• Asian Pacific Journal of Cancer Prevention
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• 제17권2호
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• pp.703-712
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• 2016

Monoclonal antibodies with specific antigens have been widely used as targeted therapy for cancer. Hep88 mAb is a monoclonal antibody which shows specific binding with anti-cancer effects against the HepG2 cell line. However, its mechanisms of action are still not completely understood. We examined cell cycling and apoptosis by flow cytometry and mRNA expression of factors involved in apoptosis and paraptosis in Hep88 mAb-treated HepG2 cells by real-time PCR. The cell-cycle analysis demonstrated that growth-inhibitory activity was associated with G2/M cell cycle arrest. Hep88 mAb induced a significant increase in apoptotic cell populations in a dose- and time-dependent manner. The mRNA expression results also suggested that the process triggered by Hep88 mAb involved up-regulation of tumor suppressor p53, pro-apoptotic Bax, Cathepsin B, Caspase-3 and Caspase-9, with a decrease of anti-apoptotic Bcl-2 - thus confirming paraptosis and apoptosis programmed cell death. These findings represent new insights into the molecular mechanisms underlying the anti-cancer properties of Hep88 mAb in liver cancer cells.

• 대한수의학회지
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• 제48권1호
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• pp.53-60
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• 2008

We have investigated efficiency of a recombinant subunit Pasteurella multocida toxin (PMT) that was mixed with a vaccine consisted of inactivated whole cells of Bordetella bronchiseptica, P. multocida (types A and D). For verification of the efficacy of the vaccine, all experimental pigs (suckling piglets, sow and gilts) in the three farms were vaccinated. Antibody titers against B. bronchiseptica and P. multocida type A of the vaccinated pigs by microplate agglutination were significantly higher than those of the control pigs (p < 0.05). Similar patterns were observed in the analysis of anti- PMT neutralizing antibody by serum neutralizing method using Vero cell (p < 0.05). Anti- P. multocida type D antibody titer of the vaccinated sows and gilts by ELISA showed significant differences with those of the non-vaccinated pigs (p < 0.05). Although antibody titers increased, it was unable to find out the difference in the clinical signs between the vaccinated and non-vaccinated pigs. However, the increase in body weight of the vaccinated piglets was observed in comparison with the non-vaccinated piglets on a farm. At slaughtering of the pigs, pathological lesions in the turbinate bones of the vaccinated pigs were significantly lower than those of the non-vaccinated pigs (p < 0.001). These results suggested that efficacy of the vaccine in pigs demonstrated to protect against atrophic rhinitis in Korea.

• 대한의생명과학회지
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• 제9권3호
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• pp.145-150
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• 2003

In the intestinal mucosa, mast cells are thought to be responsible for the expulsion of parasites. We investigated the relationship of worm expulsion and mast cells in C3H/HeN and BALB/c mice infected with Echinostoma hortense. In addition, we examined whether the worm recovery rate was associated with the strain of mice, and whether a toluidine stain and immunohistochemistry using the c-kit antibody was effective in the detection of mast cells. In order to investigate the mucosal immune response of C3H/HeN and BALB/c mice, each mouse was infected orally with 30 E. hortense metacercariae. Then, the number of mucosal mast cells and worm recovery rates was observed in experimentally infected mouse strains between 1 week and 8 weeks post infection (PI). Mucosal mast cells were increased in 3 weeks P.I. in C3H/HeN and BALB/c mice. On the other hand, only mucosal goblet cells and worm recovery rates correlated in C3H/HeN mice (P=0.0482). Worm recoveries in C3H/HeN mice were 65.7$\pm$5.6, 53.3$\pm$5.4 and 6.7$\pm$0.6 in week 1, 2, and 3 P.I. and strongly decreased in week 3 P.I. Worm recoveries in BALB/c mice were 23.0$\pm$2.5, 10.0$\pm$1.0, and 6.7$\pm$0.6% in week 1, 2, and 3 P.I. and gradually decreased from week 1 P.I. to week 3 P.I. Worm recoveries in C3H/HeN mice were significantly higher than in BALB/c mice (P<0.00l). The number of mast cells in C3H/HeN and BALB/c mice using the anti-c-kit antibody reached to a peak in week 3 P.I. and recovered as normal level in week 5 P.I. and 6 P.I. The number in E. hortense-infected C3H/HeN mice (P=0.0015) was higher than in E. hortense-infected BALB/c mice (P=0.01) compared with the control group. There were significant differences in the number of mast cells among regions of the intestine in in C3H/HeN mice (P<0.05) but not in BALB/c mice (P>0.05). Immunohistochemistry using the anti-c-kit antibody was significant method as an examination of the number of mast cells (P=0.0002). In conclusion, the present study demonstrated that mast cells play an important role in worm recovery, and immunohistochemistry using the anti-c-kit antibody was superior to toluidine stain as an examination of mast cells.

