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Gregory J Gores - One of the best experts on this subject based on the ideXlab platform.
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free fatty acids sensitise hepatocytes to trail mediated cytotoxicity
Gut, 2007Co-Authors: Harmeet Malhi, Steven F Bronk, Fernando J Barreyro, Hajime Isomoto, Gregory J GoresAbstract:Background: Elevated circulating free fatty acids (FFA) contribute to the development of hepatic steatosis and promote hepatocyte apoptosis by incompletely defined mechanisms. Although the Death ligand TRAIL has been implicated in a variety of pathological liver diseases, the role of TRAIL in mediating apoptosis of FFA induced steatotic hepatocytes is unknown. Aim: We examined TRAIL cytotoxicity in an in vitro model of hepatocyte steatosis induced by FFA. Methods: Hepatocytes (Huh 7 cells, HepG2 cells, and primary rat hepatocytes) were rendered steatotic by incubation with oleic acid. Apoptosis was assessed morphologically and biochemically by caspase activity. TRAIL Receptor regulation was examined using immunoblot analysis and siRNA for targeted knockdown. c-jun N-terminal kinase (JNK) inhibition was attained with SP600125. Results: Oleic acid sensitised the cells to TRAIL but not TNF-α cytotoxicity. FFA sensitisation to TRAIL occurred at much lower concentrations than required for FFA mediated sensitisation to Fas, or FFA induced lipoapoptosis. Oleic acid treatment led to upregulation of the cognate TRAIL Receptor Death Receptor 5 (DR5) but not Death Receptor 4 (DR4). The upregulation of DR5 was JNK dependent. siRNA targeted knockdown of either DR5 or DR4 demonstrated that DR5 was responsible for FFA sensitisation to TRAIL killing. DR5 expression was enhanced in steatotic human liver samples. Conclusion: Our results suggest that FFA induced hepatocyte steatosis sensitises to TRAIL by a DR5 mediated JNK dependent mechanism.
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mcl 1 mediates tumor necrosis factor related apoptosis inducing ligand resistance in human cholangiocarcinoma cells
Cancer Research, 2004Co-Authors: Makiko Taniai, Annette Grambihler, Hajime Higuchi, Nate Werneburg, Steve F Bronk, Daniel J Farrugia, Scott H Kaufmann, Gregory J GoresAbstract:Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL Receptor 1/Death Receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy Receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x L , and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x L , and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell Death but also identify two strategies for circumventing this resistance.
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hepatitis b virus enhances tumor necrosis factor related apoptosis inducing ligand trail cytotoxicity by increasing trail r1 Death Receptor 4 expression
Journal of Hepatology, 2003Co-Authors: Harry L A Janssen, Hajime Higuchi, Ahmad S Abdulkarim, Gregory J GoresAbstract:Background/Aims: Apoptosis by Death Receptors, such as Fas and tumor necrosis factor (TNF)-alpha Receptor-1, play a significant role in the pathogenesis of hepatitis B virus (HBV)-infections. Although liver also expresses Death Receptors for TNF-related apoptosis-inducing ligand (TRAIL), information is lacking regarding the effects of HBV on apoptosis by TRAIL. Thus, the aims of this study were to examine the effects of HBV replication on TRAIL cytotoxicity. Methods: Hep G2 and Hep G2.215 cells, the latter which is stably transfected with HBV, were employed for these studies. Results: TRAIL-mediated cell killing was concentration-dependent and greater in Hep G2.2.15 cells at all doses as compared to the parent cell line, Hep G2 cells. Cell Death by apoptosis was confirmed by demonstrating caspase activation and inhibition of cell killing by a caspase inhibitor, zVAD-fmk. TRAIL-R1/DR4 protein expression was enhanced in Hep G2.2.15 cells as compared to Hep G2 cells. Lamivudine treatment reduced TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression in Hep G2.2.15 cells. In Hep G2 cells transfected with the HBV-encoded X antigen (HBxAg), sensitivity to TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression were both increased. Conclusions: TRAIL-induced apoptosis is enhanced by the level of HBV replication in human hepatocytes, in part, by HBxAg-dependent upregulation of TRAIL-R1/DR4.
