Publications by authors named "Sukmook Lee"

37 Publications

Cell Surface GRP94 as a Novel Emerging Therapeutic Target for Monoclonal Antibody Cancer Therapy.

Cells 2021 Mar 17;10(3). Epub 2021 Mar 17.

Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 02707, Korea.

Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family. In physiological conditions, it plays a vital role in regulating biological functions, including chaperoning cellular proteins in the ER lumen, maintaining calcium homeostasis, and modulating immune system function. Recently, several reports have shown the functional role and clinical relevance of GRP94 overexpression in the progression and metastasis of several cancers. Therefore, the current review highlights GRP94's physiological and pathophysiological roles in normal and cancer cells. Additionally, the unmet medical needs of small chemical inhibitors and the current development status of monoclonal antibodies specifically targeting GRP94 will be discussed to emphasize the importance of cell surface GRP94 as an emerging therapeutic target in monoclonal antibody therapy for cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells10030670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002708PMC
March 2021

Recent Advances in Monoclonal Antibody Therapy for Colorectal Cancers.

Biomedicines 2021 Jan 5;9(1). Epub 2021 Jan 5.

Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 02707, Korea.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. Recent advances in recombinant DNA technology have led to the development of numerous therapeutic antibodies as major sources of blockbuster drugs for CRC therapy. Simultaneously, increasing numbers of therapeutic targets in CRC have been identified. In this review, we first highlight the physiological and pathophysiological roles and signaling mechanisms of currently known and emerging therapeutic targets, including growth factors and their receptors as well as immune checkpoint proteins, in CRC. Additionally, we discuss the current status of monoclonal antibodies in clinical development and approved by US Food and Drug Administration for CRC therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines9010039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824816PMC
January 2021

TSPAN8 as a Novel Emerging Therapeutic Target in Cancer for Monoclonal Antibody Therapy.

Biomolecules 2020 03 3;10(3). Epub 2020 Mar 3.

Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 02707, Korea.

Tetraspanin 8 (TSPAN8) is a member of the tetraspanin superfamily that forms TSPAN8-mediated protein complexes by interacting with themselves and other various cellular signaling molecules. These protein complexes help build tetraspanin-enriched microdomains (TEMs) that efficiently mediate intracellular signal transduction. In physiological conditions, TSPAN8 plays a vital role in the regulation of biological functions, including leukocyte trafficking, angiogenesis and wound repair. Recently, reports have increasingly shown the functional role and clinical relevance of TSPAN8 overexpression in the progression and metastasis of several cancers. In this review, we will highlight the physiological and pathophysiological roles of TSPAN8 in normal and cancer cells. Additionally, we will cover the current status of monoclonal antibodies specifically targeting TSPAN8 and the importance of TSPAN8 as an emerging therapeutic target in cancers for monoclonal antibody therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10030388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175299PMC
March 2020

Antibody-Based Targeting of Cell Surface GRP94 Specifically Inhibits Cetuximab-Resistant Colorectal Cancer Growth.

Biomolecules 2019 11 1;9(11). Epub 2019 Nov 1.

Biopharmaceutical Chemistry Major, School of Applied Chemistry, Kookmin University, Seoul 02707, Korea.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. Cetuximab, a human/mouse chimeric monoclonal antibody, is effective in a limited number of CRC patients because of cetuximab resistance. This study aimed to identify novel therapeutic targets in cetuximab-resistant CRC in order to improve clinical outcomes. Through phage display technology, we isolated a fully human antibody strongly binding to the cetuximab-resistant HCT116 cell surface and identified the target antigen as glucose-regulated protein 94 (GRP94) using proteomic analysis. Short interfering RNA-mediated GRP94 knockdown showed that GRP94 plays a key role in HCT116 cell growth. In vitro functional studies revealed that the GRP94-blocking antibody we developed strongly inhibits the growth of various cetuximab-resistant CRC cell lines. We also demonstrated that GRP94 immunoglobulin G monotherapy significantly reduces HCT116 cell growth more potently compared to cetuximab, without severe toxicity in vivo. Therefore, cell surface GRP94 might be a potential novel therapeutic target in cetuximab-resistant CRC, and antibody-based targeting of GRP94 might be an effective strategy to suppress GRP94-expressing cetuximab-resistant CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom9110681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920916PMC
November 2019

Humanized Anti-hepatocyte Growth Factor Monoclonal Antibody (YYB-101) Inhibits Ovarian Cancer Progression.

