Synthetic Chenodeoxycholic Acid Derivative HS-1200-Induced Apoptosis of Human Oral Squamous Carcinoma Cells

김인령,손현진,곽현호,김규천,박봉수,최원철,고명연,안용우,Kim, In-Ryoung,Sohn, Hyeon-Jin,Kim, Gyoo-Cheon,Kwak, Hyun-Ho,Park, Bong-Soo,Choi, Won-Chul,Ko, Myung-Yun,Ahn, Yong-Woo Korean Academy of Orofacial Pain and Oral Medicine 2007 Journal of Oral Medicine and Pain Vol.32 No.3

Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. Previous studies have 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 induced effects on YD9 human oral squamous carcinoma cells. The present study was done to examine the synthetic bile acid derivatives(HS-1199, HS-1200) induced apoptosis on YD9 cells and such these apoptosis events. We administered them in culture to YD9 cells. Tested YD9 cells showed several lines of apoptotic manifestation such as activation of caspase-3, degradation of DFF, production of poly (ADP-ribose) polymerase(PARP) cleavage(HS-1200 only), DNA degradation(HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential(HS-1200 only) and the release of cytochrome c and AIF to cytosol. Between two synthetic CDCA derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199. Therefore HS-1200 was demonstrated to have the most efficient antitumor effect. Taken collectively, we demonstrated that a synthetic CDCA derivative HS-1200 induced caspases-dependent apoptosis via mitochondrial pathway in human oral sqauamous carcinoma cells in vitro. Our data therefore provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human orall squamous carcinoma from its poweful apoptosis-inducing activity.

Mechanism Underlying a Proteasome Inhibitor, Lactacystin-Induced Apoptosis on SCC25 Human Tongue Squamous Cell Carcinoma Cells

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.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in YD9 Human Oral Squamous Carcinoma Cells

Jae-Hyoun Park(박재현),Gyoo-Cheon Kim(김규천),Hyun-Ho Kwak(곽현호),In-Ryoung Kim(김인령),Seung-Eun Lee(이승은),Jin Chung(정진),Hae-Ryoun Park(박혜련),Sang-Hun Shin(신상훈),Soo-Hyun Choi(조수현),Chul-Hoon Kim(김철훈),Chang-Ok Nam( 대한체질인류학회 2008 대한체질인류학회지 Vol.21 No.1

식물인 Pistiacia lentiscus L. var. Chia.는 그리이스 키오스 섬의 남부지방에서만 서식하며, Chios gum mastic (CGM)으로 알려져 있는 수지를 만들어 낸다. Pistacia lentiscus 나무의 줄기와 잎에서 추출한 천연물질인 CGM은 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 치료약으로 광범히 하게 사용되어 왔었다. 본 연구는 사람구강편평상피암종세포(YD9 cells)에서 CGM의 세포독성과 성장억제 효과, 그리고 세포주기의 변형과 세포자멸사(apoptosis)에 대한 분자생물학적 기전을 알기 위해서 수행하였다. YD9 세포와 사람정상각화세포(HaCaT)의 생존률 측정은 MTT법을 시행하였고, YD9 세포의 성장억제를 확인하기 위해서는 clonogenic assay를 사용하였다. 세포자멸사가 유도되는 YD9 세포를 관찰하기 위해서 hoechst 염색법과 DNA 전기영동법을 사용하였다. 그리고 YD9 세포에 CGM을 적용한 후, Western blot 분석, 세포면역화학염색, 공점레이저주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하였다. CGM으로 처리된 YD9 세포는 용량 의존적인 세포 성장억제와 세포자멸사에 의한 세포죽음을 보였고, caspase-9, caspase-3, PARP 그리고 DFF45 (ICAD)의 파괴와 분절의 생성, DNA의 조각남, 핵 응축, 사립체막전위의 감소, Bax와 Bcl-2의 분율의 변화, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동과 같은 세포자멸사 증거를 보였다. Flow cytometry 분석에서는 cyclin D1, cyclin D3, Cdk2 그리고 Cdk4의 발현의 감소와 p21<SUP>WAF1/CIP1</SUP>와 p53의 발현 증가와 관계있는 것으로 보여지는 G1 세포주기 정지를 보였다. 이러한 결과는 CGM이 세포주기 관련 단백질들의 변형을 유도한 G1 세포주기정지와 사립체와 caspase 경로를 통한 세포자멸사를 유도함을 명확하게 증명하고 있다. 이와 같은 강력한 세포주기 정지와 세포자멸사 유도능은 CGM이 사람구강편평세포암종의 새로운 치료전략으로서의 가능성을 높여 준다고 생각한다. Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21<SUP>WAF1/CIP1</SUP> and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.