Yong-sung Kim - One of the best experts on this subject based on the ideXlab platform.
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ay4 an agonistic anti Death Receptor 4 mab induces apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Endocrine-related Cancer, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Chulho KimAbstract:Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 MAB, AY4, and demonstrated the antitumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with the reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. AY4 induced Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an antioxidant (N-acetyl cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the downregulation of the anti-apoptotic protein, Bcl-xL (BCL2L1). To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the overexpression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. By contrast, Bcl-xL knockdown using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells and rendered the cells sensitive to AY4-induced cell Death. The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy.
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abstract 2946 ay4 an agonistic anti Death Receptor 4 monoclonal antibody induces Death Receptor 4 mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Cancer Research, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Chulho KimAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 monoclonal antibody, AY4, and demonstrated the anti-tumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. Methods: The effects of AY4 in Thyroid cell lines (C-643, U-HTH7, HTH83 and SW1736) was studied on apoptosis using MTT assy, Annexin V assay and Western blot analysis and was examed on the production of reactive oxygen species with DCFDA staining. HTH83 and C-643 cells were transfected with Bcl-xL siRNA using Lipofectamine 2000. Results: AY4 induced cell Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an anti-oxidant (N-acetyl-cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the down-regulation of the anti-apoptotic protein, Bcl-xL. To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the over-expression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. In contrast, Bcl-xL knock-down using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells, and rendered the cells sensitive to AY4-induced cell Death.. Conclusion: The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy. Keywords: Death Receptor 4, agonistic antibody, apoptosis, ROS, anaplastic thyroid carcinoma (ATC) Citation Format: Bok-Soon Lee, Hyun-Young Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Yong- Sung Kim, Chul-Ho Kim. AY4, an agonistic anti-Death Receptor 4 monoclonal antibody, induces Death Receptor 4-mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of Bcl-xL with reactive oxygen species generation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2013-2946
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an agonistic antibody to human Death Receptor 4 induces apoptotic cell Death in head and neck cancer cells through mitochondrial ros generation
Cancer Letters, 2012Co-Authors: Boksoon Lee, Eunsil Sung, Yong-sung Kim, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Young Chang Lim, Chulho KimAbstract:The proapoptotic Death Receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell Death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell Death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell Death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.
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abstract 4772 a novel agonist antibody to human Death Receptor 4 ay4 induces apoptotic cell Death in head and neck cancer through ros generation in vitroandin vivo
Cancer Research, 2011Co-Authors: Chulho Kim, Eunsil Sung, Yong-sung Kim, Boksoon Lee, Sung Un Kang, Hye Sook Hwang, Yoon Woo Koh, Hyun Jun Hong, Mi Ran Ryu, Jae Wook KimAbstract:Background : The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) Receptors Death Receptor (DR) 4 and DR5 are attractive targets to develop the Receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell Death-inducing activity. Here, we describe the characterization of a novel agonistic mAb (AY4) against human DR4 and apoptosis through reactive oxygen species (ROS) production in head and neck cancer model. Methods : The effects of AY4 in head and neck cell lines (KB, SNU899, HN9, FaDu) was studied on apoptosis using MTT assy, TUNEL, Annexin V assay and western blotting and was examed on the production of reactive oxygen species with DCFDA staining, intracellular signaling and animal model. In addition, we analyzed the anticancer effect of AY4 in nude mice xenograft model. In addition, drug toxicity (embryotoxicity and neurotoxicity) of AY4 were investigated in vivo zebrafish model. Results: The DR4 was expressed in head and neck cancer cell lines by Flow cytometry (FACS) and western blotting. AY4 inhibited cell growth in KB and HN9 excluding SNU899 and FaDu and caused cell Death via caspase-dependent apoptosis. The apoptotic cell Death by AY4 decreased by N-acetylcysteine (NAC), an antioxidant, suggesting ROS generation involved in the cell Death. Moreover, NAC and AY4 cotreatment augmented the reduced expression of antiapoptotic molecules, Bcl-X L and XIAP. In vivo administration of AY4 significantly inhibited tumor growth of head and neck cancer preestablished in athymic nude mice. In addition, in a zebrafish model used for toxicity testing, a considerable dose of AY4 did not result in embryotoxicity or neurotoxicity. Conclusion : Our results demonstrates the effect of AY4 on apoptosis and tumor inhibition via ROS production and provide potential use of AY4 as an anticancer therapeutic agent in head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2011-4772
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engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Changhan Lee, Kyungjin Park, Eunsil Sung, Aeyung Kim, Jida Choi, Jeongsun Kim, Myunghee Kwon, Soohyun Kim, Yong-sung KimAbstract:Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human Death Receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell Death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
Chulho Kim - One of the best experts on this subject based on the ideXlab platform.