• 대한기관식도과학회:학술대회논문집
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• 대한기관식도과학회 1993년도 제27차 학술대회 초록집
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• pp.84-84
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• 1993

17번 염색체의 단완에 위치하여 종양의 억제에 관여하는 p53유전자의 돌연변이로 인한 불활성 p53단백의 과도발현이 유방암과 대장암을 비롯한 인체의 여러 암에 있어서 보고되었으며 일부 암에서는 예후인자로서의 가능성이 제시되었다. 저자들은 비교적 완만히 성장하지만 국소재발 및 원격전이가 잦은 두경부 영역의 선양낭포암에 있어서 p53 단백 발현 양상 및 예후 인자로의 가능성을 알아보고자 1982년부터 1991년 사이에 서울대학병원에서 병리조직학적으로 진단받은 환자 중 추적관찰이 가능하였고 병리조직을 얻을 수 있었던 23례에 대해 p53 단백에 대한 단클론항체를 이용하여 면역 조직화학적 염색을 시행하여 다음과 같은 결과를 얻었다. 총 23례 중 p53단백의 양성 발현을 보인 경우는 7fP(34%)이었으며, p53단백의 양성 발현과 국소재발 또는 원격전이와의 사이에 유의한 상관관계는 없었다.

• 대한한방종양학회지
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• 제6권1호
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• pp.67-79
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• 2000

Objective : Hedyotis diffusa has been used as an anticancer agent for several decades in oriental medicine. We test whether the methanol extract of the herb affects transcriptional activation factors including $NF-{\kappa}B$ and AP-1. Methods : 1. HL-60 cells were treated with various concentrations(from 200 to $50{\mu}g/ml$) of methanol extract and $H_2O$ extract($200{\mu}g/ml$)of hedyotis diffusa, After 48h later, the cells were tested for viability by MTT assay. 2. The HL-60 cells were treated with $200{\mu}g/ml$ of methanol extract for the indicated periods. First. Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$ and AP-1. Second. Nuclear extracts were isolated and reacted with p50, p65. c-rel pan-Jun, c-Jun, JunB. JunD antibody on ice for 30min. Finally The cell lysates were prepared and analyzed by western blotting using anti-Fas, anti-FasL and anti-p53 antibody. Results : 1. The methanol extract decreases the viability of human lymphoid origin leukemia HL-60 cells in a dose-dependent manner. 2. $NF-{\kappa}B$ is rapidly activated by the addition of the methanol extract, reaches a peak at 30min and gradually returns to resting level. We confirm that $NF-{\kappa}B$ is a heterodimer mainly composed of p65 subunit with c-Rel. 3. Transcriptional activation of AP-1 is detected at 30min and reaches a maximum at 1hr after stimulation of the cells with the methanol extract. AP-1 is mainly composed with Jur-D and partially Jug-B proteins. 4. the methanol extract of Hedyotis diffusa induces the expression of Fas, Fas ligand and p53 proteins of HL-60 cells in a time dependent fashion. Conclusions : These results suggest that the methanol extract of Hedyotis diffusa exerts anticancer effects to induce the death of human leukomic HL-60 cells via activation of trascriptional factors such as $NF-{\kappa}B$ and AP-1, increase in expression of Fas mediated signalling proteins, and induction of tumor suppressor gene. p53.

• 대한수의학회지
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• 제39권3호
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• pp.593-601
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• 1999

This study was designed to evaluate the effect of cyclophosphamide(CY) on diethylnitrosamine(DEN)-induced hepatic tumors in rats. Thirty five male or female Sprague Dawley rats were continiously given water containing 0.01% DEN for 10 weeks and then were given with CY 25mg/rat/day in water for 3, 5, 7 or 9 days. The livers of rats were removed and fixed in 10% buffered neutral formalin. he appearances of positive cells by immunohistochemical methods using proliferating cell nuclear antigen (PCNA) antibody, p53 antibody and apoptotic kit were investigated. The livers of rats given with CY were grossly brilliant, red-brown color, flexible, and thin border, and stainability of the liver cells were restored microscopically, and the vaccuolated and degenerated regions were differentiated from restored regions. These restored findings also were advanced in control group because of no DEN treatment but tended to be less avanced. In immunohistochemistry, positive cells to PCNA antibody appeared more numerous in control groups than that of CY treated groups. Appearance of positive cells in CY-treated group for 7 days and for 9 days were more numerous than those of CY-treated groups for 3 days and for 5 days, respectively. So these findings suggested that CY suppressed cell proliferations and effects of these action were decreased with CY-treated days. The numbers of positive cells to PCNA antibody were more prominent in hepatocellular carcinoma regions and cholangiocarcinoma regions, and then were ranked as order of large liver cell regions and normal liver cell regions. Also the numbers of the positive cells by apoptotic kit tended to be higher in hepatocellular carcinoma regions and cholangiocarcinoma regions but not uniformly in order in all regions and were much less numbers than those of PCNA positive cells. So immunohistochemical methods using PCNA antibody together than using apoptotic kit alone when anti-carcinogen experiments. Rats with positive cells by p53 antibody were 11 of 15 rats(73.4%) in control groups and 12 of 18 rats(66.7%) in CY treated group, respectively. These positive cells appeared focally in early vacuole-occurring regions and were very low in numbers.