Apoptotic Effect of co-treatment with HS-1200 and Cisplatin on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

김덕한,김인령,박봉수,안용우,정성희,Kim, Duk-Han,Kim, In-Ryoung,Park, Bong-Soo,Ahn, Yong-Woo,Jeong, Sung-Hee The Korean Academy of Orofacial Pain and Oral Medi 2013 Journal of Oral Medicine and Pain Vol.38 No.3

담즙산은 지방의 흡수와 콜레스테롤의 항상성을 조절하는 유전자의 전사에 관여하는 필수 콜레스테롤의 생성물이다. 담즙산 합성유도체인 HS-1200이 여러 가지 암세포에서 세포자멸사(apoptosis)를 유도한다는 것이 알려져 있다. 본 연구는 사람혀 편평세포암종세포(SCC25 cells)에서 담즙산 합성유도체인 HS-1200과 대표적인 항암제인 cisplatin의 병용처리 후 세포자멸사 증가효과가 있는지 알아보기 위해 수행하였다. HS-1200과 cisplatin의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 MTT법을 시행하였고, 세포자멸사의 유도와 증가를 알기 위해서는 DNA 전기영동법, Hoechst 염색법, DNA hypoploidy법 을 사용하였다. 그리고 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위해서 Western blot 분석과 면역형광 염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 본 연구에서는 HS-1200과 cisplatin을 병용처리한 SCC25 세포에서 핵의 농축, DNA분절, MMP와 proteasome 활성도의 감소, Bax의 증가와 Bcl-2의 감소, DNA양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40(CAD)의 핵으로의 이동, caspase-9, caspase-7, caspase-3, PARP 그리고 DFF45(ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 반면에 상기 물질들에 단독처리 된 SCC25 세포에서는 세포자멸사 현상이 거의 없었다. 24시간 동안 $25{\mu}M$의 HS-1200, $4{\mu}g/ml$의 cisplatin 을 각기 단독처리 한 결과에서는 세포자멸사를 거의 유도하지 못했으나, 병용처리한 결과에는 아주 탁월하고 명확한 세포자멸사의 유도를 보였다. 그러므로 본 실험결과는 HS-1200과 cisplatin 의 병용요법이 사람구강편평세포암종 환자를 위해 새로운 치료전략으로서의 가능성을 보여준다고 생각한다. Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol homeostasis. Recently it have been identified the synthetic chenodeoxycholic acid (CDCA) derivatives HS-1200 and cisplatin showed apoptisis-inducing activity on various cancer cells in vivo and in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with HS-1200 and cisplatin on human tongue squamous cell carcinoma cells (SCC25 cells). To investigate whether the co-treatment with HS-1200 and cisplatin compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis DNA hypoploidy. Westen blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, co-treatment with HS-1200 and cisplatin on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, reduction of MMP and proteasome activity, the increase of Bax and the decrease of Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated SCC25 cells did not show these patterns. Although the single treatment of $25{\mu}M$ HS-1200 and $4{\mu}g/ml$ cisplatin for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that the combination therapy with HS-1200 and cisplatin could be considered as a novel therapeutic strategy for human squamous cell carcinoma.

Apoptotic Effect of Co-Treatment with Valproic Acid and 17AAG on Human Osteosarcoma Cells

박준영,박세진,김인령,박봉수,정성희,고명연,안용우,Park, Jun-Young,Park, Se-Jin,Kim, In-Ryoung,Park, Bong-Soo,Jeong, Sung-Hee,Ko, Myung-Yun,Ahn, Yong-Woo Korean Academy of Orofacial Pain and Oral Medicine 2011 Journal of Oral Medicine and Pain Vol.36 No.1

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.