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ay4 an agonistic anti Death Receptor 4 mab induces apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Endocrine-related Cancer, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Chulho KimAbstract:Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 MAB, AY4, and demonstrated the antitumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with the reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. AY4 induced Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an antioxidant (N-acetyl cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the downregulation of the anti-apoptotic protein, Bcl-xL (BCL2L1). To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the overexpression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. By contrast, Bcl-xL knockdown using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells and rendered the cells sensitive to AY4-induced cell Death. The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy.
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abstract 2946 ay4 an agonistic anti Death Receptor 4 monoclonal antibody induces Death Receptor 4 mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Cancer Research, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Chulho KimAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 monoclonal antibody, AY4, and demonstrated the anti-tumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. Methods: The effects of AY4 in Thyroid cell lines (C-643, U-HTH7, HTH83 and SW1736) was studied on apoptosis using MTT assy, Annexin V assay and Western blot analysis and was examed on the production of reactive oxygen species with DCFDA staining. HTH83 and C-643 cells were transfected with Bcl-xL siRNA using Lipofectamine 2000. Results: AY4 induced cell Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an anti-oxidant (N-acetyl-cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the down-regulation of the anti-apoptotic protein, Bcl-xL. To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the over-expression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. In contrast, Bcl-xL knock-down using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells, and rendered the cells sensitive to AY4-induced cell Death.. Conclusion: The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy. Keywords: Death Receptor 4, agonistic antibody, apoptosis, ROS, anaplastic thyroid carcinoma (ATC) Citation Format: Bok-Soon Lee, Hyun-Young Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Yong- Sung Kim, Chul-Ho Kim. AY4, an agonistic anti-Death Receptor 4 monoclonal antibody, induces Death Receptor 4-mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of Bcl-xL with reactive oxygen species generation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2013-2946
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an agonistic antibody to human Death Receptor 4 induces apoptotic cell Death in head and neck cancer cells through mitochondrial ros generation
Cancer Letters, 2012Co-Authors: Boksoon Lee, Eunsil Sung, Yong-sung Kim, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Young Chang Lim, Chulho KimAbstract:The proapoptotic Death Receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell Death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell Death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell Death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.
