Gartanin Induces Apoptosis in Oral Squamous Cell Carcinoma Cell Lines through Mitochondrial Signaling Pathway

김인령,유수빈,강해미,박봉수 대한구강해부학회 2015 대한구강해부학회지 Vol.36 No.1

Apoptosis is a physiological process of the controlled elimination of unhealthy or damaged cells1). When cells were undergoing apoptosis, it would be show nuclear condensation, DNA fragmentation, membrane blebbing through various signaling pathway 2, 3). Mitochondria signaling pathway is among the apoptosis pathway. Mitochondria play an important role in the regulation of cell apoptosis. Changes in the mitochondria membrane potential (MMP) are considered an early event in apoptosis and many pro-apoptotic proteins can be released from the mitochondria into the cytoplasm when the MMP is damaged4). In recent studies, reported that apoptosis might play an important role in various diseases. Especially of cancer is closely associated with apoptosis. So, it is important to study the anti-cancer capacities of natural compounds for the development of anticancer drugs without side effects5). Oral cancer is the most common type of human cancers in worldwide6), and oral squamous cell carcinoma (OSCC) arises from the mucosa of oral cavity frequently7, 8). The major risk factors of oral cancer are tobacco, alcohol and high risk Human Papilloma Virus (HPV) infection. The overall survival rate has not changed in recent years, despite extensive research on the biological and molecular aspects of OSCC9). Many tropical fruits have interesting molecular biological activities with potential treatment applications. Garcinia mangostana (mangosteen) has a long history of usage as a medicinal plant in southeast asia10, 11). This tropical plant is famous for its nutritious and flavorful fruits. The pericarps of Garcinia mangostana contain numerous xanthones with chemical diversity12). Several studies have shown that typical magosteen products, α-mangostin, β-mangostin, γ-mangostin, and gartanin have various effects in diseases such as anti-oxidant, anti-inflammatory, anti-allergic, anti-bacterial, anti-cancer via cell cycle arrest and apoptosis10, 11, 13, 14). Although recently mangosteen products, research has been actively promoted but effect in the oral cavity cancer, there is insufficient. In this study, we evaluated the anti-cancer effects of gartanin, one of the major xanthones from the mangosteen fruit in OSCC cell lines.

Mechanism underlying Chios gum mastic-induced cell cycle arrest and apoptosis of the G361 human melanoma cell line

김인령,강해미,유수빈,박봉수 대한구강해부학회 2017 대한구강해부학회지 Vol.38 No.1

Chios gum mastic (CGM) is a resin extracted from the stem and leaves of Pistacia lentiscus L. var. chia. It has been used as a traditional medicine in many Mediterranean countries. Recently, numerous researches have demonstrated that CGM induces cell cycle arrest and apoptosis in many cancer cells. In the present study, an alteration of the cell cycle and induction of apoptosis by CGM treatment on malignant melanoma was investigated. CGM treatment showed the inhibition of cell viability in a dose- and time-dependent manner on the G631 melanoma cell line. Apoptotic hallmarks, such as nuclear condensation and DNA fragmentation, were also identified in CGM-treated cells. Several lines of apoptotic manifestation were demonstrated. The proapoptotic factor Bax was increased in a time-dependent manner, leading to MMP loss, proteasome activity reduction, AIF translocation, and cytochrome c release. Activation of caspases, such as caspase-9, -7, and -3, led to the cleavage of PARP and DFF45 (ICAD). DFF40 (CAD) was translocated into the nucleus from the cytoplasm of the CGM-treated G361 cells. CGM halted the cell cycle progression by G1 arrest and the reduction in the level of cyclin-dependent kinases (CDKs). Accumulation of p53 protein was not observed in cells treated with CGM for 24–72 h. The level of p27KIP1 was increased by CGM treatment only for an initial 24-h period. Cyclin D1, D3, and E protein levels were diminished in CGM-treated cells. Therefore, it is possible that CGM treatment could serve as a novel therapeutic strategy against human melanoma.

Eugenol에 의해 세포자멸사를 보이는 세포 배양된 다양한암세포들에서의 phospho-ser 15-p53의 이동

김인령,김동진,이병구,조재범,김규천,곽현호,최원철,박봉수 대한체질인류학회 2007 해부·생물인류학 (Anat Biol Anthropol) Vol.20 No.3

