Apoptotic Response of Human Oral Squamous Carcinoma Cells to Etoposide

김규천,이경덕,박재현,김덕한,박정길,박준상,박봉수,Kim, Gyoo-Cheon,Lee, Kyoung-Duk,Park, Jae-Hyun,Kim, Duk-Han,Park, Jeong-Kil,Park, June-Sang,Park, Bong-Soo Korean Academy of Orofacial Pain and Oral Medicine 2005 Journal of Oral Medicine and Pain Vol.30 No.2

항암제의 연구는 화학물질에 민감한 암세포를 죽음에 이르게 하는 세포자멸사와 같은 다양한 세포기능에 초점을 맞추어 왔다. 그러나 약물이 유도한 세포의 죽음에 있어서 핵심적인 분자적 기작은 아직 잘 이해되지 않고 있다. Etoposide는 폐암과 고환암에 사용되는 항암제로서, 본 연구는 etoposide가 사람구강편평상피암종세포(OSC9)에도 세포독성효과와 세포자멸사를 일으키는지를 알아보기 위해 실행하였다. 이 실험에서 etoposide는 농도와 시간 의존적으로 OSC9 세포의 생존율를 현저하게 저해시켰다. TUNEL 염색과 Hoechst 염색을 이용한 핵의 형태학적 관찰에서는 etoposide에 의해 핵이 응축되고 분절되었다. p53의 발현은 48 시간에 증가했으며, etoposide 처리로 인해 caspase-3의 활성을 관찰할 수 있었으며, 그 기질에 해당되는 PARP 단백질은 116-kDa과 89-kDa으로 분절되었다. 위의 결과들은 OSC9 세포에서 etoposide가 유도한 세포자멸사는 caspase-3의 활성과 관련됨을 설명하고 있다.

Pseudomonas aeruginosa Exotoxin A Induces Apoptosis in Chemoresistant YD-9 Human Oral Squamous Carcinoma Cell Line Via Accumulation of p53 and Activation of Caspases

Gyoo-Cheon Kim(김규천),Young-Gi Gil(길영기) 한국생명과학회 2009 생명과학회지 Vol.19 No.8

구강편평상피암종은 말기에서 종종 화학치료요법제들이 유도하는 세포자멸사에 저항성을 보인다. 박테리아의 독에 대한 진전된 이해는 암치료에 대한 새로운 치료전략으로 제기되어지고 있다. 본 연구는 Pseudomonas aeruginosa exotoxin A (PEA)가 세포자멸사 기작을 통해 항암제에 저항성을 보이는 YD-9 구강편평상피암종의 생존율을 현격하게 떨어뜨림을 설명하고 있다. 세포자멸사현상은 핵의 형태학적 변화와 DNA 분절 생성을 통해 입증되었다. PEA는 caspase-3, -6, -9의 분절과 활성화를 일으켰다. 그리고 이러한 반응들은 caspase의 기질에 해당하는 poly (ADP-ribose) polymerase (PARP), DFF45, 그리고 lamin A의 단백질 분해를 야기했다. 사립체 막전위 감소, cytochromec와 Smac/DIABLO의 사립체로부터 세포질로의 유리, 그리고 AIF의 사립체에서 핵으로 이동 등이 관찰되었다. p53, p21 그리고 14-3-3γ는 증가되는 반면 cyclin B와 cdc2는 감소되었다. 이상의 결과들을 종합해 보면 PEA는 caspase를 활성화시키고, 사립체에 변화를 야기시키고 더 나아가서 세포주기 유전자를 조절함으로써 항암제에 대한 강한 저항성을 보이는 YD-9 세포에서 세포자멸사를 유도한다. Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO from mitochondria to cytosol, and translocation of AIF into nucleus were shown. While p53, p21 and 14-3-3γ were upregulated, cyclin B and cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.

Apoptotic Effect of Combination Treatment with a Proteasome Inhibitor, Lactacystin, and a Histone Deacetylase Inhibitor, Trichostatin A, on MCF-7 Cells

김규천(Gyoo-Cheon Kim),신유정(Yoo-Jung Shin),김인령(In-Ryeon Kim),이무형(Moo-Hyung Lee),황영섭(Young-Seob Hwang),길영기(Young-Gi Gil),박봉수(Bong-Soo Park) 대한해부학회 2006 Anatomy & Cell Biology Vol.39 No.6