Front Oncol 2019 9;9:571. Epub 2019 Jul 9.

Research Institute, National Cancer Center, Goyang-si, South Korea.

Current chemotherapy regimens have certain limitations in improving the survival rates of patients with advanced ovarian cancer. Hepatocyte growth factor (HGF) is important in ovarian cancer cell migration and invasion. This study assessed the effects of YYB-101, a humanized monoclonal anti-HGF antibody, on the growth and metastasis of ovarian cancer cells. YYB-101 suppressed the phosphorylation of the HGF receptor c-MET and inhibited the migration and invasion of SKOV3 and A2780 ovarian cancer cells. Moreover, the combination of YYB-101 and paclitaxel synergistically inhibited tumor growth in an ovarian cancer mouse xenograft model and significantly increased the overall survival (OS) rate compared with either paclitaxel or YYB-101 alone. Taken together, these findings suggest that YYB-101 has therapeutic potential in ovarian cancer when combined with conventional chemotherapy agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2019.00571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631954PMC
July 2019

Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer.

Int J Mol Sci 2018 Apr 2;19(4). Epub 2018 Apr 2.

Research Center, Scripps Korea Antibody Institute, Chuncheon 200-701, Korea.

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that triggers the expression of inflammatory molecules, including other cytokines and cell adhesion molecules. TNFα induces the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 was originally identified as a cell adhesion molecule that helps regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. Recent evidence suggests that VCAM-1 is closely associated with the progression of various immunological disorders, including rheumatoid arthritis, asthma, transplant rejection, and cancer. This review covers the role and relevance of VCAM-1 in inflammation, and also highlights the emerging potential of VCAM-1 as a novel therapeutic target in immunological disorders and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19041057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979609PMC
April 2018

Inhibition of VEGF-dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a.

Mol Oncol 2018 03 26;12(3):356-372. Epub 2018 Jan 26.

Scripps Korea Antibody Institute, Chuncheon, South Korea.

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1878-0261.12169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830631PMC
March 2018

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

Adv Healthc Mater 2018 02 1;7(4). Epub 2017 Dec 1.

Interdisciplinary Program in Nano-Science and Technology, Seoul National University, Seoul, 08826, Republic of Korea.

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201700870DOI Listing
February 2018

CLEC14a-HSP70-1A interaction regulates HSP70-1A-induced angiogenesis.

Sci Rep 2017 09 6;7(1):10666. Epub 2017 Sep 6.

Research Center, Scripps Korea Antibody Institute, Chuncheon, South Korea.

CLEC14a (C-type lectin domain family 14 member) is a tumor endothelial cell marker protein that is known to play an important role in tumor angiogenesis, but the basic molecular mechanisms underlying this function have not yet been clearly elucidated. In this study, using various proteomic tools, we isolated a 70-kDa protein that interacts with the C-type lectin-like domain of CLEC14a (CLEC14a-CTLD) and identified it as heat shock protein 70-1A (HSP70-1A). Co-immunoprecipitation showed that HSP70-1A and CLEC14a interact on endothelial cells. In vitro binding analyses identified that HSP70-1A specifically associates with the region between amino acids 43 and 69 of CLEC14a-CTLD. Competitive blocking experiments indicated that this interacting region of CLEC14a-CTLD significantly inhibits HSP70-1A-induced extracellular signal-regulated kinase (ERK) phosphorylation and endothelial tube formation by directly inhibiting CLEC14a-CTLD-mediated endothelial cell-cell contacts. Our data suggest that the specific interaction of HSP70-1A with CLEC14a may play a critical role in HSP70-1A-induced angiogenesis and that the HSP70-1A-interacting region of CLEC14a-CTLD may be a useful tool for inhibiting HSP70-1A-induced angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-11118-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587741PMC
September 2017

A Review of Anti-Angiogenic Targets for Monoclonal Antibody Cancer Therapy.