Mechanism Underlying NaF-induced Apoptosis and Cell Cycle Arrest on G361 Human Melanoma Cell Line

김도균(Do-Kyun Kim),손현진(Hyeon-Jin Sohn),김인령(In-Ryoung Kim),김규천(Gyoo-Cheon Kim),박봉수(Bong-Soo Park),곽현호(Hyun-Ho Kwak) 대한체질인류학회 2011 해부·생물인류학 (Anat Biol Anthropol) Vol.24 No.4

불화나트륨(sodium fluoride, NaF)은 치아우식증을 예방하기 위한 치과용 물질로 널리 사용되어 왔다. 치아우식증 예방을 위한 불소의 장기간 섭취가 인체에 미치는 영향에 대해 알려진 것이 거의 없고, 과복용 시 심각하고 치명적인 독성을 일으킬 수 있다는 보고가 있으나 불소의 안전성은 여전히 논쟁요소로 남아 있다. 그러나 불소는 구강건강을 위한 주요 물질로서 인정받고 있는 것이 사실이다. NaF은 몇 가지의 정상세포들에서 세포자멸사를 유도하고, 얼마간의 암세포에서 세포주기 정지와 세포자멸사를 유도한다고 알려져 있다. 이 연구의 목표는 사람구강편평상피세포암종세포(G361 세포)에 NaF 적용 후, 세포자멸사의 유도와 세포자멸사에 대한 분자생물학적 기전을 밝히는 것이다. 사람흑색종세포의 생존율은 MTT법으로, G36l 세포의 성장억제능의 측정은 clonogenic assay를 사용하였다. NaF 적용시, G361 세포에서 세포자멸사가 유도되는 것을 확인하기 위해서 Hemacolor 염색법, Hoechst 염색법, DNA 전기영동법 및 TUNEL법을 사용하였다. 그리고 G361 세포에 NaF을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이져주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하였다. NaF로 처리된 G361 세포는 시간 및 용량의존적인 세포생존율 감소, 용량의존적인 성장억제 및 세포자멸사에 의한 세포죽음을 보였다. 그리고 NaF가 적용된 G361 세포에서 핵농축, DNA 조각남, 사립체막전위와 proteasome 활성도의 감소, DNA양의 감소, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40(CAD)의 핵으로의 이동, Bax와 Bcl-2의 분율 변화, 그리고 caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C, DFF45(ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 더 나아가서 NaF에 처리된 G361 세포에서 G1 세포주기에 관계하는 Cyclin D1, Cyclin D3, Cdk2 그리고 Cdk4의 발현의 감소와 p53의 탁월한 발현의 증가를 보였다. 이 연구를 통하여 NaF가 G1 세포주기정지 단백질의 변형 그리고 proteasome, 사립체 및 caspase 경로의 연속 반응을 통해 G361 세포의 세포자멸사를 유도함을 명확하게 증명하였다. Fluoride is widely used in dentistry to prevent dental caries, even though the safety of fluoride is a controversial issue. There are no known adverse effects of long-term fluoride ingestion for caries prevention, but an overdose can cause serious acute toxicity. Nevertheless it is accepted that fluoride is an important material for oral health. This study was undertaken to investigate the modulation of cell cycle-related proteins and apoptosis induction underlying mechanism by NaF treatment on G361 human melanoma cell line. The viability of G361 cells and the growth inhibition of G361 cells were assessed by MTT assay and clonogenic assay respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to observe G361 cells undergoing apoptosis. G361 cells were treated with NaF, and Western blotting, immunocytochemistry, con-focal microscopy, FACScan flow cytometry, MMP activity and proteasome activity were performed. NaF treatment in G361 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. And tested G361 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, a significant shift of Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF resulted in down-regulation of the G1 cell cycle-related proteins, and up-regulation of p53. Taken collectively, our present findings demonstrate that NaF strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins and induces apoptosis via proteasome, mitochondria and cas-pase cascades in G361 cells.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Synthetic Chenodeoxycholic Acid Derivative, HS-1200, on Human Osteosarcoma Cells

