• Environmental Analysis Health and Toxicology
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• 제26권
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• pp.5.1-5.11
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• 2011

Objectives: In order to identify the possibility of slender bitterling (SB) (Acheilognathus yamatsutae) being used as a test species for estrogenic endocrine disrupting chemicals (EEDCs), we carried out the cloning and sequence characterization of the estrogen receptor (ER). Methods: The ER from a slender bitterling was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (5'-RACE and 3'-RACE) and T-vector cloning. The expression of ER mRNA was also analyzed in six tissues (brain, liver, kidney, gill, gonad, and intestines) by real-time PCR. Results: We obtained an ER from the slender bitterling. The SB ER cDNA was 2189 base pairs (bp) in length and contained a 1707 bp open reading frame that encoded 568 amino acid residues. The SB ER amino acid sequence clustered in a monophyletic group with the $ER{\alpha}$ of other fish, and was more closely related to zebrafish $ER{\alpha}$(88% identity) than to the $ER{\alpha}$ of other fish. The SB ER cDNA was divided into A/B, C, D, E and F domains. The SB ER has conserved important sequences for ER functions, such as the DNA binding domain (D domain), which are consistent with those of other teleosts. Conclusions: The ER of the slender bitterling could provide basic information in toxicological studies of EEDCs in the slender bitterling.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3223-3228
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• 2016

Reports indicate that 15-deoxy-delta-12,14-prostaglandin-J2 (15d-PGJ2) has anticancer activities, but its mechanisms of action have yet to be fully elucidated. We therefore investigated the effects of 15d-PGJ2 on the human breast cancer cell lines, MCF-7 (estrogen receptor $ER{\alpha}+/ER{\beta}+$) and MDA-MB-231 ($ER{\alpha}-/ER{\beta}+$). Cellular proliferation and cytotoxicity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays while apoptosis was determined by fluorescence microscopy and flow cytometry using annexin V-propidium iodide (PI) staining. ER expression was determined by Western blotting. Intracellular calcium was stained with Fluo-4 AM while intracellular caspase activities were detected with Caspase-$FLICA(R)$ and measured by flow cytometry. We showed that 15d-PGJ2 caused a significant increase in apoptosis in MCF-7 and MDA-MB-231 cells. $ER{\alpha}$ protein expression was reduced in treated MCF-7 cells but pre-incubation with the $ER{\alpha}$ inhibitor' ICI 182 780' did not affect the percentage of apoptotic cells. The expression of $ER{\beta}$ was unchanged in both cell lines. In addition, 15d-PGJ2 increased intracellular calcium ($Ca^{2+}$) staining and caspase 8, 9 and 3/7 activities. We therefore conclude that 15d-PGJ2 induces caspase-dependent apoptosis that is associated with an influx of intracellular $Ca^{2+}$ with no involvement of ER signaling.

• Journal of Nutrition and Health
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• 제52권2호
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• pp.176-184
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• 2019

Purpose: Protein overloading in the endoplasmic reticulum (ER) leads to endoplasmic reticulum stress, which exacerbates various disease conditions. Emodin, an anthraquinone compound, is known to have several health benefits. The effect of emodin against palmitic acid (PA) - induced ER stress in HepG2 cells was investigated. Methods: HepG2 cells were treated with varying concentrations of palmitic acid to determine the working concentration that induced ER stress. ER stress associated genes such as ATF4, XBP1s, CHOP and GRP78 were checked using RT- PCR. In addition, the expression levels of unfolded protein response (UPR) associated proteins such as $IRE1{\alpha}$, $eIF2{\alpha}$ and CHOP were checked using immunoblotting to confirm the induction of ER stress. The effect of emodin on ER stress was analyzed by treating HepG2 cells with $750{\mu}M$ palmitic acid and varying concentrations of emodin, then analyzing the expression of UPR associated genes. Results: It was evident from the mRNA and protein expression results that palmitic acid significantly increased the expression of UPR associated genes and thereby induced ER stress. Subsequent treatment with emodin reduced the mRNA expression of ATF4, GRP78, and XBP1s. Furthermore, the protein levels of $p-IRE1{\alpha}$, $p-eIF2{\alpha}$ and CHOP were also reduced by the treatment of emodin. Analysis of sirtuin mRNA expression showed that emodin increased the levels of SIRT4 and SIRT7, indicating a possible role in decreasing the expression of UPR-related genes. Conclusion: Altogether, the results suggest that emodin could exert a protective effect against fatty acid-induced ER stress and could be an agent for the management of various ER stress related diseases.

