A liver‐specific gene expression panel predicts the differentiation status of <i>in vitro</i> hepatocyte models

Kim, Dae&#x2010,Soo,Ryu, Jea&#x2010,Woon,Son, Mi‐,Young,Oh, Jung&#x2010,Hwa,Chung, Kyung&#x2010,Sook,Lee, Sugi,Lee, Jeong&#x2010,Ju,Ahn, Jun&#x2010,Ho,Min, Ju&#x2010,Sik,Ahn, Jiwon,Kang, Hyun Mi John Wiley and Sons Inc. 2017 Hepatology Vol.66 No.5

<P>Alternative cell sources, such as three‐dimensional organoids and induced pluripotent stem cell–derived cells, might provide a potentially effective approach for both drug development applications and clinical transplantation. For example, the development of cell sources for liver cell–based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end‐stage liver disease. Differentiated liver cells and three‐dimensional organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver‐specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a liver‐specific gene expression panel (LiGEP) algorithm that presents the degree of liver similarity as a “percentage.” We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of <I>in vivo</I> liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, three‐dimensional cultured HepaRG cells and human pluripotent stem cell–derived hepatocyte‐like cells showed liver similarity scores of 59.14% and 32%, respectively, although general liver‐specific markers were detected. <I>Conclusion</I>: Our study describes a quantitative and predictive model for differentiated samples, particularly liver‐specific cells or organoids; and this model can be further expanded to various tissue‐specific organoids; our LiGEP can provide useful information and insights regarding the differentiation status of <I>in vitro</I> liver models. (H<SMALL>EPATOLOGY</SMALL> 2017;66:1662–1674).</P>

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Kim, Mi Young,Copland, Alastair,Nayak, Kaustuv,Chandele, Anmol,Ahmed, Muhammad S.,Zhang, Qibo,Diogo, Gil R.,Paul, Matthew J.,Hofmann, Sven,Yang, Moon&#x2010,Sik,Jang, Yong&#x2010,Suk,Ma, Julian K&#x20 BLACKWELL 2018 PLANT BIOTECHNOLOGY JOURNAL Vol.16 No.7

<P><B>Summary</B></P><P>Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue <I>ex vivo</I> as evidenced by antigen‐specific CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.</P>

3′‐Sialyllactose as an inhibitor of p65 phosphorylation ameliorates the progression of experimental rheumatoid arthritis

Kang, Li&#x2010,Jung,Kwon, Eun&#x2010,Soo,Lee, Kwang Min,Cho, Chanmi,Lee, Jae&#x2010,In,Ryu, Young Bae,Youm, Tae Hyun,Jeon, Jimin,Cho, Mi Ra,Jeong, Seon&#x2010,Yong,Lee, Sang&#x2010,Rae,Kim, Wook,Yang John Wiley and Sons Inc. 2018 British journal of pharmacology Vol.175 No.23

<P><B>Background and Purpose</B></P><P>3′‐Sialyllactose (3′‐SL) is a safe compound that is present in high levels in human milk. Although it has anti‐inflammatory properties and supports immune homeostasis, its effect on collagen‐induced arthritis (CIA) is unknown. In this study, we investigated the prophylactic and therapeutic effect of 3′‐SL on the progression of rheumatoid arthritis (RA) in <I>in vitro</I> and <I>in vivo</I> models.</P><P><B>Experimental Approach</B></P><P>The anti‐arthritic effect of 3′‐SL was analysed with fibroblast‐like synoviocytes <I>in vitro</I> and an <I>in vivo</I> mouse model of CIA. RT‐PCR, Western blotting and ELISA were performed to evaluate its effects <I>in vitro</I>. Histological analysis of ankle and knee joints of mice with CIA was performed using immunohistochemistry, as well as safranin‐O and haematoxylin staining.</P><P><B>Key Results</B></P><P>3′‐SL markedly alleviated the severity of CIA in the mice by reducing paw swelling, clinical scores, incidence rate, serum levels of inflammatory cytokines and autoantibody production. Moreover, 3′‐SL reduced synovitis and pannus formation and suppressed cartilage destruction by blocking secretion of chemokines, pro‐inflammatory cytokines, https://en.wikipedia.org/wiki/Matrix_metalloproteinases and osteoclastogenesis <I>via</I> NF‐κB signalling. Notably, phosphorylation of p65, which is a key protein in the NF‐κB signalling pathway, was totally blocked by 3′‐SL in the RA models.</P><P><B>Conclusions and Implications</B></P><P>3′‐SL ameliorated pathogenesis of CIA by suppressing catabolic factor expression, proliferation of inflammatory immune cells and osteoclastogenesis. These effects were mediated <I>via</I> blockade of the NF‐κB signalling pathway. Therefore, 3′‐SL exerted prophylactic and therapeutic effects and could be a novel therapeutic agent for the treatment of RA.</P>