Int J Mol Sci 2017 Aug 17;18(8). Epub 2017 Aug 17.

Research Center, Scripps Korea Antibody Institute, Chuncheon 200-701, Korea.

Tumor angiogenesis is a key event that governs tumor progression and metastasis. It is controlled by the complicated and coordinated actions of pro-angiogenic factors and their receptors that become upregulated during tumorigenesis. Over the past several decades, vascular endothelial growth factor (VEGF) signaling has been identified as a central axis in tumor angiogenesis. The remarkable advent of recombinant antibody technology has led to the development of bevacizumab, a humanized antibody that targets VEGF and is a leading clinical therapy to suppress tumor angiogenesis. However, despite the clinical efficacy of bevacizumab, its significant side effects and drug resistance have raised concerns necessitating the identification of novel drug targets and development of novel therapeutics to combat tumor angiogenesis. This review will highlight the role and relevance of VEGF and other potential therapeutic targets and their receptors in angiogenesis. Simultaneously, we will also cover the current status of monoclonal antibodies being developed to target these candidates for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms18081786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578174PMC
August 2017

A Human Antibody That Binds to the Sixth Ig-Like Domain of VCAM-1 Blocks Lung Cancer Cell Migration In Vitro.

Int J Mol Sci 2017 Mar 6;18(3). Epub 2017 Mar 6.

Research Center, Scripps Korea Antibody Institute, Chuncheon 24341, Korea.

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung tissue, and high VCAM-1 expression correlated with poor survival in lung cancer patients. VCAM-1 knockdown reduced migration of A549 human lung cancer cells into Matrigel, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1 mediated A549 cell migration into Matrigel. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed the migration of A549 and NCI-H1299 lung cancer cell lines into Matrigel. Taken together, these results suggest that VCAM-1-D6 is a key domain for regulating VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms18030566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372582PMC
March 2017

Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis.

Exp Mol Med 2017 02 17;49(2):e294. Epub 2017 Feb 17.

Laboratory of Molecular Cancer Therapeutics, Research Center, Scripps Korea Antibody Institute, Chuncheon, Republic of Korea.

Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell-cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emm.2016.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336558PMC
February 2017

Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons.

Sci Rep 2017 01 31;7:41620. Epub 2017 Jan 31.

Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul, 02447, South Korea.

The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep41620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282494PMC
January 2017

Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

J Immunol Methods 2016 09 8;436:22-8. Epub 2016 Jun 8.

Laboratory of Molecular Cancer Therapeutics, Scripps Korea Antibody Institute, Hyoja-2-dong, Chuncheon-si, Gangwon-do 200-701, South Korea. Electronic address:

It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2016.06.001DOI Listing
September 2016

Heat shock protein 70-1A is a novel angiogenic regulator.

Biochem Biophys Res Commun 2016 Jan 30;469(2):222-8. Epub 2015 Nov 30.

Scripps Korea Antibody Institute, Hyoja-2-dong, Chuncheon-si, Gangwon-do, 200-701, South Korea. Electronic address:

Heat shock protein 70-1A (HSP70-1A) is a stress-inducible protein that provides an essential intracellular molecular chaperone function; however, the mechanism of HSP70-1A in angiogenesis has not been clarified. Herein, HSP70-1A gene silencing implicated this protein in angiogenesis. Additionally, recombinant human HSP70-1A (rhHSP70-1A) was able to stimulate human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and microvessel formation in vivo similarly to recombinant human vascular endothelial growth factor (rhVEGF). Furthermore, rhHSP70-1A was tightly bound to the surface of HUVECs and participated in extracellular signal-related kinase (ERK)-dependent angiogenesis. Together, these results implicate HSP70-1A as a novel angiogenic regulator.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.11.125DOI Listing
January 2016

Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

Biochem Biophys Res Commun 2015 Dec 10;468(4):774-80. Epub 2015 Nov 10.