Eugenol (4-allyl-2-methoxyphenol), a major ingredient of herbs such as clove and Magnoliae Flos, is known to induce apoptosis in mast cells via p53 pathway. This study was undertaken to examine the in vivo effect of eugenol and the molecular mechanism underlying eugenol-induced apoptosis in several cancer cells with different p53 status. Effect of eugenol on mesenteric mast cells was tested using a rat anaphylaxis model. And TUNEL staining was conducted to observe the cells undergoing apoptosis. Several cancer cells were treated with eugenol, and Western blotting, immunocytochemistry, confocal microscopy and mitochondrial fractionation were conducted. Eugenol induced apoptosis in mast cells of mesentery in vivo, decreasing the density of mast cells. Although eugenol did not increase the expression level of p53, it caused the translocation of p53 into mitochondria and subsequent release of cytochrome c. Eugenol increased the level of phospho-ser 15-p53 in several cancer cells with wild type p53 but not in the cells with mutant p53 or p53 deficient cancer cell. In cancer cells with wild type p53, p53 translocated into mitochondria was phosphorylated on ser 15. In conclusion, eugenol induces apoptosis in cancer cells with wild type p53 via the translocation of phospho-ser 15- p53. Furthermore our data suggest that the anticancer effect on cancer cells with wild type p53 may be involved with the pharmacological effect of eugenol regulating apoptosis via a phospho-ser 15-p53 dependent fashion. 신이(Magnoliae flos)나 정향(clove) 같은 풀잎의 주성분인 eugenol (4-allyl-2-methoxyphenol)은 p53 단백 질 경로를 통해서 비만세포의 세포자멸사(apoptosis)를 유도한다고 알려져 있다. 본 연구는 eugenol의 생체내에서 의 효과와 다른 유형의 p53 단백질을 발현하는 여러 가지의 암세포에서의 eugenol 유도 세포자멸사의 분자생물학 적인 기전을 알기 위해서 수행되었다. 배안 비만세포에 대한 eugenol의 효과를 알기 위해서 쥐을 이용한 아나필락시스 실험을 시행하였고 세포자멸 사를 일으키는 비만세포를 관찰하기 위해서는 TUNEL 염색법을 사용하였다. 그리고 여러 가지의 암세포들에 eugenol을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이저주사현미경 검경, 사립체분리 등을 시행하 였다. 생체내에서의 실험에서 eugenol은 창자간막(mesentery)의 비만세포에 세포자멸사를 유도하여, 비만세포의 밀집 도를 감소시켰다. 비록 eugenol이 p53의 발현을 증가시키지는 못했지만, eugenol은 p53을 사립체로 이동시키고, 이 동된 후 연이어서 cytochrome c를 사립체에서 세포질로 유리시키는 것이 관찰되었다. 그리고 eugenol은 wild type p53을 가지고 있는 암세포들에서는 phospho-ser 15-p53을 증가시켰으나, mutant p53 혹은 p53 결핍 암세포들에서 는 phospho-ser 15-p53의 증가는 없었다. 더 나아가서 wild type p53을 가지고 있는 암세포에서 사립체로 이동한 p53이 ser 15에 인산화되는 것이 관찰되었다. 본 연구는 eugenol이 wild type p53을 가진 암세포에서 phospho-ser 15-p53의 세포내 위치이동을 통해 세포자멸 사를 유도하다는 증거를 제시하였다. 더 나아가서 wild type p53을 가진 암세포의 항암효과는 phospho-ser 15-p53 의 의존적 방법을 통한 세포자멸사를 유도하는 eugenol의 약리학적 효과가 관계할 수 있다는 것을 추정할 수 있 다.

CGM과 cisplatin의 병용처리가 사람흑색종서|포에 미치는 세포자멸사 효과에 대한 연구

김인령,박봉수 대한구강해부학회 2013 대한구강해부학회지 Vol.34 No.1

Chios gum mastic (CGM) 은 그리이 스 키오스 섬에서만 자생하는 pjsüada lenüscus L. var. Chia. 의 잎과 줄기로부터 얻어지는 식물성 수지 이며 , 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 위궤양 십이지장궤양 등의 민간 치료약재로서 사용되어져 왔다 최근 CGM이 암세포에서 세포주기 정지와 세 포자별사 (apoptosis) 를 유도한다는 몇 가지 연구가 발표되었으나, 정확한 기전은 밝혀지지 않았다. 본 연구는 사람흑색종세포 (G361 cells) 에서 천연물질인 CGM과 대 표적인 항암제인 cisplatin의 병용처리 후 세 포자멸사 증가효과가 있는지 알아보기 위해수행하였다. HS- 1200과 cisplatin의 병용처리가 단독처리에 비해서 효과적인 세 포생존율 감소가 있는지 확인하기 위해서 MTT법을 시행하였고, 세 포자별사의 유도와 증가를 알기 위해서는 DNA 전기영동법, Hoechst 염색법, DNA hypoploidy법 을 사용하였다. 그리고 세 포자멸사에 관계하는 단백질의 발현 변화외- 세 포내에서의 이 동을 밝혀내기 위해서 Western blot 분석과 면역형광염색법을 수행하였다 더 나아가서proteasome 활성도와 사럽체막 전위 변화를 측정하였다. 본 연구에서는 CGM과 cisplatin을 병용처리한 G361 세포에서 핵의 농축, DNA 분절, MMP와 proteasome 활성도의 감소, Bax의 증가와 Bcl-2의 감소, DNA양의감소, cytochrome c의 세 포질로의 유리 , AIF와 DFF40(CAD) 의 핵으로의 이 동, caspase- 9, caspase-7 , caspase- 3, PARP 그리고 DFF45 (I CAD) 의 활성화와같은 다양한 세 포자별사 증거를 보였다. 반면에 상기 물질들에 단독처리 된 G361 세 포에서 는 세 포지별사 현상이 없었다 . 24시간 동안 40 M의 CGM , 2μg/ml의 cisplatin 을 각기 단독처리 한 결과에서는 세 포자멸사를 거의 유도하지 못했으나, 맹용처리한 결과에 는 아주 탁월하고 명확한 세 포자별사의 유도를 보였다 그러므로 본 실험결과는 CGM과 cisplatin 의 병용요법이 사람흑색종환자를 위해 새로운 치료전략으로서의 가능성을 보여준다고 생각한다.

