치료 작용점으로서의 Epigenetic 조절자

김군도 대한암예방학회 2005 Journal of cancer prevention Vol.10 No.1

Epigenetic processes required DNA methylation and histone modification which are regulated by several classes of enzymes in the cells. Imbalances in histone acetylation and DNA methylation lead to an aberrant behavior of the cells in morphology, cell cycle, differentiation, and carcinogenesis. Thus, the enzymes regulating the reactions can be considered as a therapeutic target of epigenetic modification. Clinical trials designed to evaluate inhibitors of DNA methyltransferases and histone deacetylases at phase I or phase II are showing encouraging results in cancer patients. A growing number of genes that are silenced by epigenetic inactivation can be used as biomarkers to monitor the efficacy of epigentically- effective drugs. (Cancer Prev Res 10, 1-5, 2005)

안토시아닌이 Vascular Endothelial Growth Factor 매개의 혈관형성에 미치는 영향에 대한 연구

김군도,김화미,김지연 대한암예방학회 2010 Journal of cancer prevention Vol.15 No.1

Angiogenesis, the process of new blood vessel growth from pre-existing capillaries, is involved in physiological and pathological processes such as tumor growth and metastasis. Thus, suppression of abnormal angiogenesis may provide therapeutic strategies for the treatment of angiogenesis-dependent disorders, like as skin cancer, psoriasis, and other reactive skin disorders. There is increasing evidence that polyphenols found in natural products may have a beneficial preventive effect on cardiovascular disease and cancer by mechanisms including factor of angiogneisis that have not yet been elucidated. In the present study, we investigated the antiangiogenic activities of anthocyanin, a class of polyphenols present at high levels in dietary black bean, in both in vitro and in vivo models. In vitro studies were performed using cultured human umbilical vein endothelial cells (HUVEC). After inducing angiogenesis by vascular endothelial cell growth factor (VEGF) on those cultured cells, we analyzed dose-dependent changes before- and after-anthocyanin treatment. In vivo, Chorioallantoic membrane (CAM) assay was done to check developmental effect on embryo vasculature. Dietary anthocyanin from black bean had statistically significant inhibitions on endothelial cell migration and tube formation in dose dependant manner, and also inhibited embryological vascular development. We suggest that an anthocyanin derived from diet has a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment. (Cancer Prev Res 15, 60-68, 2010)

부산 수영공공하수처리시설에서 분리된 광범위 항균제 베타락 탐 분해효소(Extended-Spectrum ${\beta}$-Lactamase, ESBL) 유형

김군도,이훈구,Kim, Gun-Do,Lee, Hun-Ku 한국미생물학회 2010 미생물학회지 Vol.46 No.1

본 연구는 부산 환경관리공단 수영사업소 산하 수영공공하수종말처리장 하수로부터 광범위 베타락탐 분해효소(ESBL, extended-spectrum ${\beta}$-lactamase)의 유형을 파악하기 위하여 이루어졌다. 수영공공하수처리시설은 부산광역시의 동북부 생활하수와 수세식변기의 오수가 표준활성슬러지공법으로 처리되며 일 생활하수 처리량은 총 550,000톤이다. 이중디스크 확산 검사, 제 3세대 세파계열 항균제에 대한 최소억제농도 시험을 통하여 14균주를 선별하였다. Indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylase-dihydrolase 시험과 당 발효 시험 등 생화학 검사를 통하여 동정한 결과 Escherichia coli (10균주), Klebsiella pneumoniae (4균주)가 동정되었다. 이를 전달균주로, sodium azide에 내성을 가진 피전달 균주인 E. coli J53에 교차접합을 시켜 11균주 (E. coli 9균주, K.pneumoniae 2균주)가 접합이 이루어졌다. 접합자중 2균주는 ESBL 유전자를 전달받지 못하였고 9균주는 부모로부터 ESBL 유전자를 전달받았다. 등전점, 유전자서열과 단백질서열 분석 등을 통하여 피전달균주인 E. coli J53에 전달된 유전자유형은 TEM형의 모형인 TEM-1과 SHV-12형으로 규명되었다. The study performed to identify the type of ESBL against strains which are producing extendedspectrum ${\beta}$-lactamases and isolated from sewage in Suyeong sewage disposal plant, Busan Environmental Corporation. By the standard activated sludge method, Suyeong sewage disposal plant purify living and lavatory sewage gathering from the northeast Busan and the facility purify total 550,000 tons of living sewage disposal a day. 14 strains were isolated by double disk synergy test and the third generation cepha-antibiotics test. Indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylasedihydrolase and sugar-fermentation tests identified as Klebsiella pneumoniae (n=4) and Escherichia coli (n=10). Plasmid-mediated transmission test against isolated 14 strains proved 11 strains transmitted resistance to recipient E. coli J53 (sodium $azide^R$, $ceftazidime^S$). 9 strains of conjugant were expressed ESBL genes transferred from parental strain but 2 conjugants did not expressed. The type of ESBL from each strain was determined by isoelectric focusing points, DNA and amino acids sequencing. The results indicated that the types of ESBL transmitted to recipient E. coli J53 were TEM-1, the parental TEM type and SHV-12 type.

