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백철중,김규천,김인령,이승은,곽현호,박봉수,태일호,고명연,안용우,Baek, Chul-Jung,Kim, Gyoo-Cheon,Kim, In-Ryoung,Lee, Seung-Eun,Kwak, Hyun-Ho,Park, Bong-Soo,Tae, Il-Ho,Ko, Myung-Yun,Ahn, Yong-Woo Korean Academy of Orofacial Pain and Oral Medicine 2009 Journal of Oral Medicine and Pain Vol.34 No.3
Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.
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Synthetic Chenodeoxycholic Acid Derivative HS-1200-induced Apoptosis of Human Melanoma Cells
Chul-Jung Baek(백철중),Ji-Hak Min(민지학),Seong-Hyeok Moon(문성혁),In-Ryoung Kim(김인령),Seung-Eun Lee(이승은),Duk-Han Kim(김덕한),Gyoo-Cheon Kim(김규천),Hyun-Ho Kwak(곽현호),Bong-Soo Park(박봉수) 대한체질인류학회 2007 대한체질인류학회지 Vol.20 No.4
담즙산과 합성담즙산유도체가 여러 종류의 암세포에 세포자멸사(apoptosis)를 유도하며, 항암효과가 있다고 알려져 있다. 또한 합성 chenodeoxycholic acid (CDCA) 유도체가 여러 가지 암세포에 유도한 세포자멸사 연구들이 보고되어 왔다. 하지만 아직까지 사람흑색종세포에 합성 CDCA 유도체가 유도한 세포자멸사 연구는 보고되지 않았다. 그래서 본 연구는 합성 CDCA 유도체인 HS-1199와 HS-1200이 사람흑색종세포(G361 세포)에 세포자멸사 효과와 세포자멸사 기작을 밝혀내기 위해서 수행되었다. 합성 CDCA에 처리된 G361 세포의 생존율을 확인하기 위해서 MTT 방법을 사용하였고, 세포자멸사 유도 검증은 DNA 전기영동법과 Hoechst 염색을 이용하였다. 세포자멸사에 관계하는 단백질의 발현 변화와 세포 내에서 이동을 밝혀내기 위해서 Western bot 분석과 면역형광염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 합성 CDCA 유도체로 처리된 G361 세포에서 caspase-3, DFF, PARP의 파괴, caspase-3 (HS-1200 only), PARP, DFF의 분절화, DNA 조각남(HS-1200 only), 핵 응축, proteasome 활성화의 감소, 사립체막전위(MMP)의 감소, 그리고 cytochrome c와 AIF의 사립체에서 세포질로의 유리와 같은 다양한 세포자멸사의 증거를 보였다. 두 개의 합성 CDCA 유도체 중에서 HS-1200이 HS-1199보다 더욱 강한 세포자멸사 효과를 보였다. 본 연구는 CDCA 유도체인 HS-1200이 사람흑색종세포에서 proteasome, 사립체 그리고 caspase 경로을 통해서 세포자멸사를 유도하는 것을 증명하였다. 이러한 결과는 HS-1200이 사람흑색종의 새로운 치료적 전략으로 응용될 수 있다고 생각한다. Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. It wasn’t discovered those materials have apoptosis-inducing effects on G361 human melanoma cells. The present study was done to examine the synthetic bile acid derivatives, HS-1199 and HS- 1200, induced apoptosis on G361 cells and such these apoptosis events. The viability of G361 cells was assessed by the MTT assay. Induction of apoptosis was confirmed by DNA electrophoresis and Hoechst staining. Westen blot analysis and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. Tested G361 cells showed several lines of apoptotic manifestation such as activation of caspase-3, DFF and PARP, DNA degradation (HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential, and the release of cytochrome c and AIF to cytosol. Between two synthetic derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199 did. Taken collectively, we here demonstrated for the first time that synthetic CDCA dedrivatives induce apoptosis of human melanoma cells through the proteasome, mitochondria and caspase pathway. Therefore our data provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human melanoma cells from its powerful apoptosis-inducing activity.
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Induction of Apoptosis by Genistein in Human Melanoma Cells
허영주,길영기,백철중,김인령,조운복,곽현호,박봉수,김규천 대한해부학회 2008 Anatomy & Cell Biology Vol.41 No.4
Genistein is a naturally occurring isoflavone that has been identified predominantly in soybean. It has been found that genistein can inhibit the growth of various cancer cell lines. Melanoma continues to increase in incidence in many parts of the world and remains among the top six cancers as a cause of death and morbidity. Understanding and overcoming resistance mechanism(s) of melanoma to apoptosis would therefore facilitate identification of new therapeutic targets and development of new treatments. This study was undertaken to investigate whether genistein induced apoptosis on human melanoma cells (G361). Genistein had a significant dose- and time-dependent inhibitory effect on the viability of G361 cells. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by hoechst staining, and DNA electrophoresis. p53 levels were not altered by genistein treatment. Genistein treatment induced caspase-3 cleavage and activation. Poly (ADP-ribose)- polymerase (PARP) and DNA fragmentation factor 45 (DFF45), which are caspase-3 substrates, were cleaved during genistein-induced apoptosis. It was found that the caspase-6 substrate lamin A was cleaved, whose cleavage has been reported to be necessary for complete condensation of DNA during apoptosis. The expression level and phosphorylation of focal adhesion kinase (FAK) were reduced by genistein treatment. These results suggest that genistein may constitute a potential antitumor compound against melanoma occurring at oral mucosa and skin.
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