Laboratory of Molecular Cancer Therapeutics, Scripps Korea Antibody Institute, Chuncheon, South Korea. Electronic address:

Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.11.031DOI Listing
December 2015

An antibody to the sixth Ig-like domain of VCAM-1 inhibits leukocyte transendothelial migration without affecting adhesion.

J Immunol 2012 Nov 1;189(9):4592-601. Epub 2012 Oct 1.

Cancer Research Institute, Xenotransplantation Research Center, College of Medicine, Seoul National University, Seoul 110-799, Korea.

VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511-599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α-stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1-mediated inflammation and could be a useful tool in treating inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1103803DOI Listing
November 2012

Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion.

PLoS One 2012 13;7(4):e35100. Epub 2012 Apr 13.

Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Chongno-gu, Seoul, Republic of Korea.

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035100PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325980PMC
August 2012

Preparation and in vitro evaluation of anti-VCAM-1-Fab'-conjugated liposomes for the targeted delivery of the poorly water-soluble drug celecoxib.

J Microencapsul 2011 ;28(3):220-7

Department of Bioscience and Biotechnology, Sejong University, 98 Gunja-Dong, Gwangjin-gu, Seoul 143-747, Korea.

When an inflammatory stimulus is given, vascular endothelial cells express various cell adhesion molecules including the vascular cell adhesion molecule (VCAM)-1. In this study, the possibility of specifically delivering anti-inflammatory drugs to activated endothelial cells by utilizing VCAM-1 as a target receptor was explored by loading celecoxib, a selective cyclooxygenase-2 inhibitor, into liposomes coupled to the Fab' fragment against VCAM-1. Anti-VCAM-1-Fab'-conjugated liposomes were prepared by forming an amide linkage between amino groups of Fab' and the carboxylic group of glutaryl-N-phosphatidylethanolamine in liposomes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linker in the presence of sulpho-N-hydroxysuccinimide. The coupling of Fab' to phospholipids constituting liposomes was confirmed by SDS-PAGE analysis. Under our optimized conjugation conditions, 130.0 µg Fab' was coupled to 1 µmol liposomes. Immunoblotting analysis showed that VCAM-1 protein expression could be induced by incubating human umbilical vein endothelial cells (HUVEC) with TNF-α. Confocal laser microsopy analysis revealed that Fab' conjugation to liposomes selectively increased liposomal uptake in TNF-α-pre-stimulated (VCAM-1-expressed) HUVECs, but not in cells without VCAM-1 expression. The concentration of celecoxib loaded in Fab'-conjugated liposomes was 281.1 ± 29 µg/mL, suggesting that liposomal loading also helped to overcome the limitations in celecoxib administration caused by its poor water solubility. Celecoxib loaded in Fab'-conjugated liposomes inhibited prostaglandin E₂ (PGE₂) production induced by TNF-α-pre-stimulation more efficiently than when loaded in conventional liposomes. Therefore, Fab'-conjugated liposomes served as a drug delivery system with dual functions: targeted delivery and solubilizing capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/02652048.2011.552989DOI Listing
May 2011

Aurintricarboxylic acid inhibits the nuclear factor-κB-dependent expression of intercellular cell adhesion molecule-1 and endothelial cell selectin on activated human endothelial cells.

Blood Coagul Fibrinolysis 2011 Mar;22(2):132-9

Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.

