• Genomics & Informatics
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• 제19권4호
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• pp.39.1-39.8
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• 2021

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)-positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) 'biological process' terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including 'cell cycle' (cdc2, rik1, pas1, and leo1), 'signaling' (sck2, oga1, and cki3), and 'vesicle-mediated transport' (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the 'signaling' GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

• 대한본초학회지
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• 제35권3호
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• pp.1-8
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• 2020

Objectives : In this study, we examined the estrogenic activities and anti-osteo clastogenesis effects of PCE17, a mixture of PE (an extract of Puerariae Flos), and CE (an extract of Citri Unshius Pericarpium). Methods : The estrogenic effect of PCE17, PE and CE were examined by ER-β/ERE reporter gene assay and proliferation assay in 293 T and MCF-7 cells. The expression of estrogen-responsive gene and protein were checked by Real Time-PCR (RT-PCR) and Western blotting in MCF-7 cells. Inhibitory effect of PCE17, PE and CE on RANKL-induced osteoclast differentiation were evaluated by TRAP staining and RT-PCR in primary osteoclast precursors from rat bone marrow cells. Results : PCE17 and PE bind to ERs (estrogen receptors) and show estrogenic activities in 293T cells. They also stimulated the proliferation of MCF-7 cells and increased the expression of ER response gene, pS2. Tectorigenin, an active ingredient of PE, shows similar estrogenic activities in MCF-7 cells. PCE17 and CE inhibited RANKL-induced osteoclastogenesis in rat primary osteoclast precursor cells and down-regulated the osteoclast-specific genes of Nfatc1, Ctsk, and Acp5. Conclusions : In conclusion, PCE17 may have therapeutic potential in cases of menopause and osteoporosis.

• 생명과학회지
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• 제13권3호
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• pp.314-323
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• 2003

지금까지 estrogen 호르몬이 유방암의 발생과 진전에 관여한다는 사실을 뒷받침 할 수 있는 여러 가지 증거가 제시되어 왔지만, 암화에 관여하고 있는 estrogen의 분자생물학적 작용기전에 대해서는 아직 명확히 알려져 있지 않다. 유방암 세포의 발암현상은 다양한 핵 수용체들과 이들 각각의 신호전달경로들 사이의 기능적 상호교류 (Functional Cross-talk)에 의해 조절되는 것으로 추측되고 있다. 그러므로, 유방암세포의 성장과 증식에 관여하는 신호전달경로중에서 estrogen의 작용을 조절할 수 있는 핵 수용체를 밝혀내고, 이러한 수용체와 estrogen receptor사이에 어떠한 기능적 상호교류가 일어나는지를 규명하는 것은 암화와 관련된 estrogen의 분자생물학적 작용기전을 이해하는 데 중요한 진전을 가져올 수 있다. 따라서 본 연구의 목적은 유방암세포 내에서 estrogen의 작용이 xenobiotic nuclear receptor에 의해 조절되는지를 규명하기 위하여, 두 종류의 유방암세포인 estrogen receptor가 발현되는 MCF-7 세포와 ER의 발현이 일어나지 않는 MDA-MB-231세포를 배양하여, 에스트로겐에 의해 전사가 촉진되는 보고유전자의 발현이 CAR, SXR, 그리고 PPAR${\gamma}$에 의해서 어떻게 영향을 받는지를 조사하고 그 결과를 간암세포에서의 반응과 비교 분석하였다. 최근에 보고된 연구결과와 일치하게, xenobiotic nuclear receptor가 간세포에서 일어나는 에스트로겐 수용체의 신호전달경로에 영향을 줄 수 있음을 확인하였다. PPAR${\gamma}$를 제외한 핵 수용체 CAR와 SXR은 ER의 전사활성 효과를 현저하게 또는 어느 정도 각각 감소시켰다. Hep G2세포에서와는 달리 유방암세포에서는 조사된 세가지의 xenobiotic 핵 수용체가 ER의 전사활성에 그다지 영향을 미치지 않거나 유방암세포의 종류와 각각의 수용체에 따라서 다소 촉진하는 경향을 나타내었다. MCF-7 세포에서는 CAR와 PPAR${\gamma}$을 제외한 SXR이 ER의 transactivation 효과를 약간 촉진한 반면 MDA-MB-231세포는 SXR을 제외한 CAR와 PPAR${\gamma}$에 의해 ER의 transactivation 효과가 약간 증가되는 경향을 보였다. 이러한 결과는 유방암세포에서는 CAR, SXR, PPAR${\gamma}$과 같은 xenobiotic nuclear receptor에 의한 ER transactivation 효과가 간암세포와는 다르게 나타나며, 유방암의 종류에 따라서 endogenous CAR, SXR, PPAR${\gamma}$수용체가 다르게 발현됨으로써 이들에 대한 반응이 서로 상이한 특징을 나타낼 수 있을 것으로 사료된다. 따라서 estrogen receptor에 의해 매개되는 estrogn의 전사활성조절기전이 표적세포에 따라 다른 경로를 포함 할 수 있음을 시사한다.