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abstract 4772 a novel agonist antibody to human Death Receptor 4 ay4 induces apoptotic cell Death in head and neck cancer through ros generation in vitroandin vivo
Cancer Research, 2011Co-Authors: Chulho Kim, Eunsil Sung, Yong-sung Kim, Boksoon Lee, Sung Un Kang, Hye Sook Hwang, Yoon Woo Koh, Hyun Jun Hong, Mi Ran Ryu, Jae Wook KimAbstract:Background : The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) Receptors Death Receptor (DR) 4 and DR5 are attractive targets to develop the Receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell Death-inducing activity. Here, we describe the characterization of a novel agonistic mAb (AY4) against human DR4 and apoptosis through reactive oxygen species (ROS) production in head and neck cancer model. Methods : The effects of AY4 in head and neck cell lines (KB, SNU899, HN9, FaDu) was studied on apoptosis using MTT assy, TUNEL, Annexin V assay and western blotting and was examed on the production of reactive oxygen species with DCFDA staining, intracellular signaling and animal model. In addition, we analyzed the anticancer effect of AY4 in nude mice xenograft model. In addition, drug toxicity (embryotoxicity and neurotoxicity) of AY4 were investigated in vivo zebrafish model. Results: The DR4 was expressed in head and neck cancer cell lines by Flow cytometry (FACS) and western blotting. AY4 inhibited cell growth in KB and HN9 excluding SNU899 and FaDu and caused cell Death via caspase-dependent apoptosis. The apoptotic cell Death by AY4 decreased by N-acetylcysteine (NAC), an antioxidant, suggesting ROS generation involved in the cell Death. Moreover, NAC and AY4 cotreatment augmented the reduced expression of antiapoptotic molecules, Bcl-X L and XIAP. In vivo administration of AY4 significantly inhibited tumor growth of head and neck cancer preestablished in athymic nude mice. In addition, in a zebrafish model used for toxicity testing, a considerable dose of AY4 did not result in embryotoxicity or neurotoxicity. Conclusion : Our results demonstrates the effect of AY4 on apoptosis and tumor inhibition via ROS production and provide potential use of AY4 as an anticancer therapeutic agent in head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2011-4772
Boksoon Lee - One of the best experts on this subject based on the ideXlab platform.
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ay4 an agonistic anti Death Receptor 4 mab induces apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Endocrine-related Cancer, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Chulho KimAbstract:Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 MAB, AY4, and demonstrated the antitumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with the reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. AY4 induced Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an antioxidant (N-acetyl cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the downregulation of the anti-apoptotic protein, Bcl-xL (BCL2L1). To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the overexpression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. By contrast, Bcl-xL knockdown using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells and rendered the cells sensitive to AY4-induced cell Death. The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy.
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abstract 2946 ay4 an agonistic anti Death Receptor 4 monoclonal antibody induces Death Receptor 4 mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of bcl xl with reactive oxygen species generation
Cancer Research, 2013Co-Authors: Boksoon Lee, Yong-sung Kim, Hyunyoung Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Chulho KimAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-Death Receptor 4 monoclonal antibody, AY4, and demonstrated the anti-tumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. Methods: The effects of AY4 in Thyroid cell lines (C-643, U-HTH7, HTH83 and SW1736) was studied on apoptosis using MTT assy, Annexin V assay and Western blot analysis and was examed on the production of reactive oxygen species with DCFDA staining. HTH83 and C-643 cells were transfected with Bcl-xL siRNA using Lipofectamine 2000. Results: AY4 induced cell Death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an anti-oxidant (N-acetyl-cysteine, NAC) followed by AY4 treatment. The cell Death was blocked by NAC. AY4-induced cell Death was accompanied by the down-regulation of the anti-apoptotic protein, Bcl-xL. To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the over-expression of Bcl-xL appeared to decrease AY4-mediated cell Death by blocking ROS generation in U-HTH 7 cells. In contrast, Bcl-xL knock-down using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells, and rendered the cells sensitive to AY4-induced cell Death.. Conclusion: The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy. Keywords: Death Receptor 4, agonistic antibody, apoptosis, ROS, anaplastic thyroid carcinoma (ATC) Citation Format: Bok-Soon Lee, Hyun-Young Cha, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Sam Kang, Jae Won Chang, Yong- Sung Kim, Chul-Ho Kim. AY4, an agonistic anti-Death Receptor 4 monoclonal antibody, induces Death Receptor 4-mediated apoptotic cell Death in anaplastic thyroid cancer cells via downregulation of Bcl-xL with reactive oxygen species generation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2013-2946
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an agonistic antibody to human Death Receptor 4 induces apoptotic cell Death in head and neck cancer cells through mitochondrial ros generation
Cancer Letters, 2012Co-Authors: Boksoon Lee, Eunsil Sung, Yong-sung Kim, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Young Chang Lim, Chulho KimAbstract:The proapoptotic Death Receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell Death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell Death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell Death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.