Inhibition of VEGF ‐dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC 14a

Kim, Taek&#x2010,Keun,Park, Chang Sik,Jang, Jihye,Kim, Mi Ra,Na, Hee&#x2010,Jun,Lee, Kangseung,Kim, Hyun Jung,Heo, Kyun,Yoo, Byong Chul,Kim, Young&#x2010,Myeong,Lee, Je&#x2010,Wook,Kim, Su Jin,Kim, Eu John Wiley and Sons Inc. 2018 MOLECULAR ONCOLOGY Vol.12 No.3

<P>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 <I>in vitro</I> and <I>in vivo</I> 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.</P>

Anti‐inflammatory mechanism of ginsenoside Rh1 in lipopolysaccharide‐stimulated microglia: critical role of the protein kinase A pathway and hemeoxygenase‐1 expression

Jung, Ji&#x2010,Sun,Shin, Jin A.,Park, Eun&#x2010,Mi,Lee, Jung&#x2010,Eun,Kang, Young&#x2010,Sook,Min, Sung&#x2010,Won,Kim, Dong&#x2010,Hyun,Hyun, Jin&#x2010,Won,Shin, Chan&#x2010,Young,Kim, Hee&#x201 Blackwell Publishing Ltd 2010 Journal of Neurochemistry Vol.115 No.6

<P> <I>J. Neurochem.</I> (2010) <B>115,</B> 1668–1680.</P><P><B>Abstract</B></P><P>Microglia activation plays a pivotal role in neurodegenerative diseases, and thus controlling microglial activation has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we showed that ginsenoside Rh1 inhibited inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐inflammatory cytokine expression in lipopolysaccharide (LPS)‐stimulated microglia, while Rh1 increased anti‐inflammatory IL‐10 and hemeoxygenase‐1 (HO‐1) expression. Suppression of microglial activation by Rh1 was also observed in the mouse brain following treatment with LPS. Subsequent mechanistic studies revealed that Rh1 inhibited LPS‐induced MAPK phosphorylation and nuclear factor‐κB (NF‐κB)‐mediated transcription without affecting NF‐κB DNA binding. As the increase of pCREB (cAMP responsive element‐binding protein) is known to result in suppression of NF‐κB‐mediated transcription, we examined whether Rh1 increased pCREB levels. As expected, Rh1 increased pCREB, which was shown to be related to the anti‐inflammatory effect of Rh1 because pre‐treatment with protein kinase A inhibitors attenuated the Rh1‐mediated inhibition of nitric oxide production and the up‐regulation of IL‐10 and HO‐1. Furthermore, treatment of HO‐1 shRNA attenuated Rh1‐mediated inhibition of nitric oxide and reactive oxygen species production. Through this study, we have demonstrated that protein kinase A and its downstream effector, HO‐1, play a critical role in the anti‐inflammatory mechanism of Rh1 by modulating pro‐ and anti‐inflammatory molecules in activated microglia.</P>

Functional intronic variant of <i>SLC5A10</i> affects <i>DRG2</i> expression and survival outcomes of early‐stage non‐small‐cell lung cancer

Hong, Mi Jeong,Yoo, Seung Soo,Choi, Jin Eun,Kang, Hyo&#x2010,Gyoung,Do, Sook Kyung,Lee, Jang Hyuck,Lee, Won Kee,Lee, Jaehee,Lee, Shin Yup,Cha, Seung Ick,Kim, Chang Ho,Lee, Eung Bae,Cho, Sukki,Jheon, S John Wiley and Sons Inc. 2018 Cancer Science Vol.109 No.12

