• Journal of Acupuncture Research
• /
• v.29 no.1
• /
• pp.47-59
• /
• 2012

목적 : 이 연구는 봉독이 사람의 난소암 세포인 SKOV3와 PA-1에서 death receptor의 발현을 높여 세포자멸사를 촉진함으로써 암세포의 성장을 억제하는지 밝히고자 하였다. 방법 : 난소암의 세포자멸사의 관찰에는 DAPI, TUNEL staining assay를 시행하였으며, 세포자멸사 조절 단백질의 변동 관찰에는 western blot analysis를 시행하였고, 난소암 세포에서 death receptor의 변화를 관찰하기 위해 RT-PCR analysis를 시행하였다. 결과 : 1. DAPI, TUNEL staining assay 결과, 봉독은 투여량에 따라 세포자멸사의 유도를 통해 SKOV3와 PA-1 난소암세포의 증식을 억제하였고, 세포자멸사와 동반하여 DR4와 DR6의 발현이 두 암세포 모두에서 증가하였고, DR3의 출현은 PA-1 세포에서 증가하였다. 2. Death Receptor의 발현 증가에 따라 caspase-3, 8, 9 and Bax를 포함하는 세포자멸사 촉진 단백질의 발현이 동반하여 상승하였고 JAK2, STAT3의 인산화와 Bcl-2의 발현은 억제되었다. 3. siRNA 처리 시 봉독에 의한 DR3, DR4, DR6 발현증가와 STAT3의 활성억제가 역전되었다. 결론 : 이러한 결과는 봉독이 난소암 세포에서 DR3, DR4, DR6의 증가와 JAK2/STAT3 pathway의 억제를 통하여 세포자멸사를 유발한다는 것을 시사하며, 난소암의 예방과 치료에 효과적으로 활용될 수 있을 것으로 기대된다.

• Journal of Acupuncture Research
• /
• v.30 no.1
• /
• pp.57-70
• /
• 2013

This study was to investigated the effects of the bee venom on inhibition of cell growth via upregulation of death receptor expression in the A549 human lung cancer cells. Bee venom(1-5 ${\mu}g$/ml) inhibited the growth of A549 lung cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of TNFR1, Fas, death receptors(DR) 3, 4 and 6 was increased in the cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, -9 and Bax was concomitantly increased, but the expression of Bcl-2, NF-${\kappa}B$ were inhibited by treatment with bee venom in A549 cells. Moreover, deletion of DR3, DR4 by small interfering RNA significantly reversed bee venom-induced cell growth inhibitory effect, whereas Apo3L strengthened anti-proliferative effect of bee venom through enhancement of DR3 expression. These results suggest that bee venom should exert anti-tumor effect through induction of apoptotic cell death in lung cancer cells via enhancement of death receptor expression, and that bee venom could be a promising agent for preventing and treating lung cancer.

• Journal of Acupuncture Research
• /
• v.31 no.2
• /
• pp.75-85
• /
• 2014

Objectives : We investigated whether bee venom(BV) inhibit cell growth through enhancement of death receptor expressions in the human cervix cancer C33A cells. Methods : BV($1{\sim}5{\mu}g/ml$) inhibited the growth of cervix cancer C33A cells by the induction of apoptotic cell death in a dose dependent manner. Results : Consistent with apoptotic cell death, expression of Fas, death receptor(DR) 3, 4, 5 and 6 was increased concentration dependently in the cells. Moreover, Fas, DR3 and DR6 revealed more sensitivity to BV. Thus, We reconfirmed whether they actually play a critical role in anti-proliferation of cervix cancer C33A cells. Consecutively, expression of DR downstream pro-apoptotic proteins including caspase-8, -3, -9 was upregulated and Bax was concomitantly overwhelmed the expression of Bcl-2. NF-${\kappa}B$ were also inhibited by treatment with BV in C33A cells. Conclusions : These results suggest that BV could exert anti-tumor effect through induction of apoptotic cell death in human cervix cancer C33A cells via enhancement of death receptor expression, and that BV could be a promising agent for preventing and treating cervix cancer.

• Journal of Acupuncture Research
• /
• v.31 no.1
• /
• pp.121-130
• /
• 2014

Objectives : I investigated whether bee venom inhibit cell growth through enhancement of death receptor expressions in the human lung cancer cells, NCI-H460. Methods : Bee venom(1-5 ${\mu}g/ml$) inhibited the growth of NCI-H460 lung cancer cells by the induction of apoptotic cell death in a dose dependent manner. Results : Consistent with apoptotic cell death, expression of TNF-R1, TNF-R2, FAS, death receptors(DR) 3, 4, 5 and 6 was increased in the cells. Expression of DR downstream pro-apoptotic proteins including Caspase-8, -3, -9 was upregulated and Bax was concomitantly overwhelmed the expression of Bcl-2. NF-kB were inhibited by treatment with bee venom in NCI-H460 cells through TNF response change led by TNF-R1 and TNF-R2. Conclusions : These results suggest that bee venom should exert anti-tumor effect through induction of apoptotic cell death in NCI-H460 human lung cancer cells via enhancement of death receptor expression, and that bee venom could be a promising agent for preventing and treating lung cancer.