• 한국가축번식학회지
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• 제14권2호
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• pp.101-106
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• 1990

These mxperiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro fertilization and following development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6mm of diameter. Bovine oocytes were matured in vitro for 24~26hrs in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$ and subsequently cultured in medium containing cumulus cell antibody for 1 hour. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hrs in BO solution 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and then cultured for 7 days. The results obtained in these experiments were summarized as follows : 1. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cells, the fertilization rate of cumulus intact and removed oocytes was ranged to 45.0 to 53.7%. These value is slightly lower than that(64.3%) of follicular oocytes not treated with the antibody, and increased frequency of both male and female pronuclear formation was found in cumulus intact oocytes cultred in medium without the antibody(p<0.05). 2. The fertilization rate of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cells was ranged 45.0 to 52.5%, significantly lowre than that(62.8%) of oocytes cultured in antibody free medium, and increased frequency of ova with male and female pronuclei was found when cumulus cells were present(p<0.05). 3. The rates of cumulus cell intact and removed oocytes developed to 8-, 16-cell and morula or blastocyst after treatment of intact and solubilized cumulus cell antibody were ranged 7.1 to 14.5, 2.9 to 5.9 and 1.5 to 2.9%, respectively, slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results of this stduy indicate that cumulus cells promote not only normal fertilization with proper pronuclear formation, but embryo development and that the beneficial effect of cumulus cell to the pronuclear formation and embryo development is blocked by the action of antibody to cumulus cell.

• Tuberculosis and Respiratory Diseases
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• 제65권6호
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• pp.476-486
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• 2008

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 $NF-{\kappa}B$가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 $NF-{\kappa}B$활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. $NF-{\kappa}B$ 전사활성은 luciferase reporter gene assay를 이용하였고 $I{\kappa}B{\alpha}$ 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 $NF-{\kappa}B$와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 $IgG{\kappa}B-luc$세포에서 2.45배 증가하고 NCI-H1299 $IgG{\kappa}B-luc$세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 $NF-{\kappa}B$가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 $NF-{\kappa}B$ 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다.

• 생명과학회지
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• 제28권1호
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• pp.37-42
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• 2018

NAG-1 단백질은 TGF-${\beta}$ superfamily 유전자로서 암세포의 apoptosis를 유도하고 항암 활성과 관련이 있는 것으로 알려져 있다. 본 연구에서는 프로폴리스 유래의 파이토케미칼 CAPE (caffeic acid phenethyl ester)에 의한 항암유전자 NAG-1의 발현과 발현조절에 대해 연구하였다. 인간 대장암 세포주 HCT116에서 CAPE의 처리에 의해 농도의존적, 시간의존적으로 NAG-1의 발현이 증가됨을 확인하였다. 게다가, 다른 대장암 세포주인 LOVO 세포주에서도 농도의존적으로 NAG-1의 발현이 증가됨을 확인하였다. p53-null HCT116세포주를 이용한 실험에서 CAPE에 의한 NAG-1의 발현은 전사조절인자인 p53에 의존하지 않음을 증명하였다. 또한, 3가지 종류의 NAG-1 프로모터 construct를 이용한 실험에서, cis-element 후보가 -474와 -1,086사이에 있음을 증명하였다. CAPE에 의해 전사조절인자인 ATF3와 CREB의 발현이 변화되는 지를 확인한 결과, CREB은 전혀 발현이 증가되지 않는 반면 ATF3는 CAPE 처리에 의해 농도의존적으로 발현이 증가함을 확인하였다. 그리고, pCG-ATF3와 pCREB의 cotransfection 실험에서 CREB은 NAG-1의 발현에 영향을 못 미치는 반면, ATF3의 과대발현에 의해 NAG-1의 발현이 증가됨을 확인하였다. 결론적으로, CAPE에 의한 NAG-1의 발현은 주로 전사조절인자인 ATF3를 경유하여 일어남을 시사한다.

• Journal of Applied Biological Chemistry
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• 제53권2호
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• pp.112-115
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• 2010

To analyze the surface properties of bacteriophytochrome (BphP), five (2B8, 2C11, 3B2, 3D2, 3H7) anti-BphP monoclonal antibodies were produced by using full-length of BphP of Deinococcus radiodurnas. 2B8 and 2C11 preferentially recognized the epitopes at N-terminal region of BphP, whereas 3B2, 3D2 and 3H7 showed preferential affinities to the epitopes of C-terminal region of BphP.