Publications by authors named "Hee Min Yoo"

39 Publications

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Molecules 2021 Aug 16;26(16). Epub 2021 Aug 16.

Biometrology Group, Korea Research Institute of Standards and Science, Daejeon 34113, Korea.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.
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http://dx.doi.org/10.3390/molecules26164951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399869PMC
August 2021

Anti-inflammatory effect of Ailanthus altissima (Mill.) Swingle leaves in lipopolysaccharide-stimulated astrocytes.

J Ethnopharmacol 2021 Jul 13:114258. Epub 2021 Jul 13.

Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 06974, Republic of Korea. Electronic address:

Ethnopharmacological Relevance: Activated astrocytes are involved in the progression of neurodegenerative diseases. Traditionally, Ailanthus altissima (Mill.) Swingle, widely distributed in East Asia, has been used as a medicine for the treatment of fever, gastric diseases, and inflammation. Although A. altissima has been reported to play an anti-inflammatory role in peripheral tissues or cells, its role in the central nervous system (CNS) remains unclear.

Aim Of The Study: In the present study, we investigated the anti-inflammatory effects and mechanism of action of A. altissima in primary astrocytes stimulated by lipopolysaccharide (LPS).

Materials And Methods: A nitrite assay was used to measure nitric oxide (NO) production, and the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to determine cytotoxicity. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) were determined with western blotting. Reverse-transcription PCR was used to assess the expression of inflammatory cytokines. The levels of reactive oxygen species were measured using 2,7-dichlorodihydrofluorescein diacetate. Luciferase assay and immunocytochemistry were used for assessing nuclear factor-kappa B (NF-κB) transcription and p65 localization, respectively. Memory and social interaction were analyzed using the Y-maze and three-chamber tests, respectively.

Results: The ethanol extract of A. altissima leaves (AAE) inhibited iNOS and COX-2 expression in LPS-stimulated astrocytes. Moreover, AAE reduced the transcription of various proinflammatory mediators, hindered NF-κB activation, and suppressed extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation without p38 activation. Ultra-high performance liquid chromatography with mass spectrometry analysis revealed that AAE comprised ethyl gallate, quercetin, and kaempferol, along with luteolin, which has anti-inflammatory properties, and repressed LPS-induced nitrite levels and the nuclear translocation of p65. Finally, oral administration of AAE attenuated LPS-induced memory and social impairment in mice and repressed LPS-induced ERK and JNK activation in the cortices of mice.

Conclusion: AAE could have therapeutic uses in the treatment of neuroinflammatory diseases via suppression of astrocyte activation.
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http://dx.doi.org/10.1016/j.jep.2021.114258DOI Listing
July 2021

Nucleic Acid Testing of SARS-CoV-2.

Int J Mol Sci 2021 Jun 7;22(11). Epub 2021 Jun 7.

Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.
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http://dx.doi.org/10.3390/ijms22116150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201071PMC
June 2021

Extracellular Vesicles from Thapsigargin-Treated Mesenchymal Stem Cells Ameliorated Experimental Colitis via Enhanced Immunomodulatory Properties.

Biomedicines 2021 Feb 18;9(2). Epub 2021 Feb 18.

Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea.

Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton's jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis.
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http://dx.doi.org/10.3390/biomedicines9020209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922639PMC
February 2021

Anticancer Activity of Lesbicoumestan in Jurkat Cells via Inhibition of Oxidative Stress-Mediated Apoptosis and MALT1 Protease.

Molecules 2021 Jan 2;26(1). Epub 2021 Jan 2.

Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

This study explores the potential anticancer effects of lesbicoumestan from against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-κB pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-κB pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter's active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.
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http://dx.doi.org/10.3390/molecules26010185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794876PMC
January 2021

1-Methoxylespeflorin G11 Protects HT22 Cells from Glutamate-Induced Cell Death through Inhibition of ROS Production and Apoptosis.

J Microbiol Biotechnol 2021 Feb;31(2):217-225

College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 61186, Republic of Korea.