Activation of the vascular endothelium and increased adhesion of circulating leukocytes to the activated endothelium are important events in inflammation and coagulation. Aurintricarboxylic acid (ATA), a triphenylmethyl dye compound, is known to inhibit platelet adhesion by interfering with the binding of von Willebrand factor to platelet glycoprotein Ib. However, the effect of ATA on the inflammatory response of endothelial cells has not yet been investigated. Here, we investigated the functional role and molecular mechanism of ATA on the activation of human endothelial cells. ATA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1), and endothelial cell selectin (E-selectin) was upregulated on human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-α or lipopolysaccharide (LPS). We also observed the inhibitory effect of ATA on LPS-induced mRNA expression of ICAM-1 and E-selectin. Furthermore, ATA inhibited the binding of leukocytes to activated HUVECs. ATA significantly inhibited the nuclear translocation of nuclear factor-κB (NF-κB) and degradation of IκB on activated HUVECs, suggesting that ATA inhibits NF-κB signaling. Finally, three NF-κB inhibitors effectively inhibited the expressions of ICAM-1 and E-selectin on activated endothelial cells. The present data suggest that ATA exerts beneficial effect in various inflammation conditions through inhibition of adhesion molecule expression in activated endothelial cells and the resulting inhibition of leukocytes tissue accumulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MBC.0b013e32834356b6DOI Listing
March 2011

Irreversible inactivation of glutathione peroxidase 1 and reversible inactivation of peroxiredoxin II by H2O2 in red blood cells.

Antioxid Redox Signal 2010 Jun;12(11):1235-46

Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.

Catalase, glutathione peroxidase1 (GPx1), and peroxiredoxin (Prx) II are the principal enzymes responsible for peroxide elimination in RBC. We have now evaluated the relative roles of these enzymes by studying inactivation of GPx1 and Prx II in human RBCs. Mass spectrometry revealed that treatment of GPx1 with H(2)O(2) converts the selenocysteine residue at its active site to dehydroalanine (DHA). We developed a blot method for detection of DHA-containing proteins, with which we observed that the amount of DHA-containing GPx1 increases with increasing RBC density, which is correlated with increasing RBC age. Given that the conversion of selenocysteine to DHA is irreversible, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime. Prx II is inactivated by occasional hyperoxidation of its catalytic cysteine to cysteine sulfinic acid during catalysis. We believe that the activity of sulfiredoxin in RBCs is sufficient to counteract the hyperoxidation of Prx II that occurs in the presence of the basal level of H(2)O(2) flux resulting from hemoglobin autoxidation. If the H(2)O(2) flux is increased above the basal level, however, the sulfinic Prx II begins to accumulate. In the presence of an increased H(2)O(2) flux, inhibition of catalase accelerated the accumulation of sulfinic Prx II, indicative of the protective role of catalase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ars.2009.2701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875961PMC
June 2010

Porcine aortic endothelial cell genes responsive to selected inflammatory stimulators.

J Vet Med Sci 2009 Nov;71(11):1499-508

Department of Biochemistry, Hanyang University & GenoCheck Co. Ltd, Ansan, Gyeonggi, South Korea.

Use of porcine tissues has been suggested as a promising solution for severe shortage of transplantable human organs. The immediate hurdle for xenotransplantation is acute immune/inflammatory vascular rejection of the transplant. Because endothelial cells play a key role in the initiation and the amplification of inflammation, alteration of gene expression in human endothelial cells, by various inflammatory stimulators has been studied extensively. However, transcriptional changes induced by human and other inflammatory stimulators in porcine endothelial cells have thus far not been studied. In this study, we treated porcine endothelial cells with human tumor necrosis factor (TNF)-alpha, porcine interferon (IFN)-gamma, H(2)O(2) and lypopolysaccharide (LPS) and profiled transcriptional change at 1 hr, 6 hr and 24 hr, using pig oligonucleotide 13K microarray. We found that mRNA species such as chemokine (C-X-C motif) ligand 6 (CXCL6) and Cathepsin S were significantly induced in porcine endothelial cells, as was previously reported with human endothelial cell. We also found that mRNA species including secreted frizzled-related protein 2 (SFRP2), radical S-adenosyl methionine domain containing 2 (RSAD2), structure specific recognition protein 1 (SSRP1) also were highly overexpressed in porcine endothelial cells. This result shows clues to understand underlying mechanisms of xenotransplantation rejection and the highly responsive porcine genes may serve as novel targets to be regulated for improving the function of grafted porcine donor organs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.001499DOI Listing
November 2009

Identification of alpha-Gal and non-Gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry.