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abstract 4772 a novel agonist antibody to human Death Receptor 4 ay4 induces apoptotic cell Death in head and neck cancer through ros generation in vitroandin vivo
Cancer Research, 2011Co-Authors: Chulho Kim, Eunsil Sung, Yong-sung Kim, Boksoon Lee, Sung Un Kang, Hye Sook Hwang, Yoon Woo Koh, Hyun Jun Hong, Mi Ran Ryu, Jae Wook KimAbstract:Background : The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) Receptors Death Receptor (DR) 4 and DR5 are attractive targets to develop the Receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell Death-inducing activity. Here, we describe the characterization of a novel agonistic mAb (AY4) against human DR4 and apoptosis through reactive oxygen species (ROS) production in head and neck cancer model. Methods : The effects of AY4 in head and neck cell lines (KB, SNU899, HN9, FaDu) was studied on apoptosis using MTT assy, TUNEL, Annexin V assay and western blotting and was examed on the production of reactive oxygen species with DCFDA staining, intracellular signaling and animal model. In addition, we analyzed the anticancer effect of AY4 in nude mice xenograft model. In addition, drug toxicity (embryotoxicity and neurotoxicity) of AY4 were investigated in vivo zebrafish model. Results: The DR4 was expressed in head and neck cancer cell lines by Flow cytometry (FACS) and western blotting. AY4 inhibited cell growth in KB and HN9 excluding SNU899 and FaDu and caused cell Death via caspase-dependent apoptosis. The apoptotic cell Death by AY4 decreased by N-acetylcysteine (NAC), an antioxidant, suggesting ROS generation involved in the cell Death. Moreover, NAC and AY4 cotreatment augmented the reduced expression of antiapoptotic molecules, Bcl-X L and XIAP. In vivo administration of AY4 significantly inhibited tumor growth of head and neck cancer preestablished in athymic nude mice. In addition, in a zebrafish model used for toxicity testing, a considerable dose of AY4 did not result in embryotoxicity or neurotoxicity. Conclusion : Our results demonstrates the effect of AY4 on apoptosis and tumor inhibition via ROS production and provide potential use of AY4 as an anticancer therapeutic agent in head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2011-4772
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an agonistic antibody to human Death Receptor 4 induces apoptotic cell Death in head and neck cancer cells through mitochondrial ros generation
Cancer Letters, 2012Co-Authors: Boksoon Lee, Eunsil Sung, Yong-sung Kim, Yoo Seob Shin, Sung Un Kang, Hye Sook Hwang, Young Chang Lim, Chulho KimAbstract:The proapoptotic Death Receptor 4 (DR4), along with DR5, is currently regarded as a promising target for development of agonistic anti-cancer agents due to its tumor-selective apoptosis-inducing ability with no significant cytotoxicity to normal cells. In this study, we examine susceptibility of various head and neck cancer (HNC) cells and mechanism of cell Death to an anti-DR4 agonistic monoclonal antibody (mAb), AY4. AY4 as a single agent induced caspase-dependent apoptotic cell Death of KB and HN9, but not in SNU899 and FaDu cell lines. AY4 treatment resulted in accumulation of intracellular reactive oxygen species (ROS) generated from mitochondria in AY4-sensitive cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) resulted in protection of AY4-sensitive cells against AY4-induced apoptosis. ROS generation induced by AY4 treatment triggered down-regulation of anti-apoptotic molecules of Bcl-xL and X-linked inhibitor of apoptosis (XIAP) without affecting the expression levels of DR4, Mcl-1, and survivin. AY4 also inhibited growth of pre-established HN9 tumors in a nude mouse xenograft model and did not show noticeable cytotoxicity in a zebrafish model. Our results provide further insight into the mechanism of DR4-mediated cell Death and potential use of AY4 mAb as an anti-cancer therapeutic agent in AY4-sensitive HNC types.
