• Reproductive and Developmental Biology
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• 제35권3호
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• pp.307-311
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG ($33.1{\pm}9.6$ vs $21.7{\pm}8.3%$). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP-treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The mRNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

• Asian Pacific Journal of Cancer Prevention
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• 제13권7호
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• pp.3471-3476
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• 2012

Background and Objective: Histone deacetylase (HDAC) inhibitors represent a promising class of potential anticancer agents for treatment of human malignancies. In this study, we investigated the effect of trichostatin A (TSA), one such HDAC inhibitor, in combination with docetaxel (TXT), a cytotoxic chemotherapy agent or erlotinib, a novel molecular target therapy drug, on lung cancer A549 cells. Methods: A549 cells were treated with TXT, erlotinib alone or in combination with TSA, respectively. Cell viability, apoptosis, and cell cycle distribution were evaluated using MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assay, Hochst33258 staining and flow cytometry. Moreover, immunofluorescent staining and Western blot analysis were employed to examine alterations of ${\alpha}$-tubulin, heat shock protein 90 (hsp90), epidermal growth factor receptor (EGFR), and caspase-3 in response to the different exogenous stimuli. Results: Compared with single-agent treatment, co-treatment of A549 cells with TSA/TXT or TSA/erlotinib synergistically inhibited cell proliferation, induced apoptosis, and caused cell cycle delay at the $G_2/M$ transition. Treatment with TSA/TXT or TSA/erlotinib led to a significant increase of cleaved caspase-3 expression, also resulting in elevated acetylation of ${\alpha}$-tubulin or hsp90 and decreased expression of EGFR, which was negatively associated with the level of acetylated hsp90. Conclusions: Synergistic anti-tumor effects are observed between TXT or erlotinib and TSA on lung cancer cells. Such combinations may provide a more effective strategy for treating human lung cancer.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.542-550
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• 2018

Heat shock protein 90 (Hsp90) is treated as a molecular therapeutic target for the prevention and treatment of cancer. Geldanamycin (GA) was the first identified natural Hsp90 inhibitor, but hepatotoxicity has limited its clinical application. Nevertheless, a new GA analog (WK-88-1) with the non-benzoquinone skeleton, obtained from genetically engineered Streptomyces hygroscopicus, was found to have anticancer activity against two human breast cancer cell lines. WK-88-1 produced concentration-dependent inhibition of cell proliferation, cell cycle arrest, and apoptosis in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cell lines. Detailed analysis showed that WK-88-1 downregulated some key cell cycle molecules (CDK1 and cyclin B1) and lead to $G_2/M$ cell cycle arrest. Further studies also showed that WK-88-1 could induce human breast cancer cell apoptosis by downregulating Hsp90 client proteins (Akt, p-Akt, IKK, c-Raf, and Bcl-2), decreasing the ATP level, increasing reactive oxygen species production, and lowering the mitochondrial membrane potential. Meanwhile, we discovered that WK-88-1 significantly decreased the levels of Her-2 and $ER-{\alpha}$ in MCF-7 cells but not in MDA-MB-231 cells. In addition, WK-88-1 significantly increased caspase-3, -8, and -9 activities and the cleavage of PARP in a concentration-dependent manner (with the exception of caspase-3 and PARP in MCF-7 cells). Taken together, our preliminary results suggest that WK-88-1 has the potential to play a role in breast cancer therapy.

• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7043-7048
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• 2014

Inhibition of heat shock protein 90 (Hsp90) leads to inappropriate processing of proteins involved in DNA damage repair pathways after DNA damage and may enhance tumor cell radio- and chemotherapy sensitivity. To investigate the potentiation of antitumor efficacy of lidamycin (LDM), an enediyne agent by the Hsp90 inhibitorgeldanamycin (GDM), and possible mechanisms, we have determined effects on ovarian cancer SKOV-3, hepatoma Bel-7402 and HepG2 cells by MTT assay, apoptosis assay, and cell cycle analysis. DNA damage was investigated with H2AX C-terminal phosphorylation (${\gamma}H2AX$) assays. We found that GDM synergistically sensitized SKOV-3 and Bel-7402 cells to the enediyne LDM, and this was accompanied by increased apoptosis. GDM pretreatment resulted in a greater LDM-induced DNA damage and reduced DNA repair as compared with LDM alone. However, in HepG2 cells GDM did not show significant sensitizing effects both in MTT assay and in DNA damage repair. Abrogation of LDM-induced $G_2/M$ arrest by GDM was found in SKOV-3 but not in HepG2 cells. Furthermore, the expression of ATM, related to DNA damage repair responses, was also decreased by GDM in SKOV-3 and Bel-7402 cells but not in HepG2 cells. These results demonstrate that Hsp90 inhibitors may potentiate the antitumor efficacy of LDM, possibly by reducing the repair of LDM-induced DNA damage.