This study aimed to investigate the neuroprotective effects of 1-methoxylespeflorin G11 (MLG), a pterocarpan, against glutamate-induced neurotoxicity in neuronal HT22 hippocampal cells. The protective effects of MLG were evaluated using MTT assay and microscopic analysis. The extent of apoptosis was studied using flow cytometric analysis performed on the damaged cells probed with annexin V/propidium iodide. Moreover, mitochondrial reactive oxygen species (ROS) were assessed using flow cytometry through MitoSOXTM Red staining. To determine mitochondrial membrane potential, staining with tetramethylrhodamine and JC-1 was performed followed by flow cytometry. The results demonstrated that MLG attenuates glutamate-induced apoptosis in HT22 cells by inhibiting intracellular ROS generation and mitochondrial dysfunction. Additionally, MLG prevented glutamate-induced apoptotic pathway in HT22 cells through upregulation of Bcl-2 and downregulation of cleaved PARP-1, AIF, and phosphorylated MAPK cascades. In addition, MLG treatment induced HO-1 expression in HT22 cells. These results suggested that MLG exhibits neuroprotective effects against glutamate-induced neurotoxicity in neuronal HT22 cells by inhibiting oxidative stress and apoptosis.
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http://dx.doi.org/10.4014/jmb.2011.11032DOI Listing
February 2021

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

J Microbiol Biotechnol 2021 03;31(3):358-367

Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (C) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.
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http://dx.doi.org/10.4014/jmb.2009.09006DOI Listing
March 2021

Inhibition of Jurkat T Cell Proliferation by Active Components of Roots Via Induced Mitochondrial Damage and Apoptosis Promotion.

J Microbiol Biotechnol 2020 Dec;30(12):1885-1895

Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea.

Houtt (RJH) is a valuable plant used in traditional medicine to treat several diseases, such as scabies and jaundice. In this study, Jurkat cell growth inhibitory extracts of roots were subjected to bioassay-guided fractionation, resulting in the isolation of three naphthalene derivatives (3-5) along with one anthraquinone (6) and two phenolic compounds (1 and 2). Among these compounds, 2-methoxystypandrone (5) exhibited potent anti-proliferative effects on Jurkat cells. Analysis by flow cytometry confirmed that 2-methoxystypandrone (5) could significantly reduce mitochondrial membrane potential and promote increased levels of mitochondrial reactive oxygen species (ROS), suggesting a strong mitochondrial depolarization effect. Real-time quantitative polymerase chain reaction (qPCR) analysis was also performed, and the results revealed that the accumulation of ROS was caused by reduced mRNA expression levels of heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, 2-methoxystypandrone (5) triggered strong apoptosis that was mediated by the arrest of the G0/G1 phase of the cell cycle. Furthermore, 2-methoxystypandrone (5) downregulated p-IκB-α, p-NF-κB p65, Bcl2, and Bcl-xl and upregulated BAX proteins. Taken together, these findings revealed that 2-methoxystypandrone (5) isolated from RJH could potentially serve as an early lead compound for leukemia treatment involving intracellular signaling by increasing mitochondrial ROS and exerting anti-proliferative effects.
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http://dx.doi.org/10.4014/jmb.2007.07018DOI Listing
December 2020

Anti-Melanogenesis Activity of 6--Isobutyrylbritannilactone from on B16F10 Melanocytes and In Vivo Zebrafish Models.

Molecules 2020 Aug 26;25(17). Epub 2020 Aug 26.