Curr Eye Res 2009 Oct;34(10):877-95

Institute of Molecular Biology and Genetics and Institute of Bioengineering, Seoul National University, Seoul, Korea.

Purpose: To investigate the expression of alpha-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry.

Methods: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immunochemical staining was added to confirm the non-Gal antigen expression in pig corneal cells.

Results: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of alpha-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed.

Conclusions: As well as alpha-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the alpha-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/02713680903184243DOI Listing
October 2009

Collapsin response mediator protein-2 regulates neurite formation by modulating tubulin GTPase activity.

Cell Signal 2009 Dec 8;21(12):1818-26. Epub 2009 Aug 8.

Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Republic of Korea.

Collapsin response mediator protein-2 (CRMP-2) plays a key role in axonal development by regulating microtubule dynamics. However, the molecular mechanisms underlying this function have not been clearly elucidated. In this study, we demonstrated that hCRMP-2, specifically amino acid residues 480-509, is essential for stimulating tubulin GTPase activity. We also found that the GTPase-activating protein (GAP) activity of hCRMP-2 was important for microtubule assembly and neurite formation in differentiated PC12 pheochromocytoma cell lines. Mutant hCRMP-2, lacking arginine residues responsible for GAP activity, inhibited microtubule assembly and neurite formation. Interestingly, we found that the N-terminal region (amino acids150-299) of hCRMP-2 had an inhibitory role on GAP activity via a direct interaction with the C-terminal region (amino acids 480-509). Our results suggest that CRMP-2 as a tubulin direct binder may be a GAP of tubulin in neurite formation and that its GAP activity may be regulated by an intramolecular interaction with an N-terminal inhibitory region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellsig.2009.07.017DOI Listing
December 2009

Qualitative and quantitative comparison of N-glycans between pig endothelial and islet cells by high-performance liquid chromatography and mass spectrometry-based strategy.

J Mass Spectrom 2009 Jul;44(7):1087-104

Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.

N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 alpha-galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one alpha-galactosylated glycans from pig islet cells. The quantity of the alpha-galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using alpha 1,3-galactosyltransferase gene-knockout (GalT-KO) pig.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jms.1587DOI Listing
July 2009

Hydrogen peroxide-induced VCAM-1 expression in pancreatic islets and beta-cells through extracellular Ca2+ influx.

Transplantation 2008 Nov;86(9):1257-66

Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.

Background: The use of porcine islets as alternatives to transplantable human islets is hampered by xenotransplant rejection. To identify molecular mechanisms that would allow subversion of xenoislet rejection, we investigated the role of H2O2 in vascular cell adhesion molecule-1 (VCAM-1) expression by porcine and mouse islets and beta-cell lines.

Methods: Porcine islets were treated with H2O2, tumor necrosis factor alpha, interferon-gamma, interleukin-1beta, and lipopolysaccharide, to assess the effects of inflammatory stimulators on VCAM-1 expression using flow cytometry. The role of Ca2+ in H2O2-induced VCAM-1 expression was investigated in beta-cell lines using an extracellular Ca2+ chelator and Ca2+-depleted media. Furthermore, H2O2-induced VCAM-1 expression was measured in beta-cells, pretreated with inhibitors of protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt. Finally, H2O2-induced VCAM-1 expression was evaluated in porcine islets and rodent beta-cell lines infected with an adenovirus encoding catalase, a H2O2-removing enzyme.

Results: H2O2 was most potent inflammatory stimulator of VCAM-1 expression in porcine islets and had the greatest effect on VCAM-1 expression by beta-cells. Signaling pathway analysis demonstrated that extracellular Ca2+ influx was critical to H2O2-mediated VCAM-1 expression; however, protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt activation were not required for VCAM-1 expression. Finally, catalase overexpression inhibited H2O2-induced VCAM-1 expression by islets and beta-cell lines.