Proteasome 저해제와 HDAC 저해제의 상호작용에 의한 상승효과가 여러 형태의 암에서 세포자멸사를 일으키는 연구가 많음에도 불구하고, 그 기작에 대해서는 거의 알려진 바가 없다. 본 연구는 proteasome 저해제인 lactacystin과 HDAC 저해제인 TSA의 병용처리가 세포자멸사 유도에 미치는 영향을 알아보고자 시행되었다. TSA를 전처리한 후 연이어 lactacystin을 처리한 군은 강한 항암효과와 핵의 응축현상을 보였다. Western blot 분석에서는 TSA와 lactacystin의 병용 처리는 Bax/Bcl-2 분율변화와 XIAP 수준의 감소 및 caspase-7의 활성화와 PARP의 분절의 결과를 얻었다. 그리고 더 나아가서 병용처리한 실험군에서 cell cycle arrest는 G2/M기에서 일어났으며, proteasome 활성은 사라졌다. 본 연구는 TSA와 lactacystin의 병용처리에 의해 일어나는 세포자멸사의 기작에 대해 설명하였고, 본 연구의 결과 자료는 생체의 항암치료전략에 도움을 줄 수 있을 거라고 생각한다. Although much information has been accumulated about the synergistic interaction of proteasome inhibitors and HDAC inhibitors to induce apoptosis in a certain type of cells, much less is known currently about the underlying mechanism. This study was undertaken to explore the combination effect of a histone deacetylase inhibitor, TSA, and a proteasome inhibitor, lactacystin, on the induction of apoptosis. Pretreatment of TSA and subsequent treatment of lactacystin showed the strong antitumor activity and nuclear condensation. Western blot assay showed that combination treatment of TSA and lactacystin increased Bax/Bcl-2 ratio and decreased level of XIAP. Activation of caspase-7 and cleavage of PARP were demonstrated after the combination treatment. In combination treatment group, cell cycle arrest was induced at G2/M phase and abolished increase in proteasome activity. This study is elucidating the mechanims whereby targeting apoptotic machineries may help in directing therapeutic strategies.

Synthetic Bile Acid Derivative HS-1200-induced Apoptosis of Human Osteosarcoma Cells

김규천(Gyoo-Cheon Kim),허용수(Young-Soo Her),박재현(Jae-Hyun Park),문용석(Yong-Suk Moon),유영현(Young-Hyun Yoo),신상훈(Sang-Hun Shin),박봉수(Bong-Soo Park) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.5

담즙산 및 합성유도체가 여러 암세포에서 세포자멸사를 유도하여 항암활성을 보인다고 밝혀졌지만 뼈육종세포에서의 세 포자멸사 유도활성은 보고된 바 없다. 본 연구는 담즙산 유도체들이 뼈육종세포에 세포자멸사를 유도하는지를 알아보고 그 기작을 연구하기 위하여 수행되었다. UDCA 합성 유도체와는 달리 CDCA 합성유도체는 뼈육종세포의 사망을 유도하였다. CDCA 유도체 중 HS-1200을 선택하여 세포자멸사 기전을 연구하였다. HS-1200은 핵의 농축, cytochrome c 방출, Bax/Bcl-xL분율 변화, caspase-3의 활성화 및 CAD와 PARP 분절 등을 관찰되었다. 비록 더 많은 연구가 필요하지만 본 시험관 연구 자료는 합성담즙산 유도체 투여가 뼈육종의 치료전략이 될 수 있음을 시사한다. Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and had anticancer effects. However, it wasn’t discovered those materials have apoptosis induced effects on osteosarcoma cells. The present study was done to examine the synthetic bile acid derivatives induced apoptosis on osteosarcoma cells and such these apoptosis events. The synthetic bile acid derivatives, chenodeoxycholic acid (CDCA) induced the cell death on human osteosarcoma (HOS) cells contrary to ursodeoxycholic acid (UDCA). HS-1200, a synthetic derivative of CDCAs, was chosen to experiment apoptosis events in HOS cells. HOS cells treated with HS-1200 showed nucleus condensation, cytochrom c release, Bax/Bcl-xL alteration, activation of caspase-3 and caspase-activated deoxyribonuclease (CAD), and degradation of poly (ADP-ribose) polymerase (PARP). Though this study needs more investigations, these in vitro data suggest that treatment of the synthetic bile acid derivatives can give medical therapy on HOS cells.