Microbiological Analysis Team, Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

A potential natural melanogenesis inhibitor was discovered in the form of a sesquiterpene isolated from the flowers of , specifically 6--isobutyrylbritannilactone (IBL). We evaluated the antimelanogenesis effects of IBL on B16F10 melanocytes and zebrafish embryos. As a result, we found that 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production was reduced in a dose-dependent manner in B16F10 cells by IBL. We also analyzed B16F10 cells that were and were not treated with IBMX, investigating the melanin concentration, tyrosinase activity, mRNA levels. We also studied the protein expressions of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRP1, and TRP2). Furthermore, we found that melanin synthesis and tyrosinase expression were also inhibited by IBL through the modulation of the following signaling pathways: ERK, phosphoinositide 3-kinase (PI3K)/AKT, and CREB. In addition, we studied antimelanogenic activity using zebrafish embryos and found that the embryos had significantly reduced pigmentation in the IBL-treated specimens compared to the untreated controls.
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http://dx.doi.org/10.3390/molecules25173887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504228PMC
August 2020

Neuroprotective Effects of Cryptotanshinone in a Direct Reprogramming Model of Parkinson's Disease.

Molecules 2020 Aug 7;25(16). Epub 2020 Aug 7.

Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea.

Parkinson's disease (PD) is a well-known age-related neurodegenerative disease. Considering the vital importance of disease modeling based on reprogramming technology, we adopted direct reprogramming to human-induced neuronal progenitor cells (hiNPCs) for in vitro assessment of potential therapeutics. In this study, we investigated the neuroprotective effects of cryptotanshinone (CTN), which has been reported to have antioxidant properties, through PD patient-derived hiNPCs (PD-iNPCs) model with induced oxidative stress and cell death by the proteasome inhibitor MG132. A cytotoxicity assay showed that CTN possesses anti-apoptotic properties in PD-hiNPCs. CTN treatment significantly reduced cellular apoptosis through mitochondrial restoration, such as the reduction in mitochondrial reactive oxygen species and increments of mitochondrial membrane potential. These effects of CTN are mediated via the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in PD-hiNPCs. Consequently, CTN could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.
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http://dx.doi.org/10.3390/molecules25163602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463464PMC
August 2020

Apoptosis in Leukemic Cells Induced by Anti-proliferative Coumarin Isolated from the Stem Bark of .

J Microbiol Biotechnol 2020 Aug;30(8):1214-1221

Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea.

Esculetin 6-O-β-D-arabinofuranosyl-(1→6)-β-D-glucopyranoside (EAG) is a coumarin glycoside isolated from the stem bark of . This study scrutinized the anti-proliferative activity of EAG on blood cancer-derived Jurkat leukemic cells. Cell viability assays in leukemic cancer cells determined that EAG possesses potent anti-proliferative effects. Moreover, treatment with EAG increased the proportion of apoptotic cells, resulted in cell cycle arrest being induced at the subG0/ G1 phase, and reduced the proportion of cells present in the S phase. In addition, mitochondrial membrane potential was reduced by EAG in Jurkat cells. Additionally, EAG triggered apoptosis that was mediated by the downregulation of BCL-XL, p-IκBα, and p-p65 expressions in addition to the upregulation of cleaved Caspase 3 and BAX expressions. These findings revealed that the toxic effect of EAG was mediated by intracellular signal transduction pathways that involved a mechanism in which reactive oxygen species (ROS) were upregulated. Thus, this study concludes that EAG could potentially serve as a therapeutic agent for leukemia.
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http://dx.doi.org/10.4014/jmb.2006.06022DOI Listing
August 2020

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells.

J Vis Exp 2020 06 13(160). Epub 2020 Jun 13.

Microbiological Analysis Team, Group for Biometrology, Korea Research Institute of Standards and Science (KRISS);

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.
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http://dx.doi.org/10.3791/61285DOI Listing
June 2020

The Antimelanogenic Effect of Inularin Isolated from Flowers of on B16F10 Melanoma Cells and Zebrafish Embryos.

J Microbiol Biotechnol 2020 May;30(5):749-752

Department of Food Science and Biotechnology, Dongguk University, Seoul 10326, Republic of Korea.

In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of , and the structure was determined using spectroscopic and chemical methods. The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated. Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells. Zebrafish embryos were used to confirm the antimelanogenic activity. Inularin significantly decreased the pigmentation of embryos compared with untreated controls.
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http://dx.doi.org/10.4014/jmb.2003.03025DOI Listing
May 2020

Iroquois Homeobox Protein 2 Identified as a Potential Biomarker for Parkinson's Disease.