<P>RegulomeDB is a new tool that can predict the regulatory function of genetic variants. We applied RegulomeDB in selecting putative functional variants and evaluated the relationship between these variants and survival outcomes of surgically resected non‐small‐cell lung cancer. Among the 244 variants studied, 14 were associated with overall survival (<I>P </I><<I> </I>0.05) in the discovery cohort and one variant (rs2257609 C>T) was replicated in the validation cohort. In the combined analysis, rs2257609 C>T was significantly associated with worse overall and disease‐free survival under a dominant model (<I>P </I>=<I> </I>2 × 10<SUP>−5</SUP> and <I>P </I>=<I> </I>0.001, respectively). rs2257609 is located in the <I>SLC5A10</I> intron, but RegulomeDB predicted that this variant affected <I>DRG2</I>, not <I>SLC5A10</I> expression. The expression level of <I>SLC5A10</I> was not different with the rs2257609 genotype. However, <I>DRG2</I> expression was different according to the rs2257609 genotype (<I>P</I><SUB>trend</SUB><SUP> </SUP>= 0.03) and was significantly higher in tumor than in non‐malignant lung tissues (<I>P </I>=<I> </I>1 × 10<SUP>−5</SUP>). Luciferase assay also showed higher promoter activity of <I>DRG2</I> in samples with the rs2257609 T allele (<I>P </I><<I> </I>0.0001). rs2257609 C>T affected <I>DRG2</I> expression and, thus, influenced the prognosis of early‐stage non‐small‐cell lung cancer. This study was approved by the Institutional Review Broad of Kyungpook National University of Hospital (Approval No. KNUMC 2014‐04‐210‐003).</P>

Downregulation of CHIP promotes ovarian cancer metastasis by inducing Snail‐mediated epithelial–mesenchymal transition

Park, Sun&#x2010,Mi,Park, Seung&#x2010,Ho,Ryu, Ki‐,Jun,Kim, In&#x2010,Kyu,Han, Hyeontak,Kim, Hyo&#x2010,Jin,Kim, Seon&#x2010,Hee,Hong, Keun&#x2010,Seok,Kim, Hyemin,Kim, Minju,Cho, Bok Im,Heo, Je Elsevier Science B.V. Amsterdam 2019 MOLECULAR ONCOLOGY Vol.13 No.5

<P>The epithelial–mesenchymal transition (EMT) plays a pivotal role in the conversion of early‐stage tumors into invasive malignancies. The transcription factor Snail, an extremely unstable protein whose subcellular levels are regulated by many E3 ubiquitin ligases, promotes EMT as well as associated pathological characteristics including migration, invasion, and metastasis. Through yeast two‐hybrid screening, we identified the carboxyl terminus of Hsc70‐interacting protein (CHIP) as a novel Snail ubiquitin ligase that interacts with Snail to induce ubiquitin‐mediated proteasomal degradation. Inhibition of CHIP expression increases Snail protein levels, induces EMT, and enhances <I>in vitro</I> migration and invasion as well as <I>in vivo</I> metastasis of ovarian cancer cells. In turn, Snail depletion abrogates all phenomena induced by CHIP depletion. Finally, Snail and CHIP expression is inversely correlated in ovarian tumor tissues. These findings establish the CHIP–Snail axis as a post‐translational mechanism of EMT and cancer metastasis regulation.</P>

TXNIP regulates AKT‐mediated cellular senescence by direct interaction under glucose‐mediated metabolic stress

Huy, Hangsak,Song, Hae Young,Kim, Mi Jeong,Kim, Won Sam,Kim, Dong Oh,Byun, Jae&#x2010,Eun,Lee, Jungwoon,Park, Young&#x2010,Jun,Kim, Tae&#x2010,Don,Yoon, Suk Ran,Choi, Eun&#x2010,Ji,Lee, Chul&#x2010,Ho John Wiley and Sons Inc. 2018 Aging cell Vol.17 No.6