Conclusion: An extracellular calcium-dependent H2O2 pathway is the critical mediator of VCAM-1 expression by pancreatic islets and beta-cells. Inhibition of this pathway by catalase overexpression in donor islets can be exploited to protect against xenoislet immune responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/TP.0b013e318188ab04DOI Listing
November 2008

GbetaL regulates TNFalpha-induced NF-kappaB signaling by directly inhibiting the activation of IkappaB kinase.

Cell Signal 2008 Nov 9;20(11):2127-33. Epub 2008 Aug 9.

Graduate School of Medicine, Korea University, 136-705 Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea.

The transcriptional activation of NF-kappaB, a critical player in both physiological and pathological cellular responses to diverse cytokines, is dependent on IKK activation. Although molecular mechanisms underlying IKK activation have been well elucidated, the processes that negatively regulate IKK activity are still largely unknown. Using yeast two-hybrid screening, we have identified GbetaL as an interacting partner of IKKbeta. In this study, we demonstrate that GbetaL interacts with IKKalpha and IKKbeta in vitro and in vivo. The C-terminal WD domains of GbetaL are required for the interaction with both the kinase domain and leucine zipper domain of IKKbeta. Overexpression of GbetaL inhibits TNFalpha-induced activation of NF-kappaB signaling, while down-regulation of GbetaL expression by small interfering RNA enhances NF-kappaB activity. GbetaL constitutively interacts with IKKbeta, and this interaction is enhanced by TNFalpha treatment. GbetaL also inhibits TNFalpha-induced phosphorylation of IKKs. Taken together, these data suggest that GbetaL is involved in the negative regulation of TNFalpha-stimulated NF-kappaB signaling through a direct interaction with IKK.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellsig.2008.08.001DOI Listing
November 2008

Lysophosphatidic acid signaling through LPA receptor subtype 1 induces colony scattering of gastrointestinal cancer cells.

J Cancer Res Clin Oncol 2009 Jan 1;135(1):45-52. Epub 2008 Jul 1.

Department of Biochemistry, College of Medicine and Cancer Research Institute, Seoul National University, Seoul, 110-799, Republic of Korea.

Purpose: Lysophosphatidic acid (LPA) is a multifunctional lipid mediator involved in triggering tumor cell invasion and metastasis, as well as malignant cell growth. LPA is also known to modulate the colony scattering of epithelial cancers, which is a prerequisite for cell invasion. However, the underlying details of how this is accomplished are not clear. Here we have investigated the roles of specific LPA receptor subtypes in cell scattering.

Methods: Gastrointestinal carcinoma cell lines were examined for cell scattering activity in response to LPA, and the expression of LPA receptor subtypes was determined by RT-PCR. The effect of down regulation of each LPA receptor in DLD1 cells was determined using a shRNA-lentivirus system. In addition, the effect of overexpression of LPA receptors on cell scattering was investigated using lentivirus expression constructs.

Results: The colonies of AGS and DLD1, but not MKN74, cells were dispersed in response to LPA. RT-PCR analysis revealed that the mRNAs of LPA1, LPA2, and LPA3 were present in AGS and DLD1 cells, but only LPA2 mRNA was detected in MKN74 cells. In DLD1 cells, the scattering activity induced by LPA was partially blocked by pretreatment with PP2 and PD98059, inhibitors of src kinase and MEK, respectively. LPA1 knockdown with shRNA decreased the degree of cell scattering induced by LPA. Knockdown of LPA2 or LPA3 had no effect on LPA-induced scattering. In addition, overexpression of LPA1 in DLD1 cells slightly decreased the response time of LPA-induced cell scattering. On the contrary, MKN74 cells expressing exogenous LPA1 did not respond to LPA by scattering.