Int J Mol Sci 2020 May 14;21(10). Epub 2020 May 14.

Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea.

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms, such as resting tremors, rigidity, and bradykinesia. According to previous reports, non-motor symptoms, notably gastrointestinal dysfunction, could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However, connecting PD to the intestine is methodologically challenging. Thus, we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human G2019S mutation, which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs, we determined that a particular gene, namely, Iroquois homeobox protein 2 (), showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD.
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http://dx.doi.org/10.3390/ijms21103455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278941PMC
May 2020

Improvement of digital PCR conditions for direct detection of KRAS mutations.

J Clin Lab Anal 2020 Aug 24;34(8):e23344. Epub 2020 Apr 24.

Center for Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon, Korea.

Background: In standard analytical conditions, an isolation step is essential for circulating tumor DNA (ctDNA) analysis. The necessity of this step becomes unclear with the development of highly sensitive detection methods. The aim of this study was to evaluate ctDNA mimetic nDNA detection as reference materials (RMs) using dPCR technologies either directly from serum or without serum.

Methods: To determine an absolute count of both mutation and wild-type bearing DNA molecules, genomic DNA (gDNA) and nucleosomal DNA (nDNA), which are similar in size to cell-free DNA, were evaluated. We tested 3 KRAS mutations in colorectal cancer cell lines.

Results: We describe the recent progress in RMs. The short DNA fragments, such as sDNA and nDNA, exhibited higher quantitative values of dPCR compared to gDNA. The efficiency between Atlantis dsDNase (AD) and Micrococcal Nuclease (MN) affects DNA quantification. Moreover, there was a significant difference in dPCR output when spiking gDNA or nDNA containing KRAS mutations into FBS compared to the dPCR output under non-FBS conditions.

Conclusion: The matrix effect crucially affects the accuracy of gDNA and nDNA level estimation in the direct detection of mimic of patient samples. The form of reference material we proposed should be optimized for various conditions to develop reference materials that can accurately measure copy number and verify the detection of KRAS mutations in the matrix.
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http://dx.doi.org/10.1002/jcla.23344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439326PMC
August 2020

Subepithelial Spread of Early Gastric Signet Ring Cell Carcinoma: How Far They Can Reach?

Dig Dis 2020 19;38(6):442-448. Epub 2020 Mar 19.

Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea.

Introduction: Although signet ring cell carcinoma (SRC) is a poorly differentiated cancer subtype, recent studies suggest that endoscopic resection can be applied in small, mucosal early gastric SRC. However, other studies report frequent positive lines at the lateral resection margin after endoscopic treatment. Subepithelial spread beneath normal mucosa can exist in SRC, and such lesions may be the cause of positive margins after endoscopic resection. Thus, we conducted a retrospective study in order to evaluate the significance of subepithelial spread in early gastric SRC.

Method: Medical records of early gastric SRC patients who underwent surgery or endoscopic resection from January 2011 to December 2016 at a single tertiary hospital (Daejeon, South Korea) were reviewed to examine subepithelial spread and clinical datum. Two expert pathologists reviewed all pathologic specimens, and only patients showing a pure SRC component were included.

Results: Eighty-six patients were initially enrolled, and subepithelial spread existed in 62 patients (72.1%). The mean distance of subepithelial spread was 1,132.1 µm, and the maximal distance was 6,000 μm. Only discoloration was significantly associated with the presence of a subepithelial spread (p < 0.05, χ2 test, and logistic regression test). Distance of subepithelial spread did not correlate with total lesion size.