Induction of Apoptosis via Autophagy on SK-Hep1 Human Hepatocellular Carcinoma Cells by Kurarinone Isolated from Sophora flavescens

김순진,최재수,김군도 대한암예방학회 2012 Journal of cancer prevention Vol.17 No.2

The dried roots of Sophora flavescens have been used in traditional medicine. Previous studies have shown that the roots of Sophora flvescens have various effects, including anti-oxidation, anti-inflammation and anti-tumor activities. In this study, we targeted the of kurarinone isolated from Sophora flavescens on induction of apoptosis in SK-Hep1 human hepatocarcinoma cells. Kurarinone inhibited the proliferation of SK-Hep1 cells in a dose-dependent manner with IC50 value of 75 μM. Western blot analysis revealed that kurarinone reduced the expression of survival-related proteins but induced apoptosis in dose-dependent manner. Induced apoptosis is reflected by not only increase of caspases, cleaved poly ADP ribose polymerase (PARP) and phosphorylated lamin A/C but also decrease of DNA fragmentation factor (DFF) 45/35. Kurarinone also induced autophagic cell death in the cells through the activation of autophagy-specific gene (Atg), Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3). These results indicate that kurarinone induces cell death by both authophagy and apoptosis through the activation of pro-apoptotic proteins and caspase-dependent pathway in SK-Hep1 cells.

Anticancer Mechanism of AP1, Abalone Peptide by Cell Cycle Arrest in Human Gastric Cancer Cells

김초원,김군도,고혜진,서정길,박남규 대한암예방학회 2013 Journal of cancer prevention Vol.18 No.1

Gastric cancer is one of the most common malignant tumors and the second cause of cancer related death in the world. Here, we evaluated the effects of abalone peptide (AP1) on cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-28 cell lines were used as human gastric cancer cells and the mechanism study showed that AP1 decreased cell population by cell cycle arrest and then led to apoptotic cell death of MKN-28 and AGS cells. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with AP1 arrested the cell cycle in G1/S phase. AP1 decreased expressions of not only CDK2 and CDK6 but also Cyclin D1 and Cyclin D3. Along with those decrease, expressions of phospho-Rb, E2F1, phospho-chk1 and Cdc25A also decreased as well. Activation of caspases-3, -6 and -7 was also observed. In conclusion, the findings in this study suggested that AP1 have potential anticancer efficacy via cell cycle arrest in G1/S phase and induction of apoptosis. Its potential role to be a candidate of anticancer agent is worth being further investigated.

건선에서 Acitretin이 Vascular endothelial growth factor의 발현에 미치는 영향에 대한 면역조직학적 연구

김지연, 김군도, 윤태영 충북대학교 의과대학 충북대학교 의학연구소 2004 忠北醫大學術誌 Vol.14 No.2

-본문참고-

Types of Extended-Spectrum β-Lactamase Produced in Enteric Bacteria Isolated from Sewage Plant Drain Water

Gun-Do Kim(김군도),Hun-Ku Lee(이훈구) 한국생명과학회 2010 생명과학회지 Vol.20 No.5

본 연구는 임상검체만이 아닌 주변 생활환경에도 광범위 베타 락탐분해효소를 생성하는 균주(extended-spectrum β-lactamase, ESBL)가 존재하는지를 확인하고 만약 존재할 경우 그 균주를 분리하고 ESBL유형을 알아보기 위하여 실시되었다. 부산 광안리 하수처리 방류수에서 이중 디스크 확산 검사 결과 양성반응을 나타낸 29균주를 선별하였다. 이중 sodium azide에 내성을 가진 피전달 균주인 Escherichia coli J5에 교차접합이 이루어진 15균주를 대상으로 indole, methyl-red, Voges-Proskauer, Simmon's citrate 시험과 decarboxylase-dihydrolase 및 여러 종류의 당 발효 시험 등 생화학 검사를 실시한 결과 Klebsiella pneumoniae(13균주)와 Escherichia coli(2균주)가 동정되었다. 등전점, PCR, 유전자서열 분석을 실시하여 ESBL 유형을 결정하였다. Klebsiella pneumoniae의 ESBL 생성유형은 SHV-12(4균주)와 SHV-12/TEM-1(9균주)의 2종류로 구분되었고, Escherichia coli의 ESBL 생성유형은 TEM-1(2균주)로 판명되었다. This study focused on typing of the extended-spectrum β-lactamase (ESBL) produced in organisms isolated from a natural environment, rather than a clinical setting. Samples were collected from drain water issuing from a sewage plant in Kwanganri (Busan, Korea). Following double disk synergy testing, 29 strains were selected as potential ESBL positive strains. Of these, 15 strains were transconjugants of the sodium azide resistant recipient strain Escherichia coli J53 and analyzed biochemically including indole, methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylase-dihydrolase and sugar- fermentation tests. The tests classified the 15 strains as Klebsiella pneumoniae (n=13) and Escherichia coli (n=2). The type of ESBL from each strain was deduced by isoelectric focusing point analysis and DNA sequencing. The results indicated that the types of ESBL were SHV-12 (n=4) and SHV-12/TEM-1 (n=9) from K. pneumoniae and TEM-1 (n=2) from E. coli strains.