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.

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.

자가 산부식 프라이머의 세포독성에 관한 실험적 연구

이창훈,김인령,김규천,김성식,순우성 대한치과교정학회 2006 대한치과교정학회지 Vol.36 No.6

자가 산부식 프라이머는 세포독성이 있는 것으로 알려져 있어 교정치료를 하는 동안 치주조직에 손상을 일으킬 수 있다. 본 연구의 목적은 자가 산부식 프라이머가 치주조직에 미치는 영향을 평가해 보고 이를 전통적인 접착법에 사용되는 프라이머와 비교하기 위하여 시행되었다. 시편은 임상에서 브라켓 접착 시 사용하는 Transbond XT Adhesive (3M Unitek, Monrovia, CA, USA)를 각각 Transbond XT Primer (3M Unitek, Monrovia, CA, USA), Clearfil SE bond (Kuraray, Osaka, Japan), Transbond Plus Self Etching Primer, Adper Prompt L-Pop (3M Unitek, Monrovia, CA, USA)과 혼합한 후 광중합하여 제작하였고, Transbond XT Adhesive를 중합한 대조군과 비교하였다. 이를 배양된 HGF-1 (Human Gingiva Fibroblast), HaCaT (Human Keratinocyte cell line), RHEK (immortalized Human Epidermal Keratinocyte)에 노출시킨 후 세포의 형태 변화를 관찰하였고, MTT assay를 시행하여 세포독성을 비교, 평가하였다. 실험결과 72시간 후 HGF-1, HaCaT, RHEK를 이용한 실험에서 모든 프라이머의 세포독성이 높게 나타나 세포 돌기의 위축, 세포 형태의 변화, 세포 수의 감소, 세포의 괴사가 관찰되었다. MTT assay실험 시 HGF-1을 이용한 실험에서 Clearfil SE Bond, Transbond XT Primer, Transbond Plus SEP, Adper Prompt L-Pop의 순으로 세포독성이 높게 나타났고, HaCaT를 이용한 실험에서 Clearfil SE Bond, Adper Prompt L-Pop, Transbond Plus SEP, Transbond XT Primer의 순으로 세포독성이 높게 나타났으며, RHEK를 이용한 실험에서 Clearfil SE Bond, Transbond XT Primer, Adper Prompt L-Pop, Transbond Plus SEP 순으로 세포독성이 높게 나타났다. 자가 산부식 프라이머는 전통적으로 사용되는 프라이머와 마찬가지로 세포독성이 유의하게 높으므로 구강내 사용시 주의가 필요하다. Objective: Several ions and components are released from self-etching primers in the oral cavity. This may cause injury to the periodontal tissues throughout orthodontic treatment. The purpose of this study was to assess the cytotoxicity of self-etching primers to HGF-1 , HaCaT, and RHEK cells. Method: Transbond XT Primer (3M Unitek, Monrovia, CA, USA), and self-etching primers, Clearfil SE Bond (Kuraray, Osaka, Japan), Transbond Plus SEP (3M Unitek, Monrovia, CA, USA), and Adper Prompt L-Pop (3M Unitek, Monrovia, CA, USA), were evaluated by MTT assay, and cellular changes were also observed, Results: In all cells after 72 hours with all primers, severe morphological changes such as atrophy and necrosis were observed. In the MTT assay using HGF-1, Clearfil SE Bond, Transbond XT Primer, Transbond Plus SEP, and Adper Prompt L-Pop were lined up in order of ascending cytotoxicity. When using HaCaT, Clearfil SE Bond, Adper Prompt L-Pop, Transbond Plus SEP, and Transbond XT Primer were lined up in order of ascending cytotoxicity. When using RHEK, Clearfil SE Bond, Transbond XT Primer, Adper Prompt L-Pop, and Transbond Plus SEP were lined up in order of ascending cytotoxicity Conclusion: The result of this study shows that care is needed because self-etching primers show cytotoxic properties similar to conventional primers.

하복부 수술에서 경막외 Bupivacaine과 Fentanyl에 의한 선행진통법이 술후 통증관리에 미치는 효과

이준학,김인령,윤채식,정은배,이기남,문준일 예수병원 1998 예수병원학술지 Vol.19 No.-

지속적 경막외진통법후 Pressure Algometer 에 의한 요통의 평가

권영은,박성희,김인령,이준학,이기남,문준일 예수병원 1997 예수병원학술지 Vol.18 No.-

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.