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abstract 4772 a novel agonist antibody to human Death Receptor 4 ay4 induces apoptotic cell Death in head and neck cancer through ros generation in vitroandin vivo
Cancer Research, 2011Co-Authors: Chulho Kim, Eunsil Sung, Yong-sung Kim, Boksoon Lee, Sung Un Kang, Hye Sook Hwang, Yoon Woo Koh, Hyun Jun Hong, Mi Ran Ryu, Jae Wook KimAbstract:Background : The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) Receptors Death Receptor (DR) 4 and DR5 are attractive targets to develop the Receptorspecific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell Death-inducing activity. Here, we describe the characterization of a novel agonistic mAb (AY4) against human DR4 and apoptosis through reactive oxygen species (ROS) production in head and neck cancer model. Methods : The effects of AY4 in head and neck cell lines (KB, SNU899, HN9, FaDu) was studied on apoptosis using MTT assy, TUNEL, Annexin V assay and western blotting and was examed on the production of reactive oxygen species with DCFDA staining, intracellular signaling and animal model. In addition, we analyzed the anticancer effect of AY4 in nude mice xenograft model. In addition, drug toxicity (embryotoxicity and neurotoxicity) of AY4 were investigated in vivo zebrafish model. Results: The DR4 was expressed in head and neck cancer cell lines by Flow cytometry (FACS) and western blotting. AY4 inhibited cell growth in KB and HN9 excluding SNU899 and FaDu and caused cell Death via caspase-dependent apoptosis. The apoptotic cell Death by AY4 decreased by N-acetylcysteine (NAC), an antioxidant, suggesting ROS generation involved in the cell Death. Moreover, NAC and AY4 cotreatment augmented the reduced expression of antiapoptotic molecules, Bcl-X L and XIAP. In vivo administration of AY4 significantly inhibited tumor growth of head and neck cancer preestablished in athymic nude mice. In addition, in a zebrafish model used for toxicity testing, a considerable dose of AY4 did not result in embryotoxicity or neurotoxicity. Conclusion : Our results demonstrates the effect of AY4 on apoptosis and tumor inhibition via ROS production and provide potential use of AY4 as an anticancer therapeutic agent in head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2011-4772
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histone deacetylase inhibitors synergistically potentiate Death Receptor 4 mediated apoptotic cell Death of human t cell acute lymphoblastic leukemia cells
Apoptosis, 2010Co-Authors: Eunsil Sung, Joon Seong Park, Junho Chung, Myunghee KwonAbstract:Cell-Death signaling through the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) Receptors, Death Receptor 4 (DR4) and DR5, has shown tumor-selective apoptotic activity. Here, we examine susceptibility of various leukemia cell lines (HL-60, U937, K562, CCRF-CEM, CEM-CM3, and THP-1) to an anti-DR4 agonistic monoclonal antibody (mAb), AY4, in comparison with TRAIL. While most of the leukemia cell lines were intrinsically resistant to AY4 or TRAIL alone, the two T-cell acute lymphoblastic leukemia (T-ALL) lines, CEM-CM3 and CCRF-CEM cells, underwent synergistic caspase-dependent apoptotic cell Death by combination of AY4 or TRAIL with a histone deacetylase inhibitor (HDACI), either suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). All of the combined treatments synergistically downregulated several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-XL, XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the Receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. Our results demonstrate that the HDACIs have synergistic effects on DR4-specific mAb AY4-mediated cell Death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.
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engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Changhan Lee, Kyungjin Park, Eunsil Sung, Aeyung Kim, Jida Choi, Jeongsun Kim, Myunghee Kwon, Soohyun Kim, Yong-sung KimAbstract:Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human Death Receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell Death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
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humanization of an agonistic anti Death Receptor 4 single chain variable fragment antibody and avidity mediated enhancement of its cell Death inducing activity
Molecular Immunology, 2010Co-Authors: Seunghyun Lee, Eunsil Sung, Dongwoon Park, Hyeran Park, Jinkyoo Kim, Yong-sung KimAbstract:Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule Death Receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) Receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell Death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell Death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell Death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family Receptors, avidity-mediated oligomerization of DR4 augments the Receptor-mediated apoptotic cell Death by promoting intracellular cell Death signaling.