The Anti-Inflammatory Effects of Persicaria thunbergii Extracts on Lipopolysaccharide-Stimulated RAW264.7 Cells

Sang-Bo Kim(김상보),Yeong Ae Seong(성영애),Hee Jae Jang(장희재),Gun-Do Kim(김군도) 한국생명과학회 2011 생명과학회지 Vol.21 No.12

다양한 식용식물에 함유되어 있는 것으로 알려진 phenolic acids의 일종인 p-coumaric acid의 항암활성을 규명하고자, 인체 급성백혈병 T 세포주인 Jurkat T 세포에 대한 p-coumaric acid의 에폽토시스 유도기전을 조사하였다. Jurkat T 세포를 p-coumaric acid (50-150 μM)로 처리한 결과, 세포독성, 에폽토시스-관련 DNA fragmentation, 및 pro-apoptotic multidomain Bcl-2 family member인 Bak의 활성화, Δψm loss, caspase-9, -3, -7, 및 -8의활성화, 그리고 PARP 분해 등의 여러 에폽토시스-관련 생화학적 현상들이 농도의존적으로 나타났다. 그러나 이러한 에폽토시스-관련 생화학적 현상들은 Jurkat T 세포에 anti-apoptotic Bcl-2 단백질을 과발현할 경우에는 나타나지 않았다. 또한 p-coumaric acid처리에 의해 유도되는 Jurkat T 세포의 에폽토시스에는 necrosis가 수반되지 않는 것으로 확인되었다. Jurkat T 세포를 pan-caspase inhibitor인 z-VAD-fmk를 전처리할 경우, p-coumaric acid 처리에 의해 유도되는 apoptotic sub-G₁ peak는 차단되어 나타나지 않았으나 Δψm loss는 여전히 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도에 caspase cascade 활성화가 필수적이며Δψm loss의 downstream 현상임을 나타낸다. 한편, FADD 및caspase-8을 함께 발현하는 Jurkat T 세포주 A3, FADD-결손 Jurkat T 세포주 I2.1, 그리고 caspase-8-결손 Jurkat T 세포주 I9.2의 p-coumaric acid의 세포독성에 대한 감수성은 서로 유사하게 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도가 Fas와 FasL간의 상호작용에 의해 개시되지 않음을 시사한다. p-Coumaric acid의 세포독성은 Jurkat T 세포에 비해 인체 정상 말초혈액 T 세포에서 훨씬 낮게 나타났다. 이러한 결과들은 p-coumaric acid 처리에 의해 유도되는 Jurkat T 세포의 에폽토시스가 Bak 활성화, Δψm loss, caspase-9, -3, -7, 및 -8로 이루어진 caspase cascade의 활성화, 그리고 PARP 분해에 의해 유도되며, 또한 anti-apoptotic 단백질인 Bcl-2의 과발현에 의해서 음성적으로 조절됨을 나타낸다. In this study, we investigated the anti-inflammation effect of Persicaria thunbergii (P. thunbergii) on RAW 264.7 murine macrophage cells. The anti-inflammatory activity of P. thunbergii was determined by measuring expression of the LPS-induced inflammatory proteins, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB), and the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Methanol extract of P. thunbergii decreased the expression of iNOS, COX-2 and NF-κB, and increased the expression of HO-1 in LPS-stimulated RAW264.7 cells. Methanol extract was fractioned by n-butanol, hexane and ethyl acetate (EtOAc) and each fraction was tested for inhibitory effects on inflammation. Among the sequential solvent fractions, the EtOAc soluble fraction was investigated by the expression of prostaglandin E2 (PGE2), and showed decreasing form to the dose-dependent manner. EtOAc extract showed the most effective inhibitory activity of the expression of iNOS, COX-2 and NF-κB, and the production of NO. The study showed that P. thunbergii has anti-inflammatory activity through the decrease of NO and inhibition of iNOS, COX-2, PGE₂ and NF-κB expression, and by the increase of HO-1 enzyme. This study needs for more investigation to find out the most effective single compound with anti-inflammatory activity.