Conclusion: Subepithelial spread of early gastric SRC occurs frequently and can reach up to 6 mm. Lesion discoloration may be associated with the presence of subepithelial spread. Our results suggest that careful decision of the margin is needed when performing endoscopic resection of early gastric SRC.
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http://dx.doi.org/10.1159/000507322DOI Listing
November 2020

Anti‑inflammatory role of Prunus persica L. Batsch methanol extract on lipopolysaccharide‑stimulated glial cells.

Mol Med Rep 2020 May 10;21(5):2030-2040. Epub 2020 Mar 10.

College of Pharmacy, Chung‑Ang University, Seoul 06974, Republic of Korea.

Glial cells are the resident immune cells of the central nervous system. Reactive glial cells release inflammatory mediators that induce neurotoxicity or aggravate neurodegeneration. Regulation of glial activation is crucial for the initiation and progression of neuropathological conditions. Constituents of the peach tree (Prunus persica L. Batsch), which has a global distribution, have been found to exert therapeutic effects in pathological conditions, such as rashes, eczema and allergies. However, the therapeutic potential of its aerial parts (leaves, fruits and twigs) remains to be elucidated. The present study aimed to evaluate the anti‑inflammatory role of P. persica methanol extract (PPB) on lipopolysaccharide (LPS)‑stimulated glial cells. High‑performance liquid chromatography coupled with tandem mass spectrometry analysis showed that PPB contained chlorogenic acid and catechin, which have antioxidant properties. Western blot and reverse transcription polymerase chain reaction results indicated that PPB reduced the transcription of various proinflammatory enzymes (nitric oxide synthase and cyclooxygenase‑2) and cytokines [tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6] in LPS‑stimulated BV2 cells. In addition, PPB inhibited the activation of NF‑κB and various mitogen‑activated protein kinases required for proinflammatory mediator transcription. Finally, nitrite measurement and immunocytochemistry results indicated that PPB also suppressed nitrite production and NF‑κB translocation in LPS‑stimulated primary astrocytes. Thus, PPB may be used as a potential therapeutic agent for neurodegenerative diseases and neurotoxicity via the suppression of glial cell activation.
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http://dx.doi.org/10.3892/mmr.2020.11016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115241PMC
May 2020

Development of a three-dimensional in vitro co-culture model to increase drug selectivity for humans.

Diabetes Obes Metab 2020 08 16;22(8):1302-1315. Epub 2020 Apr 16.

Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea.

Aim: Insulin resistance is a metabolic state where insulin sensitivity is lower than normal condition and strongly related to type 2 diabetes. However, an in vitro model mimicking insulin resistance is rare and thus screening drugs for insulin resistance severely depends on an in vivo model. Here, to increase anti-diabetic drug selectivity for humans, 3D ADMSCs and macrophages were co-cultured with in-house fabricated co-culture plates.

Material And Methods: 3D co-culture plates were designed to load ADMSCs and RAW264.7 cells containing hydrogels in separate wells while allowing cell-cell interaction with co-culturing media. Hydrogels were constructed using a 3D cell-printing system containing 20 mg/ml alginate, 0.5 mg/ml gelatin and 0.5 mg/ml type I collagen. Cells containing hydrogels in 3D co-culture plates were incubated for 10 min to allow stabilization before the experiment. 3D co-culture plates were incubated with the CaCl2 solution for 5 min to complete the cross linking of alginate hydrogel. Cells in 3D co-culture plates were cultured for up to 12 days depending on the experiment and wells containing adipocytes and macrophages were separated and used for assays.

Results: KR-1, KR-2 and KR-3 compounds were applied during differentiation (12 days) in 3D co-cultured mouse 3T3-L1 adipocytes and 3D co-cultured human ADMSCs. Glucose uptake assay using 2-DG6P and 2-NBDG and western blot analysis were performed to investigate changes of insulin resistance in the 3D co-cultured model for interspecies selectivity of drug screening. KR-1 (mouse potent enantiomer) and KR-3 (racemic mixture) showed improvement of 2-DG and 2-NBDG uptake compared with KR-2 (human potent enantiomer) in 3D co-cultured 3T3-L1 adipocytes. In connection with insulin resistance in a 3D 3T3-L1 co-cultured model, KR-1 and KR-3 showed improvement of insulin sensitivity compared to KR-2 by markedly increasing GLUT4 expression. In contrast to the result of 3D co-cultured 3T3-L1 adipocytes, KR-1 failed to significantly improve 2-DG and 2-NBDG uptake in 3D co-cultured ADMSC adipocytes. Results of 2-NBDG accumulation and western blot analysis also showed that KR-2 and KR-3 improved insulin sensitivity relatively better than KR-1.