Ji-Hak Min(민지학),Min-Jeong Kim(김민정),In-Ryoung Kim(김인령),Seung-Eun Lee(이승은),Hyun-Ho Kwak(곽현호),Gyoo-Cheon Kim(김규천),Hae-Ryoun Park(박혜련),Sang-Hun Shin(신상훈),Chul-Hoon Kim(김철훈),Na-Young Jeong(정나영),Hongsuk Suh 대한체질인류학회 2008 대한체질인류학회지 Vol.21 No.2

Chios gum mastic(CGM)은 그리이스 지역에서만 서식하는 Pistacia lenticulus 나무의 줄기와 잎에서 추출한 수지상의 천연 추출물이다. 합성 chenodeoxycholic acid(CDCA) 유E씨가 여러 가지 암세포에 유도한 세포자멸사 연구들이 보고되어져 왔다. 본 연구는 천연물질인 CGM과 항성 CDCA 유도체인 HS-1200의 병용처리가 사람골육종세포에 효과적인 상승 세포자멸사 효과가 있는지를 알기 위해서 수행되었다. CDM과 HS-1200의 병용처리가 단독처리에 비해서 효과적인 세포생존율 감소가 있는지 확인하기 위해서 MIT법을 시행하였고, 세포자멸사의 유도와 증가를 확인하기 위해서 Hoechst 염색법과 DNA 전기영동법을 사용하였다. 병용처리 때, 세포자멸사에 관계하는 단백질의 발현 변화와 세포내에서의 이동을 밝혀내기 위해서 Western bot 분석과 면역형광염색법을 수행하였다. 더 나아가서 proteasome 활성도와 사립체막 전위 변화를 측정하였다. 병용처리 된 사람골육종세포는 단독처리 된 사람골육종세포에서 거의 관찰할 수 없었던 많은 핵 응축, DNA 조각남, 사립체막 전위와 proteasome 활성도의 감소, DNA 양의 감소, cytochrome c의 세포질로의 유리, AIF와 DFF40(CAD)의 핵으로의 이동, caspase-7, caspase-3 그리고 PARP의 활성화와 같은 세포자멸사 증거를 보였다. 24시간 동안 40㎍/mL CGM과 25μM HS-1200을 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. Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently it reported that CGM induced apoptosis in a few cancer cells in vitro. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of HDS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immuno-fluorescent staining were performed to study the alterations of the expression level and translocation of apoptosis-related proteins in co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, HOS cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation whereas each single treated HOS cells did not. Although the single treatment of 40 ㎍/㎖, CGM or 25 μM HS-1200 for 24 h did not induce apoptosis, the co-treatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of CGM and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

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.

Apoptotic Effect of Co-Treatment with Chios Gum Mastic and Eugenol on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

손현진,예병호,김인령,박봉수,정성희,안용우,고명연,Sohn, Hyeon-Jin,Yea, Byeong-Ho,Kim, In-Ryoung,Park, Bong-Soo,Jeong, Sung-Hee,Ahn, Yong-Woo,Ko, Myung-Yun Korean Academy of Orofacial Pain and Oral Medicine 2011 Journal of Oral Medicine and Pain Vol.36 No.3

Eugenol (4-allyl-2-methoxyphenol) is a natural phenolic constituent extensively used in dentistry as a component of zinc oxide eugenol cement and is applied to the mouth environment. Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and natural phenolic compound, eugenol on SCC25 human tongue squamous cell carcinoma cell line. To investigate whether the co-treatment with eugenol and CGM compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by Hoechst staining, TUNEL staining and DNA hypoploidy. Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and the translocation of apoptosis-related proteins in co-treatment. In this study, co-treatment of with eugenol and CGM on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, the increase and decrease of Bax and Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-3, caspase-6 caspase-7, caspase-9, PARP, Lamin A/C and DFF45 (ICAD) whereas each single treated SCC25 cells did not show or very slightly these patterns. Although the single treatment of 40 ${\mu}g$/ml CGM and 0.5 mM eugenol for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that combination therapy with CGM and eugenol could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.