Induction of Apoptosis in Human Oral Squamous Carcinoma Cells by Extracellular Products from Pseudomonas aeruginosa

김규천(Gyoo-Cheon Kim),강현희(Hyeun-Hee Kang),김현철(Hyeon-Cheol Kim),김인령(In-Ryeon Kim),이무형(Moo-Hyung Lee),구병찬(Byung-Chan Koo),김덕한(Duk-Han Kim),민지학(Ji-Hak Min),박봉수(Bong-Soo Park) 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.4

100년 전부터 암을 가진 동물이나 사람이 박테리아에 감염되었을 때, 그 암조직이 줄어들었다는 보고가 있었다. 박테리아는 감염된 세포내에서 단백질합성 저해제와 구멍형성 단백질 분비, 죽음에 이르게 하는 내재성 단백질 분자의 활성화를 포함하는 다양한 기작을 통해서 세포자멸사를 유발할 수 있다. 본 연구는 Pseudomonas aeruginosa 균주로부터 분리한 세포밖 분비물(EPPA)이 구강편평상피암종세포(OSC9)의 세포자멸사를 일으키는지를 알아보고자 시행 하였다 EPPA는 OSC9 암세포에 농도 의존적으로, 또한 시간 의존적으로 암세포독성효과를 나타내었다. Hoechst 염색법, TUNEL 분석법 그리고 DNA 전기영동 등의 방법을 통해 염색질 응축과 핵분절 등이 확인되었다. EPPA가 처리 후 24시간 경과된 세포에서부터 caspase-3와 caspase-6의 활성형태를 관찰할 수 있었다. Caspase-3의 기질인 PARP와 DFF45 그리고 caspase-6의 기질인 lamin A의 분절형태가 관찰 되었다. 이상의 결과로 EPPA가 OSC9 암세포에서 casapse 의존성 세포자멸사를 유발시킴을 알 수 있었다. 사람구강편 평상피암종에 항암효과를 가지는 새로운 물질이 EPPA에 포함되어 있는 것으로 보인다. It was reported that cancer in humans and animals infected with microbial pathogens was regressed about 100 years ago. Bacteria are able to trigger apoptosis by a variety of mechanisms including the secretion of protein synthesis inhibitors, pore forming proteins, molecules activating the endogenous death machinery in the infected cell. This study was conducted in order to investigate whether extracellular products of Psuedomonas aeruginosa (EPPA) induce apoptosis in human oral carcinoma cells (OSC9). The EPPA showed cytotoxic effect on OSC9 cells in dose and time-dependent manner. The cell death was demonstrated to be due to apoptosis characterized by chromatin condensation and nuclear fragment. EPPA treatment induced cleavage of caspase-3 and caspase-6. The caspase substrates, PARP, DFF45 and lamin A were cleaved during EPPA-induced apoptosis. Taken together, EPPA induces apoptosis on human oral squamous carcinoma cells in caspase-dependent manner. Our data therefore provide that EPPA contains a novel antitumor agent for human oral squamous carcinoma.

인테그린 α₂와 상피성장인자수용체 차단항체의 저해작용을 통한 구강편평상피암 세포의 선택적 제거

최연식,김규천,윤식,황대석,김철훈,전영찬,변준호,신상훈,김욱규,Choi, Yeon-Sik,Kim, Gyoo-Cheon,Yoon, Sik,Hwang, Dae-Seok,Kim, Cheol-Hun,Jeon, Young-Chan,Byun, June-Ho,Shin, Sang-Hun,Kim, Uk-Kyu 대한악안면성형재건외과학회 2013 Maxillofacial Plastic Reconstructive Surgery Vol.35 No.3

Purpose: This study was to find efficacy of integrin alpha2 (${\alpha}_2$) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the selective cell death effect of anti-integrin ${\alpha}_2$ and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticles (GNP) with air plasma for selective cell death of oral SCC. Methods: Expression of integrin ${\alpha}_2$, EGFR on human SCC cells (SCC25) were examined by western blot. SCC25 cells were treated with anti-integrin ${\alpha}_2$, anti-EGFR and analysed by Hemacolor staining, immunoflorescence staining, FACS flow cytometry. Conjugated GNP with integrin ${\alpha}_2$, EGFR antibody were treated by air plasma on SCC cells. Results: Integrin ${\alpha}_2$ and EGFR were over-expressed on SCC25 cells than normal lung WI-38 cells. The cell viability rate of SCC25 cells treated with anti-integrin ${\alpha}_2$, anti-EGFR was lower than WI-38 cells. The concentration changes of nucleus, releasing cytochrome c and apoptosis inducing factor (AIF) from mitochondria to cytosol were observed. The changes of proteins related with apoptosis were observed. Increase of bax, bcl-xL, activation of caspase-3, -7, -9, and fragmentation of PARP, DFF45 and decrease of lamin A/C in SCC25 cells were observed. In FACS, increase of sub-$G_1$ and S phase was observed. Cell cycle related proteins, Such as cyclin D1, cyclin dependent kinase (CDK) 4, cyclin A, cyclin E, CDK 2, p27 were decreased. After SCC25 cells treated with conjugatged GNP-Integrin ${\alpha}_2$, GNP-EGFR, additionally air plasma, the cell death rate was significantly increased. Conclusion: Integrin ${\alpha}_2$, EGFR were over-expressed in oral SCC cells. Anti-integrin ${\alpha}_2$, anti-EGFR in SCC25 cells induced apoptosis selectively. When GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR were treated with air plasma on SCC25 cells, cancer cells were died more selectively. GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR with air plasma could be treatment choice of oral SCC.