Conclusions: Our 3D co-culture model with/without 3D co-culture plates can successfully mimic insulin resistance while allowing investigation of the effects of anti-obesity or anti-diabetic drugs on human or mouse co-culturing cell type. This 3D co-culture system may accelerate screening of drugs for insulin resistance depending on species.
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http://dx.doi.org/10.1111/dom.14033DOI Listing
August 2020

2α-Hydroxyeudesma-4,11(13)-Dien-8β,12-Olide Isolated from Induces Apoptosis in Diffuse Large B-cell Lymphoma Cells.

Biomolecules 2020 02 18;10(2). Epub 2020 Feb 18.

Center for Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

2α-Hydroxyeudesma-4,11(13)-dien-8β,12-olide (HEDO), a eudesmane-type sesquiterpene lactone belonging to large group of plant terpenoids isolated from , displays cytotoxic activity against diffuse large B cell lymphoma cells in vitro. However, the molecular mechanism of the anticancer effect remains unclear. In this study, we showed that HEDO inhibits cell growth by inducing apoptosis in lymphoma cell lines through its antiproliferative activity. HEDO increases the depolarization of mitochondrial membrane potential and upregulated intracellular reactive oxygen species (ROS). Furthermore, we examined the cell cycle effect, and our results provided evidence that the arrest of the cell cycle at the SubG0/G1 phase plays an important role in the ability of HEDO to inhibit cell growth in Ontario Cancer Institute (OCI)-LY3 lymphoma cells by preventing nuclear factor-kappa B (NF-κB) signaling. In addition, HEDO induced apoptosis by instigating the activation of Bcl-2-associated X (BAX) and cleaved caspase-3, decreasing B-cell lymphoma 2 (BCL2), B-cell lymphoma-extra large (BCL-XL), and procaspase 3 expression levels. Based on these findings, we suggest that HEDO has potential as an anticancer drug of lymphoma by inducing ROS-dependent accumulation of SubG0/G1 arrest and apoptosis in OCI-LY3 cells.
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http://dx.doi.org/10.3390/biom10020324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072307PMC
February 2020

Active Turnover of Heme in Hibernation Period in Mammals.

Front Physiol 2019 15;10:1586. Epub 2020 Jan 15.

College of Pharmacy, Ajou University, Suwon, South Korea.

Heme oxygenase (HO)-1 plays an important role during hibernation by catalyzing the degradation of heme to biliverdin/bilirubin, ferrous iron, and carbon monoxide, which activates the protective mechanisms against stress. In this context, it was important to analyze the metabolic processes of heme. Nevertheless, to date, no study has approached on biosynthesis of heme. Therefore, our study aims to understand the process of heme biosynthesis, which regulates cell survival in conditions of hypothermia and calorie restriction (CR). During hibernation, the mRNA levels of enzymes responsible for heme biosynthesis were increased in the liver tissue of a Syrian hamster model of hibernation. Moreover, heme trafficking and iron metabolism were found to be more active, as assessed based on the changes in the levels of heme transporter and ferroportin mRNA. The levels of HO-1, a powerful antioxidant, were also upregulated during hibernation. Additionally, increased levels of Sirt-1 mRNA were also observed. These enzymes are known to act as cellular metabolic sensors that activate the cytoprotective mechanisms. These results indicate that HO-1 induction, brought about by the upregulation of heme during the pre-hibernation period, may protect against external stress. Here, we describe heme catabolism during hibernation by analyzing the regulation of the key molecular players involved in heme metabolism. Therefore, this study offers a new strategy for the better regulation of intracellular heme concentrations during hypothermia and other stresses.
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http://dx.doi.org/10.3389/fphys.2019.01586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974447PMC
January 2020

iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS.