<P><B>Abstract</B></P><P>Aging is associated with an inevitable and universal loss of cell homeostasis and restricts an organism's lifespan by an increased susceptibility to diseases and tissue degeneration. The glucose uptake associated with producing energy for cell survival is one of the major causes of ROS production under physiological conditions. However, the overall mechanisms by which glucose uptake results in cellular senescence remain mysterious. In this study, we found that TXNIP deficiency accelerated the senescent phenotypes of MEF cells under high glucose condition. TXNIP<SUP>‐/‐</SUP> MEF cells showed greater induced glucose uptake and ROS levels than wild‐type cells, and <I>N</I>‐acetylcysteine (NAC) treatment rescued the cellular senescence of TXNIP<SUP>‐/‐</SUP> MEF cells. Interestingly, TXNIP<SUP>‐/‐</SUP> MEF cells showed continuous activation of AKT during long‐term subculture, and AKT signaling inhibition completely blocked the cellular senescence of TXNIP<SUP>‐/‐</SUP> MEF cells. In addition, we found that TXNIP interacted with AKT via the PH domain of AKT, and their interaction was increased by high glucose or H<SUB>2</SUB>O<SUB>2</SUB> treatment. The inhibition of AKT activity by TXNIP was confirmed using western blotting and an in vitro kinase assay. TXNIP deficiency in type 1 diabetes mice (Akita) efficiently decreased the blood glucose levels and finally increased mouse survival. However, in normal mice, TXNIP deficiency induced metabolic aging of mice and cellular senescence of kidney cells by inducing AKT activity and aging‐associated gene expression. Altogether, these results suggest that TXNIP regulates cellular senescence by inhibiting AKT pathways via a direct interaction under conditions of glucose‐derived metabolic stress.</P>

Feasibility of Bronchial Washing Fluid‐Based Approach to Early‐Stage Lung Cancer Diagnosis

Ryu, Jeong&#x2010,Seon,Lim, Jun Hyeok,Lee, Myoung Kyu,Lee, Seung Jae,Kim, Hyun&#x2010,Jung,Kim, Min Jeong,Park, Mi Hwa,Kim, Jung Soo,Nam, Hae&#x2010,Seong,Park, Nuri,Yong, Seok Joong AlphaMed Press 2019 The oncologist Vol.24 No.7

<P>The potential of circulating tumor DNA (ctDNA) detection in early stage lung cancer is explored. This study investigated whether bronchial washing, a minimally invasive procedure that yields fluids that may contain ctDNA, can reflect genetic profiles of primary tumors using next‐generation sequencing.</P><P>A blood‐based approach such as circulating tumor DNA remains challenging in diagnosis for early‐stage disease. Bronchial washing (BW) is a minimally invasive procedure that yields fluids that may contain tumor DNA. Therefore, we prospectively enrolled 12 patients with early‐stage non‐small cell lung cancer without endoscopically visible tumors. Somatic mutations were analyzed using ultra‐deep next‐generation sequencing in 48 paired specimens (primary tumor tissue, normal tissue, BW supernatant, and BW precipitate). In primary tumors, 130 missense mutations/indels (5–16 per patient) and 20 driver mutations (0–3 per patient) were found. Concordance of driver mutations between BW fluids and primary tumors was 95.0%. The allele frequencies for missense mutations/indels in BW supernatants significantly correlated with those in primary tumors and were higher than those in BW precipitates. These findings suggest that BW supernatants are reflective of tumor‐associated mutations and could be used for early‐stage lung cancer diagnosis.</P>

XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells

Lee, You&#x2010,Jin,Park, Bong&#x2010,Soo,Park, Hae&#x2010,Ryoun,Yu, Su&#x2010,Bin,Kang, Hae&#x2010,Mi,Kim, In&#x2010,Ryoung John Wiley and Sons Inc. 2017 Environmental toxicology Vol.32 No.11

<P><B>Abstract</B></P><P>Embelin is an active ingredient of traditional herbal remedies for cancer and other diseases. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. Therefore, we conducted this study to examine whether Embelin modulates autophagy in Ca9‐22. Our results showed that Embelin had anticancer activity against the Ca9‐22 human tongue squamous cell, and we observed that autophagic vacuoles were formed by MDC and AO. We also analyzed Embelin‐treated Ca9‐22 cells for the presence of biochemical markers and found that it directly affected the conversion of LC3‐II, the degradation of p62/SQSTM1, full‐length cleavage formation of ATG5‐ATG12 complex and Beline‐1, and caspase activation. Rescue experiments using an autophagy inhibitor showed Embelin‐induced cell death in Ca9‐22, confirming that autophagy acts as a pro‐death signal. Furthermore, Embelin exhibited anticancer activity against Ca9‐22 via both autophagy and apoptosis. These findings suggest that Embelin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.</P>