• 대한의생명과학회지
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• 제15권1호
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• pp.97-99
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• 2009

Geldanamycin is a benzoquinone ansamycin antibiotic that binds to cytosol HSP90 (Heat Shock Protein 90) and changes its biological function. HSP90 is involved in the intracellular important roles for the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis. To identify genes expressed during geldanamycin treatment against neurons of rats (PC12 cells), DNA microarray method was used. We have isolated 2 gene groups (up-or down-regulated genes) which are geldanamycin differentially expressed in neurons. Granzyme B is the gene most significantly increased among 204 up-regulated genes (more than 2 fold over-expression) and Chemokine (C-C motif) ligand 20 is the gene most dramatically decreased among 491 down-regulated genes (more than 2 fold down-expression). The gene increased expression of Cxc110, Cyp11a1, Gadd45a, Gja1, Gpx2, Ifua4, Inpp5e, Sox4, and Stip1 are involved stress-response gene, and Cryab, Dnaja1, Hspa1a, Hspa8, Hspca, Hspcb, Hspd1, Hspd1, and Hsph1 are strongly associated with protein folding. Cell cycle associated genes (Bc13, Brca2, Ccnf, Cdk2, Ddit3, Dusp6, E2f1, Illa, and Junb) and inflammatory response associated genes (Cc12, Cc120, Cxc12, Il23a, Nos2, Nppb, Tgfb1, Tlr2, and Tnt) are down-regulated more than 2 times by geldanamycin treatment. We found that geldanamycin is related to expression of many genes associated with stress response, protein folding, cell cycle, and inflammation by DNA microarray analysis. Further experimental molecular studies will be needed to figure out the exact biological function of various genes described above and the physiological change of neuronal cells by geldanamycin. The resulting data will give the one of the good clues for understanding of geldanamycin under molecular level in the neurons.

• Journal of Oral Medicine and Pain
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• 제36권1호
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• pp.11-20
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• 2011

Valproic acid(VPA)는 아주 잘 알려진 항경련제로서, 30년 동안 간질치료제로서 사용되어져 왔다. VPA는 1997년에 최초로 항암제의 효능이 밝혀졌으며, VPA의 항암효과는 히스톤탈아세틸화효소 억제제의 기전에 기인한다고 규명되었다. 17AAG(17-Allyamnio-17-demethoxygeldanamycin)는 HSP90의 억제제이며, HSP90은 세포증식과 세포생존에 관여하며, 최근 17AAG가 세포자멸사를 유도한다는 연구들이 보고되어지고 있다. 본 연구는 히스톤탈아세틸화효소억제제인 VPA와 HSP90 억제제인 17AAG의 병용처리가 사람골육종세포에 상승 세포자멸사 효과가 있는지를 알기 위해서 수행되었다. VPA과 17AAG의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 trypan-blue법을 시행하였고, 세포자멸사의 유도와 증가를 확인하기 위해서 Hoechst 염색법, flow cytometry(DNA hypoploidy와 MMP 측정), Western bot 분석법 그리고 면역형광염색법을 수행하였다. 병용처리 된 사람골육종세포는 단독처리 된 사람골육종세포에서 거의 관찰할 수 없었던 핵 응축과 조각남, 사립체막 전위와 DNA 양의 감소, cytochrome c의 세포질로의 유리, AIF의 핵으로의 이동, caspase-3과 caspase-7의 파괴 및 PARP의 분절화와 같은 세포자멸사 증거를 보였다. 48시간 동안 1 mM의 VPA와 0.5 ${\mu}M$ 17AAG을 각기 단독처리 한 결과에서는 세포자멸사를 유도 못했으나, 병용처리한 결과에는 아주 탁월한 세포자멸사의 유도를 보였다. 이러한 병용처리 결과는 사람골육종의 새로운 치료적 전략으로 응용될 수 있다고 생각한다.