Sci Rep 2019 11 14;9(1):16746. Epub 2019 Nov 14.

Center for Bioanalysis, Division of Chemical and Medical metrology, Korea Research Institute of Standards and Science, Daejeon, 34113, Republic of Korea.

The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.
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http://dx.doi.org/10.1038/s41598-019-53196-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856061PMC
November 2019

Antiproliferative Pterocarpans and Coumestans from .

J Nat Prod 2019 11 1;82(11):3025-3032. Epub 2019 Nov 1.

College of Pharmacy , Chonnam National University , Gwangju 61186 , Korea.

Chromatographic purification of a methanol extract of the roots of led to the isolation of four new pterocarpans (-), two new coumestans ( and ), two new arylbenzofurans ( and ), and the known pterocarpan 1-methoxyerythrabyssin II (). Their structures were identified using NMR spectroscopy, UV spectroscopy, and mass spectrometry. Cytotoxicity assays showed that compounds - exerted antiproliferative effects on blood cancer cells. Of these compounds, and induced mitochondrial depolarization and induced apoptosis in Jurkat cells. These compounds promoted cell death by inducing cell-cycle arrest at the G1 stage, reducing levels of BCL2, and increasing cleavage of PARP-1. These findings indicate that and are possible lead compounds for the treatment of human leukemia cells via intracellular signaling.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00567DOI Listing
November 2019

Differential Mechanism of ATP Production Occurs in Response to Succinylacetone in Colon Cancer Cells.

Molecules 2019 Oct 3;24(19). Epub 2019 Oct 3.

College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon 443-749, Korea.

Our aim was to verify the potential ability of succinylacetone (SA) to inhibit mitochondrial function, thereby suppressing cancer cell proliferation. SA treatment caused apoptosis in HCT116 and HT29 cells, but not in SW480 cells, with mitochondria playing a key role. We checked for dysfunctional mitochondria after SA treatment. Mitochondria of HT29 cells were swollen, indicating damage, whereas in HCT116 cells, several mitochondria had a diminished size. Damaged mitochondria decreased ATP production and induced reactive oxygen species (ROS) in the cells. To understand SA-induced reduction in ATP production, we investigated the electron transfer chains (ETC) and pyruvate dehydrogenase kinase (PDK) activity, which prevents the transfer of acetyl-CoA to the TCA (tricarboxylic acid) cycle by inhibiting PDH (pyruvate dehydrogenase) activity. In each cell line, the inhibitory mechanism of ATP by SA was different. The activity of complex III consisting of the mitochondrial ETCs in HT29 cells was decreased. In contrast, PDH activity in HCT116 cells was reduced. Nicotinamide nucleotide transhydrogenase (NNT)-removing reactive oxygen species (ROS) was upregulated in HT29 cells, but not in HCT116 cells, indicating that in HT29 cells, a defense mechanism was activated against ROS. Collectively, our study showed a differential mechanism occurs in response to SA in colon cancer cells.
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http://dx.doi.org/10.3390/molecules24193575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803967PMC
October 2019

Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Using 2D vs. 3D Culture in Colorectal Cancer Cells.

Biomolecules 2019 10 1;9(10). Epub 2019 Oct 1.

Center for Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.
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http://dx.doi.org/10.3390/biom9100557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843223PMC
October 2019

Platyphylloside Isolated from is Antiproliferative and Induces Apoptosis in Colon Cancer and Leukemic Cells.

Molecules 2019 Aug 15;24(16). Epub 2019 Aug 15.