• Toxicological Research
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• 제33권1호
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• pp.71-77
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• 2017

Hormone replacement therapy (HRT) consists of highly effective prescription medications for treating menopausal symptoms; however, these agents have exhibited side effects including the risk of estrogen-induced carcinogenesis. Therefore, interest in phytotherapy-based materials as a natural source of alternatives to estrogen therapy has increased. However, some of these herbal medicines have been reported to increase the risk of estrogen-induced cancer. Herbal formulations composed of a combination of Cynanchum wilfordii Hemsley (CW), Phlomis umbrosa Turczaninow (PU), and Angelica gigas Nakai (AG) extracts (CPAE) have been used for treating menopausal symptoms. Therefore, in this study, we aimed to examine the safety of CPAE by determining its potential adverse estrogenic activity using the Organization for Economic Cooperation and Development (OECD) test guideline 455 (TG455) in a stably transfected transcriptionally activated human estrogen receptor ${\alpha}$ ($hER{\alpha}$)-HeLa9903 cell model. We found that CPAE did not how any estrogenic activity or stimulate promoters containing estrogen response elements in MCF-7 cells. In addition, CPAE showed no significant selective activity against $hER{\alpha}$ and $hER{\beta}$, non-selective activity against the ER, or effects on ER target gene expression. Furthermore, CPAE did not significantly induce MCF-7 cell proliferation and uterine weight increase in ovariectomized rats. These results demonstrate that CPAE can be used as beneficial herbal drug for prevention and therapeutic intervention of estrogen carcinogenesis in menopausal women.

• Natural Product Sciences
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• 제25권1호
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• pp.28-33
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• 2019

A popular approach for the study of estrogen receptor ${\alpha}$ inhibition is to investigate the protein-protein interaction between the estrogen receptor (ER) and the coactivator surface. In our study, we investigated phytochemicals from Rubus coreanus that were able to disrupt $ER{\alpha}$ and coactivator interaction with an $ER{\alpha}$ antagonist. The E-screen assay and molecular docking analysis were performed to evaluate the effects of the estrogenic activity of R. coreanus extract and its constituents on the MCF-7 human breast cancer cell line. At $100{\mu}g/mL$, R. coreanus extract significantly stimulated cell proliferation ($574.57{\pm}8.56%$). Sanguiin H6, which was isolated from R. coreanus, demonstrated the strongest affinity for the $ER{\alpha}$ coactivator-binding site in molecular docking analysis, with a binding energy of -250.149. The initial results of the study indicated that sanguiin H6 contributed to the estrogenic activity of R. coreanus through the activation of the $ER{\alpha}$ coactivator-binding site.

• 대한의생명과학회지
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• 제25권1호
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• pp.23-31
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• 2019

The protein kinase $CK2{\alpha}$ (formerly Casein Kinase II) is implicated in tumorigenesis and transformation. However, the mechanisms of $CK2{\alpha}$ activation in breast cancer have yet to be elucidated. This study investigated the mechanisms of $CK2{\alpha}$ activation in estrogen signaling. Estrogen increased reactive oxygen species (ROS) production, $CK2{\alpha}$ activity, and protein expression in estrogen receptor positive ($ER^+$) MCF-7 human breast cancer cells, which were inhibited by the antioxidant N-acetyl-L-cysteine. $H_2O_2$ enhanced $CK2{\alpha}$ activity and protein expression. Human epidermal growth factor (EGF) increased ROS production, $CK2{\alpha}$ activity and protein expression in EGF receptor 2 (HER2)-overexpressing MCF-7 (MCF-7 HER2) cells, but not in MCF-7 cells. Estrogen induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The p38 inhibitor, SB202190, blocked estrogen-induced increases in ROS production, $CK2{\alpha}$ activity and $CK2{\alpha}$ protein expression. The data suggest that ROS/p38 MAPK is the key inducer of $CK2{\alpha}$ activation in response to estrogen or EGF.

• Molecules and Cells
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• 제27권3호
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• pp.351-357
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• 2009

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, $17{\beta}$-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER ${\alpha}$ as compared to $17{\beta}$-estradiol and genistein. Despite poor binding to ER ${\alpha}$, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER ${\alpha}$ at $Ser^{118}$. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

• 대한의생명과학회지
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• 제11권2호
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• pp.135-141
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• 2005

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when overexpressed. Since Jpk protein harbors a transmembrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and MitoTracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be overexpressed under ER-stress condition. In the presence of high concentration of ER-sterss inducer (10 mM), about 70 (DTT) to $95\%$ (EGTA) of cells died stronly expressing ($10\~12$ fold) Jpk. Whereas at the low concentration ($0.001\~1.0\;mM$) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.

• BMB Reports
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• 제44권9호
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• pp.595-600
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• 2011

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.106-113
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• 2006

Phthalate analogues are a plasticizer and solvent used in industry. Phthalates were classified in the category of "suspected" endocrine disruptors. The purpose of our study was to screen and elucidate the endocrine disrupting activity of seven phthalate analogues. E-screen assay was performed in MCF-7 human breast cancer cells with seven phthalate analogues. In this cell proliferation assay, benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) showed high estrogenic activity. Their relative proliferation efficiencies (RPE) were 109 and 106%, respectively. In vitro estrogen receptor (ER) binding assay, BBP, di-n-octyl phthalate (DOP) and dinonyl phthalate (DNP) showed weak relative binding affinity (RBA: 0.02%) compared to $17{\beta}-estradiol\;(E2)$ (RBA: 100%). In uterotrophic assay, E2 produced a significant increase, whereas four tested phthalate analogues had potential estrogenic effects in vitro did not increased in uterus weight in immature rats. From these results, we demonstrated that phthalate analogues exhibit weak estrogenic activity in vitro assays at high concentrations. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce a uterus weight increase in vivo. From these, we may suggest that these phthalate analogues are easily metabolized to inactive forms in vivo. Further investigation in other in vitro and in vivo experimental systems might be required.