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.

골다공증 유도 백서에서 임플란트 삽입후 칼슘과 비타민 D의 섭취가 골조직에서 혈관내피세포성장인자 수용체 발현에 미치는 영향

김호철 ( Ho Chul Kim ),권택균 ( Taek Kyun Kwon ),이재열 ( Jae Yeol Lee ),송재민 ( Jae Min Song ),정인교 ( In Kyo Chung ),김규천 ( Gyoo Cheon Kim ),신상훈 ( Sang Hun Shin ) 한국조직공학과 재생의학회 2011 조직공학과 재생의학 Vol.8 No.2s

Angiogenesis is closely assocatied with bone formation and osteoporosis is risk factor of success of implant. Recently reported, uptake of calcium and Vitamin D improve bone healing process in osteoporosis. Vascular endothelial growth factor also interact bone formation through modulation of angiogenesis. The purpose of this study was to evaluate the interaction between vascular endothelial growth factor (VEGF) and calcium with vitamin D in bone tissue around implant in osteoporosis induced rats by measuring the expression of vascular endothelial grawth factor receptors (VEGFR). Titanium screw type implants were placed into tibias of ovariectomized. The rats were sacrificed at different time interval (1,2 and 4 weeks after implantaion) for immunohistochemistry with antibody for receptor of vascular endothelial growing factor 1, 2 and 3. In this study we observed 3 type vascular endothelial growth factor (VEGFR1-3). Receptor of vascular endothelial growth factor 3 (VEGFR-3) was increased by calcium and vitamin D in bone formation process but others (receptor of vascular endothelial growth factor 1 and 2) were not shown significant difference. This study, with previously published work, shows that VEGF interact with calcium and vitamin D and especially VEGFR-3 was incresed in early stage.

가토의 측두하악관절원판 결손에서 간세포 성장인자가 치유에 미치는 영향

김복주,성화식,김철훈,김규천,황희성,신상훈,Kim, Bok-Joo,Seong, Hwa-Sik,Kim, Chul-Hoon,Kim, Gyoo-Cheon,Hwang, Hee-Sung,Shin, Sang-Hun 대한악안면성형재건외과학회 2009 Maxillofacial Plastic Reconstructive Surgery Vol.31 No.1

Purpose: The purpose of this study is to investigate the therapeutic use of Hepatocyte growth factor(Adv.CMV.HGF) in temporomandibular joint disc defect. Materials and methods: Twelve New Zealand white rabbits, weighing 2.5 - 3.0 kg, were used in this experiment. Defects(2 mm in diameter) were created in their TMJ discs. Recombinant Adv.CMV.HGF with gelatin sponge($Gelfoam^{(R)}$) as carrier was implanted in the defects. We divided the rabbits into four batches according to the duration of the implantation - of 1, 4, 8, 12 weeks - and both left and right TMJ of each rabbit in all groups were used in the research : left joints were used as experiment group and right were control group. Each batch of rabbits was killed one, four, eight and twelve weeks after the experimentation respectively, and called Group A, B, C, and D. (Group A = 1 wk, B = 4 wks, C = 8 wks, and D = 12 wks) Results: The experimental group showed a significant increase in the number of chondroblasts and active cell differentiation at the margin of the defects. Compared to the control group, in the experiment group chondroblasts increased and chondrocytes showed a columnar arrangement, which is witnessed at the time of cell differentiation. Conclusion: This study supports the case that Avd.CMV.HGF may be useful in the repair of articular disc of the rabbit TMJ.

RF 플라즈마 및 전기장의 흑색종 (G361 melanoma) 세포에 대한 사멸 효과

孫采化(Shon Chae-Hwa),金奎千(Kim Gyoo-Cheon),李海準(Lee Hae June) 대한전기학회 2007 전기학회논문지 Vol.56 No.11

Micro plasma has been recently studied to investigate the effects on various cells. We study a micro-plasma produced by a plasma needle that is operated with RF power and its effects on G361 melanoma cells. The micro plasma size ranges from sub-㎜ to several mm at a few watts of RF power. For the bio-medical treatment, low-temperature plasma is obtained and gas temperature is controlled within several tens of degrees (℃) in order not to disturb cell activities. Elementary spectroscopic studies to obtain plasma characteristics are presented for Ar and He plasma with different frequencies of RF power. Also the preliminary results of the micro plasma effects on G361 melanoma cells are presented. It was observed that the irradiation of micro plasma induces cell death through the deprivation of tyrosine phosphorylation in the G361 cells.