Center for Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea.

bark has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diarylheptanoids are the major constituents of the bark and possess various pharmacological effects. Our previous study confirmed the selective antiproliferative effect of platyphylloside (BPP) isolated from on colon cancer and leukemic cells using 60 different cancer cell lines from thr National Cancer Institution (NCI). In line with previous reports, this study focuses on the apoptotic pathway of BPP, a phenolic glycoside composed of two aromatic rings joined by a seven-carbon chain. Cytotoxicity assays in solid tumor and blood cancer cell models demonstrated that BPP possesses potent antiproliferative activity. The level of apoptosis increased with BPP treatment, causing cell cycle arrest at the G1 phase along with the downregulation of IκBα phosphorylation and BCL-2, as well as upregulation of cleaved caspase 3 and BAX proteins. In addition, BPP displayed potent mitochondrial depolarization effects in Jurkat cells. The combined findings revealed that the cytotoxic effects of BPP were mediated by intracellular signaling, possibly through a mechanism involving the upregulation of mitochondrial reactive oxygen species (ROS). Thus, BPP could be a potential multitarget therapeutic agent in leukemia and colon cancer.
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http://dx.doi.org/10.3390/molecules24162960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720625PMC
August 2019

αβ-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture.

J Pharm Sci 2019 11 24;108(11):3704-3712. Epub 2019 Jul 24.

Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Republic of Korea. Electronic address:

Integrin αvβ3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvβ3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvβ3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs ([email protected]). [email protected] was successfully delivered to αvβ3-expressing A375 cells, and compared with nontargeted [email protected], it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with [email protected] compared with those incubated with [email protected] These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.
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http://dx.doi.org/10.1016/j.xphs.2019.07.011DOI Listing
November 2019

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T Cell Leukemia/Lymphoma.

Cancer Cell 2018 08 26;34(2):286-297.e10. Epub 2018 Jul 26.

Lymphoid Malignancies Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.
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http://dx.doi.org/10.1016/j.ccell.2018.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078141PMC
August 2018

Synergistic cooperation and crosstalk between and mutations that dysregulate CD79B and surface IgM.

J Exp Med 2017 Sep 12;214(9):2759-2776. Epub 2017 Jul 12.

Australian Cancer Research Foundation Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia

and mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of and mutations individually and combined in normal activated mouse B lymphocytes. mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing decreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of In B cells chronically stimulated by self-antigen, and mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by and provide an explanation for the co-occurrence of and mutations in lymphomas.
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http://dx.doi.org/10.1084/jem.20161454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584117PMC
September 2017

The MPN domain of Caenorhabditis elegans UfSP modulates both substrate recognition and deufmylation activity.

Biochem Biophys Res Commun 2016 08 28;476(4):450-456. Epub 2016 May 28.

Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea. Electronic address:

Ubiquitin-fold modifier 1 (Ufm1) specific protease (UfSP) is a novel cysteine protease that activates Ufm1 from its precursor by processing the C-terminus to expose the conserved Gly necessary for substrate conjugation and de-conjugates Ufm1 from the substrate. There are two forms: UfSP1 and UfSP2, the later with an additional domain at the N-terminus. Ufm1 and both the conjugating and deconjugating enzymes are highly conserved. However, in Caenorhabditis elegans there is one UfSP which has extra 136 residues at the N terminus compared to UfSP2. The crystal structure of cUfSP reveals that these additional residues display a MPN fold while the rest of the structure mimics that of UfSP2. The MPN domain does not have the metalloprotease activity found in some MPN-domain containing protein, rather it is required for the recognition and deufmylation of the substrate of cUfSP, UfBP1. In addition, the MPN domain is also required for localization to the endoplasmic reticulum.
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http://dx.doi.org/10.1016/j.bbrc.2016.05.143DOI Listing
August 2016

Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma.

Cancer Cell 2016 Apr;29(4):494-507

Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 4N115, Bethesda, MD 20892, USA. Electronic address:

Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activate IκB kinase (IKK) and the classical NF-κB pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-κB activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-κB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL.
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http://dx.doi.org/10.1016/j.ccell.2016.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026033PMC
April 2016
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