• Asian Pacific Journal of Cancer Prevention
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• 제15권7호
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• pp.3311-3317
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• 2014

Background: The consequence of Rho GDP dissociation inhibitor alpha (RhoGDI${\alpha}$) activity on migration and invasion of estrogen receptor positive ($ER^+$) and negative ($ER^-$) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDI${\alpha}$ and other proteins interacting directly or indirectly with RhoGDI${\alpha}$ in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. Materials and Methods: $ER^+$ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDI${\alpha}$ using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDI${\alpha}$. Results: The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDI${\alpha}$ in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDI${\alpha}$ in MCF7, while only one protein was identified in the upregulation of RhoGDI${\alpha}$ in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-${\alpha}$ activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Conclusions: Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDI${\alpha}$ with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

• 한국환경보건학회지
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• 제30권2호
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• pp.71-78
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• 2004

This study was performed to evaluate developmental and estrogenic activity of bisphenol A (BPA) and butyl benzyl phthalate (BBP) to the second generation of Sprague-Dawley rats ingested during gestational or lactational periods. Rats were given BPA 20$\mu\textrm{g}$/kg BBP 100mg/kg of pregnancy or lactation periods. Maternal body weight and neonatal body weight were recorded. The rats were sacrificed on day 21 after birth. Reproductive organs of dam and neonate were utilized for receptor binding assay. The plasma concentrations of BPA and MBep, one of the major metabolites of BBP were analyzed with HPLC. The co-administration of BPA and BBP induced slow weight gain compared with single administration in dams. Also, such mixture induced low neonatal body weights in next generation. The dams treated with BPA and BBP during lactational periods showed significant organ weight changes in liver and spleen. The dams exposed during lactational periods showed significant organ weight changes not only in liver and spleen but also in kidney, uterus and ovary. The F1 female rats exposed during lactation periods showed significant organ weight changes in liver, spleen, ovary. The F1 male rats showed significant organ weight changes in liver, kidney, epididymis, vesicular glands, prostate. However, no clear synergistic effects of BPA and BBP were noted. There was no significantly different ER$\alpha$ expression pattern between control and treated groups. However, ER$\alpha$ expression were increased in F1 male testis and female uterus. PI male showed distinct ER$\alpha$ expression, especially in the group of lactational combined exposure. Synergistic ER$\alpha$ expression was found by combined treatment of BPA and BBP. We could not find any evidences of synergistic effects on BPA and/or BBP combined administration on dams and their fetuses, except in ER$\alpha$ expression of F1 male.

• Molecules and Cells
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• 제21권3호
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• pp.356-359
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• 2006

The transcription factor E2F1 coordinates cell cycle progression and induces apoptosis in response to DNA damage stress. Aside from DNA damage, the role of E2F1 in the endoplasmic reticulum (ER) stress signaling pathways is unclear. We found that $E2F1^{-/-}$ murine embryonic fibroblasts (MEFs) are resistant to apoptosis triggered by the ER stress inducer thapsigargin. In addition, E2F1 deficiency results in enhanced phosphorylation of eukaryotic translation initiation factor $2{\alpha}$ ($elF2{\alpha}$). These results therefore indicate that E2F1 deficiency increases phosphorylation of $elF2{\alpha}$ in response to ER stress triggered by thapsigargin, and suggest that the reduction in ER stress-induced apoptosis in E2F1-deficient cells is related to the high level of $elF2{\alpha}$ phosphorylation.

• Journal of Chest Surgery
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• 제45권1호
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• pp.1-10
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• 2012

Background: ${\alpha}$-Lipoic acid (${\alpha}$-LA) has been studied as an anticancer agent as well as a therapeutic agent for diabetes and obesity. We performed this study to evaluate the anticancer effects and mechanisms of ${\alpha}$-LA in a lung cancer cell line, A549. Materials and Methods: ${\alpha}$-LA-induced apoptosis of A549 cells was detected by fluorescence-activated cell sorting analysis and a DNA fragmentation assay. Expression of apoptosis-related genes was analyzed by western blot and reverse transcription.polymerase chain reaction analyses. Results: ${\alpha}$-LA induced apoptosis and DNA fragmentation in A549 cells in a dose- and time-dependent manner. ${\alpha}$-LA increased caspase activity and the degradation of poly (ADP-ribose) polymerase. It induced expression of endoplasmic reticulum (ER) stress-related genes, such as glucose-regulated protein 78, C/EBP-homologous protein, and the short form of X-box binding protein-1, and decreased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein. Reactive oxygen species (ROS) production was induced by ${\alpha}$-LA, and the antioxidant N-acetyl-L-cysteine decreased the ${\alpha}$-LA-induced increase in expression of apoptosis and ER stress-related proteins. Conclusion: ${\alpha}$-LA induced ER stress-mediated apoptosis in A549 cells via ROS. ${\alpha}$-LA may therefore be clinically useful for treating lung cancer.