Regulation of Cyclin D3 by Calpain Protease in Human Breast Carcinoma MDA-MB-231 Cells

최병태,김군도,최영현,Choi, Byung-Tae,Kim, Gun-Do,Choi, Yung-Hyun Korean Society of Life Science 2006 생명과학회지 Vol.16 No.4

The $Ca^{2+}-activated$ neutral protease calpain induced proteolysis has been suggested to play a role in certain cell growth regulatory proteins. Cyclin proteolysis is essential for cell cycle progression. D-type cyclins, which form an assembly with cyclin-dependent kinases (cdk4 and cdk6), are synthesized earlier in G1 of the cell cycle and seem to be induced in response to external signals that promote entry into the cell cycle. Here we show that cyclin D3 protein levels are regulated at the posttranscriptional level by calpain protease. Treatment of human breast carcinoma MDA-MB-231 cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. The specific inhibitor of the 26S proteasome, lactacystin, the lysosome inhibitors, ammonium chloride and chloroquine, and the serine protease inhibitor, phenylmethylsulfonylfluoride (PMSF), did not block the degradation of cyclin D3 by lovastatin and actinomycin D. Results of in vitro degradation of cyclin D3 by purified calpain showed that cyclin D3 protein is degraded in a $Ca^{2+}-dependent$ manner, and the half-life of cyclin D3 protein was dramatically increased in LLnL treated cells. These data suggested that cyclin D3 protein is regulated by the $Ca^{2+}-activated$ protease calpain. $Ca^{2+}$-농도 의존적으로 활성화되는 neutral protease calpain에 의한 단백질 분해는 세포의 성장을 조절하는데 중요한 단백질들의 역할에 매우 중요한 역할을 한다. Cyclin의 분해는 세포주기의 진행을 위한 필연적인 과정이다. D-type cyclins는 외부자극이나 신호에 의하여 세포주기의 G1 초기에 합성이 된 후 cyclin-dependent kinases (cdk4 및 cdk6)와의 결합하여 세포주기 S기 진입을 촉진하는 역할을 한다. 본 연구에서는 MDA-MB-231 인체 유방암세포에서 cyclin D3 단백질이 calpain protease에 의하여 전사 후 수준에서 조절 받고 있음을 제시하였다. 본 실험의 조건에서 lovastatin과 actinomycin D가 처리된 MDA-MB-231 세포에서 cyclin D3 단백질의 발현이 완전히 사라졌지만, calpain inhibitor인 LLnL의 처리에 의하여 정상 수준으로 회복되었음을 알 수 있었다. 그러나 26S proteasome의 선택적 억제제인 lactacystin, the lysosome 억제제인 ammonium chloride 및 chloroquine, serine protease 억제제인 PMSF는 동일 조건에서 lovastatin과 actinomycin D 처리에 의한 cyclin D3의 발현저하를 억제하지는 못하였다. In vitro 조건에서 순수 분리된 calpain은 cyclin D3 단백질을 $Ca^{2+}$ 농도 의존적으로 분해하였으며, cyclin D3 단백질의 half-life는 LLnL 처리에 의하여 매우 유의적으로 증가되었다. 이러한 결과는 cyclin D3 단백질이 $Ca^{2+}$에 의해 활성화 되는 protease calpain에 의해 조절됨을 보여준다.

PI3K/Akt/mTOR Mediated Anti-proliferation by the Extract of Fungus Chaetomium sp. in SK-Hep1 Human Hepatocellular Carcinoma Cells

정현일,김군도,손병화 대한암예방학회 2012 Journal of cancer prevention Vol.17 No.1

The marine environment has immeasurable diversity of the chemical and the biological compounds and is an extraordinary resource for the discovery of new anti-cancer drugs. In this study, we investigated the anti-cancer effect of the extract of marine algae-derived symbiotic fungus Chaetomium sp. on SK-Hep1human hepatocellular carcinoma cells. The extract exhibited inhibition of cell proliferation by inducing apoptosis, cell cycle arrest at G1/S phase and autophagic cell death. The results showed that apoptosis resulted from the activation of pro-apoptotic proteins (tBid, Bad, PUMA) and caspases (-3 and -8). We identified that the extract arrested cell cycle at sub-G1 phase and increased expression of p21CIP1/WAF1while reduced expression of cdc25A and E2F-1. The extract also inhibited the activation of PI3K regulatory subunit (PI3K p110α) and downstream targets such as Akt, mTOR and p70S6K. The cells treated with the extract finally showed autophagy by the up-regulation of Atg5, 7, 12 and LC3B. The results suggested that the extract of fungus Chaetomium sp. has a potential anti-cancer effect on SK-Hep1cells.