Conclusion: These results demonstrate that LPA1 mediates LPA-stimulated cell scattering of gastrointestinal carcinomas, but that activation of other intracellular pathways, besides those contributing to ERK phosphorylation, is also necessary for cell scattering in response to LPA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00432-008-0441-zDOI Listing
January 2009

Weak response of porcine C5a receptor towards human C5a in miniature pig endothelial cells and PMNs.

Xenotransplantation 2007 Nov;14(6):563-71

Cancer Research Institute, Seoul National University College of Medicine, Seoul.

Background: The anaphylatoxin C5a is a potent inflammatory molecule generated during complement activation. Although some reports have implicated C5a in xenograft rejection, to date, the molecular compatibility between human C5a and porcine C5a receptor (C5aR) has been little studied. To examine the need for pC5aR-deficient pig in xenotransplantaion, we aimed to look at the degree of direct interaction between human C5a (recipient side) and porcine endothelial cells (PECs) and porcine polymorphonuclear neutrophils (PMNs) (donor side).

Methods: Following the treatment of human C5a to isolated porcine PMNs, transmigration of PMNs was measured by Transwell system and superoxide generation by cytochrome c reduction assay. Next, the effects of human C5a on several intracellular signaling pathways were further checked; actin cytoskeletal change was observed under a confocal microscope after staining with Alexa Fluor-546-phalloidin, intracellular calcium mobilization was measured by spectrofluorophotometer. The degree of direct effect of human C5a on porcine PMNs was compared with that in human PMNs. Finally, microarray was performed to monitor the effect of human C5a on gene expression of PEC and the expression of several candidate proteins was checked by flow cytometry.

Results: We found that human C5a was able to induce chemotaxis, superoxide generation, actin cytoskeletal change, and intracellular calcium mobilization in porcine PMNs. However, higher concentration of human C5a was required to stimulate porcine PMNs in comparison with activating human PMNs. The amino acid sequences of porcine C5aR with those of human C5aR showed a sequence homology of only 67%. To elucidate the effect of human C5a to PECs, microarray analysis following the treatment of PECs with human C5a was performed. These data showed that human C5a did not significantly affect gene transcription patterns in PECs. Additionally, treatment of PECs with human C5a also did not induce protein expression of several cell adhesion molecules, including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, P-selectin, and E-selectin, or secretion of interleukin-8 from PECs.

Conclusions: These results suggest that human C5a may play a minor role on PEC activation possibly due to molecular incompatibility across the species barrier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1399-3089.2007.00421.xDOI Listing
November 2007

Hydrogen peroxide increases human leukocyte adhesion to porcine aortic endothelial cells via NFkappaB-dependent up-regulation of VCAM-1.

Int Immunol 2007 Dec 29;19(12):1349-59. Epub 2007 Oct 29.

Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.

Although a severe shortage of organs in transplantation can be overcome by using xenotransplantation of porcine donor organs, profound immune rejection to xenogeneic antigens remains a main obstacle. To elucidate the role of hydrogen peroxide (H(2)O(2)) on xenogeneic immune responses, we investigated its effects on porcine aortic endothelial cells (PAECs). We found that H(2)O(2) can specifically induce vascular cell adhesion molecule-1 (VCAM-1) expression on PAECs, but little on human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs). Furthermore, we further confirmed that H(2)O(2) induces activation of NFkappaB in PAECs, but not in HAECs. Interestingly, cell adhesion assay showed that U937, human promonocytic leukocyte, can adhere to PAECs in an H(2)O(2)-dependent manner and by using a neutralizing assay with anti-VCAM-1-specific antibodies, we also found that the interaction is mediated primarily by VCAM-1. Finally, we also demonstrated that up-regulation of VCAM-1 expression on PAECs by reactive oxygen species-producing HL-60, human leukemic neutrophil cells, could be significantly diminished by over-expressing an H(2)O(2)-removing catalase. In summary, our results suggest that NFkappaB-dependent porcine VCAM-1 expression by H(2)O(2) may promote interaction of human leukocyte to PAECs, and thus may play an important role on inducing xenogeneic immune responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/intimm/dxm104DOI Listing
December 2007