• BMB Reports
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• 제51권12호
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• pp.660-665
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• 2018

Human epidermal growth factor receptor 2 (HER2) inhibitors, such as trastuzumab and lapatinib are used to treat HER2-positive breast and gastric cancers. However, as with other targeted therapies, intrinsic or acquired resistance to HER2 inhibitors presents unresolved therapeutic problems for HER2-positive gastric cancer. The present study describes investigations with AUY922, a heat shock protein 90 (HSP90) inhibitor, in primary lapatinib-resistant (ESO26 and OE33) and lapatinib-sensitive gastric cancer cells (OE19, N87, and SNU-216) harboring HER2 amplification/over-expression. In order to investigate whether AUY922 could overcome intrinsic and acquired resistance to HER2 inhibitors in HER2-positive gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (OE19/LR and N87/LR) by continuous exposure to lapatinib in vitro. We found that activation of HER2 and protein kinase B (AKT) were key factors in inducing intrinsic and acquired lapatinib-resistant gastric cancer cell lines, and that AUY922 effectively suppressed activation of both HER2 and AKT in acquired lapatinib-resistant gastric cancer cell lines. In conclusion, AUY922 showed a synergistic anti-cancer effect with lapatinib and sensitized gastric cancer cells with intrinsic resistance to lapatinib. Dual inhibition of the HSP90 and HER2 signaling pathways could represent a potent therapeutic strategy to treat HER2-positive gastric cancer with intrinsic and acquired resistance to lapatinib.

• 한국동물생명공학회지
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• 제34권3호
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• pp.240-246
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• 2019

The aim of this study was to investigate effect of heat stress on expression levels of plasminogen activators (PAs) related mRNAs and proteins, and changes of PAs activity in porcine endometrial explants. The endometrial explants (200 ± 50 mg) were isolated from middle part of uterine horn at follicular phase (Day 19-21) and were pre-incubated in serum-free culture medium at 38.5℃ in 5% CO₂ for 18 h. Then, the tissues were transferred into fresh medium and were cultured at different temperature (38.5, 39.5, 40.5 or 41.5℃) for 24 h. The expression level of urokinase-type PA (uPA), type-1 PA inhibitor (PAI-1), type-2 PAI (PAI-2), and heat shock protein-90 (HSP-90) mRNA were analysis by reverse-transcription PCR and proteins were measured by western blotting. The supernatant were used for measurement of PAs activity. In results, mRNA and protein levels of HSP-90 was higher in 41.5℃ treatment groups than other treatment groups (p < 0.05). The expression of uPA, PAI-1, and PAI-2 mRNA were slightly increased by heat stress, however, there were no significant difference. Heat stress condition suppressed expression of active uPA and PAI-2 proteins (p < 0.05), whereas PAI-1 protein was increased (p < 0.01). Although PAI-1 protein was increased and active uPA was decreased, PAs activity was greatly enhanced by exposure of heat stress (p < 0.05). These results suggest that heat stress condition could change intrauterine microenvironment through regulation of PAs activity and other factors regarding with activation of PAs might be regulate by heat stress. Therefore, more studies regarding with regulatory mechanism of PAs activation are needed.

• 생명과학회지
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• 제21권1호
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• pp.44-55
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• 2011

본 연구는 복분자의 수용성 추출물(RCE) 이 배양된 제대정맥내피세포에서 내피세포 NO 합성 효소(eNOS)의 발현과 활성에 미치는 효과를 연구하고, 이러한 RCE의 효과가 어떤 신호전달 과정을 거치는지를 밝히기 위한 것이다. 연구에 따르면 RCE가 제대정맥내피세포에서 NO의 생성을 증가시키는데 이는 iNOS 보다는 eNOS의 활성화에 의한 것임을 이들의 특이 억제제를 사용한 실험으로 확인할 수 있었다. 나아가 eNOS에 의한 NO 생성 증가는 이 효소의 활성 증가뿐만 아니라 mRNA 수준에서의 발현증가에도 기인함을 확인할 수 있었다. PKC-특이억제제인 RO-317549는 RCE에 의한 NO 생성의 증가에 별다른 영향을 주지 않았으나, 에스트로젠 수용체-특이 억제제인 Tamoxifen, ERK-특이 억제제인 PD98059와 PI3K/Akt-특이 억제제인 LY-294002는 제대정맥내피세포에서 RCE에 의해 증가된 NO 생성을 억제하였으며, 이는 두 저해제가 eNOS의 활성형인 pSer1177의 양을 감소시키며, 특히 PD98059는 비활성형인 pThr495의 양도 증가시키기 때문임을 알 수 있었다. 이상의 결과로써, 복분자 수추출물은 사람의 제대정맥내피세포에서 NO 생성을 증가시키며, 이는 eNOS의 발현을 증가시킬 뿐만 아니라, ERK와 PI3K/Akt의 신호전달과정을 거쳐서 eNOS의 활성도 증가시키기 때문임을 확인할 수 있었다.