• Journal of Acupuncture Research
• /
• v.31 no.2
• /
• pp.87-98
• /
• 2014

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in $HT_{29}$ human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.6
• /
• pp.2889-2892
• /
• 2014

Death receptor 4 (TRAIL-R1 or DR4) polymorphisms have been associated with cancer risk, but findings have been inconsistent. To estimate the relationship in detail, a meta-analysis was here performed. A search of PubMed was conducted to investigate the association between DR4 C626G, A683C and A1322G polymorphisms and cancer risk, using odds ratios (ORs) with 95% confidence intervals. The results suggested that DR4 C626G and A683C polymorphisms were indeed associated with cancer risk (for C626G, dominant model, OR 0.991, 95%CI 0.866-1.133, p=0.015; for A683C, additive model, OR=1.140, 95%CI: 0.948-1.370, p=0.028; dominant model, OR=1.156, 95%CI: 0.950-1.406, p=0.080) in the Caucasian subgroup. However, the association was not significant between DR4 polymorphism A1322G with cancer risk in Caucasians (For A1322G, additive model: OR 1.085, 95%CI 0.931-1.289, p=0.217; dominant model: OR 1.379, 95%CI 0.934-2.035, p=0.311; recessive model: OR 1.026, 95%CI 0.831-1.268 p=0.429.). In summary, our finding suggests that DR4 polymorphism C626G and A683 rather than A1322G are associated with cancer risk in Caucasians.

• Tuberculosis and Respiratory Diseases
• /
• v.64 no.4
• /
• pp.278-284
• /
• 2008

Background: TRAIL is a promising anticancer agent which induces selective tumor cell death due to a unique receptor system that includes death receptors and decoy receptors. DR5 TRAIL receptor is an originally identified p53-regulated death receptor gene that was induced, by doxorubicine, only in cells with a wild-type p53 status. We investigated that focused on the correlation between the DR5 and p53 expressions in non-small cell lung cancer (NSCLC). Methods: Immunohistochemical analysis, with using avidin-biotinylated horseradish peroxidase complex, was carried out in 89 surgically resected NSCLC formalin-fixed paraffin-embedded tissue sections. As primary antibodies, we used anti-DR5 polyclonal antibody and anti-p53 monoclonal antibody. A negative control was processed with each slide. The positive tumor cells were quantified twice and these values were expressed as percentage of the total number of tumor cells, and the intensity of immunostaining was expressed. The analysis of the DR5 expression was done separately in tumor area and in a nearby region of normal tissue. Results: The DR5 expression was high in the bronchial epithelium (89% of cases) but this was almost absent in type I & II pneumocytes, lymphocytes and smooth muscle cells. High DR5 expression rate in tumor was seen in 28% (15/53) of squamous cell carcinomas, in 47% (15/32) of adenocarcinomas and, in 50% (2/4) of large cell carcinomas. The DR5 expression did not show any statistical significance relationship with the T stage, N stage, or survival. However, the DR5 expression showed significant inverse correlation with the p53 expression. (p< 0.01). Conclusion: We demonstrated that the DR5 expression in NSCLC via immunohistochemical analysis is relatively tumor-specific except for that in the normal bronchial epithelium and it is significantly dependent on the p53 status. This might be in vivo evidence for the significance of the DR5 gene as a p53 downstream gene.

• Clinical and Experimental Pediatrics
• /
• v.53 no.4
• /
• pp.525-531
• /
• 2010

Purpose : Expression levels of tumor necrosis factor (TNF)-${\alpha}$ expression on the mucosa of the small intestine is increased in patients with villous atrophy in food protein-induced enterocolitis syndrome (FPIES). TNF-${\alpha}$ has been reported to induce apoptotic cell death in the epithelial cells. We studied the TNF family and TNF-receptor family apoptosis on the duodenal mucosa to investigate their roles in the pathogenesis of FPIES. Methods : Fifteen infants diagnosed as having FPIES using standard oral challenge test and 5 controls were included. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was performed to identify the apoptotic cell death bodies. Immunohistochemical staining of TNF-${\alpha}$, Fas ligand (FasL) for TNF family and TNF-related apoptosis-including ligand (TRAIL) receptor 1 (DR4), TRAIL receptor 2 (DR5), and Fas for TNF-receptor family were performed to determine the apoptotic mechanisms. Results : $TUNEL^+$ was significantly more highly expressed in the duodenal mucosa of FPIES patients than in controls ($P$-0.043). TNF-${\alpha}$ ($P$=0.0001) and DR4 ($P$=0.003) were significantly more highly expressed in FPIES patients than in controls. Expression levels of FasL, Fas, and DR5 were low in both groups and were not significantly different between the 2 groups. Conclusion : These results suggest that FPIES pathogenesis is induced by apoptosis, and that TNF-${\alpha}$ expression and DR4 pathway may have an important role in apoptosis.

• Biomolecules & Therapeutics
• /
• v.20 no.6
• /
• pp.538-543
• /
• 2012

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-${\kappa}B$ (NF-${\kappa}B$) inactivation and $G_2$/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.

• Korean Journal of Microbiology
• /
• v.54 no.4
• /
• pp.330-342
• /
• 2018

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.