• The Korean Journal of Physiology and Pharmacology
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• 제15권6호
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• pp.345-351
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• 2011

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-$1{\alpha}$) and phosphoreukaryotic initiation factor alpha (p-eIF-$2{\alpha}$) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-$2{\alpha}$ as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

• Clinical and Experimental Pediatrics
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• 제52권1호
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• pp.111-118
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• 2009

목 적 : 어린시기에 genistein과 같은 식물성 에스트로겐의 섭취가 사회적 관심사로 대두되고 있다. 본 연구에서는 어린 쥐에서 genistein에 노출이 사춘기 개시 및 생식기관에 미치는 영향을 알아 보았다. 방 법 : 이유기(3주령) 암컷 흰쥐를 저용량 genistein (10 mg/kg/day), 고용량 genistein (100 mg/kg/day), 대조군의 세 그룹 (각 그룹 당 n=6)으로 나누고 첫 번째 질구 개방이 확인되는 날까지 농도별로 각각 경구 투여하였다. 질구 개방일을 확인하고 생식 기관의 무게를 측정하며 난소와 자궁에서 $ER{\alpha}$, $ER{\beta}$, PR 유전자들의 발현양상을 RT-PCR을 이용해 비교하였고, 난소와 자궁의 구조적 이상을 확인하기 위해 조직학적 분석을 실시하였다. 결 과 : 고용량 genistein 투여군은 저용량군 및 대조군에 비해 질구 개방일이 유의하게 촉진되었다. RT-PCR결과, $ER{\alpha}$, $ER{\beta}$, PR의 전사활성은 genistein에 노출된 쥐들의 난소와 자궁에서 유의하게 증가하였다. 그라프 난포와 황체는 genistein 투여군의 난소에서만 발견되었고, 대조군의 난소에서는 1차, 2차 난포들과 작은 미성숙 난포들만이 관찰되었다. Genistein 처리군의 자궁에서도 내막층 근막층 및 상피층이 과다성장상태였으나 대조군에서는 모든 세포층과 분비선이 미약하게 발달하였다. 결 론 : 결론적으로, 사춘기 이전 시기에 비교적 단기간의 genistein 노출이라도 미성숙 암컷 흰쥐에서 생식 내분비 활성을 일으켜 조기 사춘기와 성 스테로이드 호르몬 수용체의 발현 양상 변화를 초래할 수 있으며, genistein의 노출이 아동기 성성숙에 미치는 영향에 대한 더욱 많은 연구가 필요할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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• pp.337-343
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• 2002

Objectives: To investigate the distribution of $ER{\alpha}$, $ER{\beta}$, c-fos and c-jun in the uterine myoma and myometrium in oder to know how the tamoxifen cause the growth of myoma. Methods: Myoma and myometrial tissue were obtained from the postmenopausal women treated with tamoxifen in the patients with breast cancer and in the premenopausal patients, who were undergoing myoma of uterus from 1998 through 2000. The espression of each gene was quantitated with quantitative RT-PCR. Results: The expression of $ER{\alpha}$ was slightly increased in the myoma than the myometrium in the proliferative phase, and was slightly decreased in the myometrium than the myoma in the secretory phase. However it was not significant statistically. In the postmemopausal women treated with tamoxifen, $ER{\alpha}$ was expressed in all myoma and myome1rial tissues and the expression was not statistically significant. The expression ofER~ was slightly increased in the myome1rium than the leiomyoma in the proliferative and secretory phase, but it was not significant statistically. In the postmemopausal women treated with tamoxifen, the expression of ER~ was significantly incresed in the myome1rium than the leiomyoma. The expression of c-fos was significantly increased in the myome1rium than the leiomyoma in the proliferative and secretory phase. In the postmemopausal women treated with tamoxifen, the expression of c-fos was slightly increased in the leiomyoma than the myomelrium, however, it was not statistically significant. Conclusion: Tamoxifen may cause the growth of leiomyoma by $ER{\alpha}$ with AP-l pathway reducing the counteraction of 6$ER{\beta}$ to $ER{\alpha}$.