the $ER{\beta}$ expression tended to recur, but there was no clinical significance (p=0.084). Conclusion: The $ER{\beta}$ expression may be a predictive marker of a poor response to endocrine therapy in breast cancer patients, although this needs to be confirmed in additional studies.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.65-70
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• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.164-164
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• 2002

에스트로젠을 처리한 송사리의 간으로부터 choriogenin vitellogenin estrogen receptor의 발현량을 전사수준에서 Real-time을 사용하여 정량.비교하였다. 시험어종으로는 부화 후 5개월 이상된 성숙한 수컷 송사리(Oryzias latipes)를(체중 약 250mg/마리)를 사용하여 17$\beta$-estradiol(25ppt, 50ppt, 100ppt)에 24시간 노출시켰다. Fluorescence dye는 choriogenin vitellogenin estrogen receptor의 경우 FAM (6-carboxyfluorescein)을 사용하였으며, $\beta$-actin의 경우는 VIC를 사용하였다. 프로브에 사용하는 quencher dye는 TAMRA(6-carboxy-N',N',N',N'-tetramethyl rhodamine)을 사용하였다. Internal control로 사용된 $\beta$-actin은 17$\beta$-estradiol의 농도에 상관 없이 0~10pM 범위에서 일정하게 발현됨을 보여주었다. vitellogenin choriogenin L 및 choriogenin H는 17$\beta$-estradiol의 농도에 의존하여 발현이 증가되는 용량-반응양상(Dose-dependent)을 나타내었다. 반면, estrogen receptor는 모든 처리군에서 $10^{-2}$pM 정도로 발혐됨에 따라 본 시험농도의 17$\beta$-estradiol에 의해서는 거의 유도발현이 되지 않음을 보여주었다. choriogenin L, choriogenin H, vitellogenin I 및 estrogen receptor 발현감수성을 비교한 결과, 25ppt 및 50ppt의 17$\beta$-esoadiol 농도에서는 ChgL > ChgH > VTG I >ER의 순으로 감수성이 높았으며, 100ppt 노출에서는 ChgL > VTG I > Chg H > ER의 순으로 감수성이 높게 나타났다. 결론적으로 choriogenin이 에스트로젠물질에 의한 가장 민감한 생체지표임을 알 수 있었다.

• Journal of Life Science
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• v.28 no.3
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• pp.307-313
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• 2018

Alopecia cause psychological stress due to their effect on appearance. Thus, the global market size of the alopecia treatment products are growing quickly. Timosaponin A III is the well known active ingredient of Anemarrhenae Rhizoma. In this study, we investigated and compared the binding affinity of timosaponin A III with finasteride (5-beta reductase antagonist) and minoxidil (androgen receptor antagonist) on the target protein active site by in silico computational docking studies. The three dimensional crystallographic structure of 5-beta reductase (PDB ID : 3G1R) and androgen receptor (PDB ID: 4K7A) was obtained from PDB database. In silico computational autodocking analysis was performed using PyRx, Autodock Vina, Discovery Studio Version 4.5, and NX-QuickPharm option based on scoring functions. The timosaponin A III showed optimum binding affinity (docking energy) with 5-beta reductase as -12.20 kcal/mol as compared to the finasteride (-11.70 kcal/mol) and with androgen receptor as -9.00 kcal/mol as compared to the minoxidil (-7.40 kcal/mol). The centroid X, Y, Z grid position of the timosaponin A III on the 5-beta reductase was similar (overlap) to the finasteride, but the X, Y, Z centroid grid of the timosaponin A III on the androgen receptor was significantly far from the minoxidil centroid position. These results significantly indicated that timosaponin A III could be more potent antagonist to the 5-beta reductase and androgen receptor. Therefore, the extract of Anemarrhenae Rhizoma or timosaponin A III containing biomaterials can substitute the finasteride and minoxidil and can be applied to the alopecia protecting product and related industrial fields.