Publications by authors named "Yen-Hua Huang"

129 Publications

Clinical implications on HBV preS/S mutations and the effects of preS2 deletion on mitochondria, liver fibrosis, and cancer development.

Hepatology 2021 Mar 5. Epub 2021 Mar 5.

Translational Research Division, Medical Research Department, Taipei Veterans General Hospital, Taiwan, ROC, Taipei.

Background & Aims: PreS mutants of HBV have been reported to be associated with hepatocellular carcinoma (HCC). We conducted a longitudinal study of the role of HBV preS mutations on development of HCC, particularly in chronic hepatitis B (CHB) patients having low HBV DNA or ALT levels, and investigated effects of secretion-defective preS2 deletion mutant (preS2ΔMT) on hepatocyte damage in vitro and liver fibrosis in vivo.

Approach And Results: Association of preS mutations with HCC in 343 CHB patients was evaluated by retrospective case-control follow-up study. Effects of preS2ΔMT on HBsAg retention, ER stress, calcium accumulation, mitochondrial dysfunction, and liver fibrosis were examined. Multivariate analysis revealed significant association of preS mutations with HCC (HR: 3.210, 95% CI: 1.072-9.613; p=0.037) including cases with low HBV DNA or ALT levels (HR: 2.790, 95% CI: 1.133-6.873; p=0.026). Antiviral therapy reduced HCC risk, including cases with preS mutations. preS2ΔMT expression promoted HBsAg retention in ER and unfolded protein response (UPR). TEM examination, MitoTracker staining, real-time ATP assay, and calcium staining of preS2ΔMT-expressing cells revealed aberrant ER and mitochondrial ultrastructure, reduction of mitochondrial membrane potential and ATP production, and calcium overload. Serum HBV secretion levels were ~100-fold lower in preS2ΔMT-infected hu-FRG mice than in WT HBV-infected mice. preS2ΔMT-infected mice displayed upregulation of UPR and caspase-3, and enhanced liver fibrosis.

Conclusions: PreS mutations were significantly associated with HCC development in CHB patients, including those low HBV DNA or ALT levels. Antiviral therapy reduced HCC occurrence in CHB patients, including those with preS mutations. Intracellular accumulation of mutated HBsAg induced or promoted ER stress, calcium overload, mitochondrial dysfunction, impaired energy metabolism, liver fibrosis, and HCC.
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http://dx.doi.org/10.1002/hep.31789DOI Listing
March 2021

The Role of IGF/IGF-1R Signaling in Hepatocellular Carcinomas: Stemness-Related Properties and Drug Resistance.

Int J Mol Sci 2021 Feb 16;22(4). Epub 2021 Feb 16.

International PhD Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

Insulin-like Growth Factor (IGF)/IGF-1 Receptor (IGF-1R) signaling is known to regulate stem cell pluripotency and differentiation to trigger cell proliferation, organ development, and tissue regeneration during embryonic development. Unbalanced IGF/IGF-1R signaling can promote cancer cell proliferation and activate cancer reprogramming in tumor tissues, especially in the liver. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, with a high incidence and mortality rate in Asia. Most patients with advanced HCC develop tyrosine kinase inhibitor (TKI)-refractoriness after receiving TKI treatment. Dysregulation of IGF/IGF-1R signaling in HCC may activate expression of cancer stemness that leads to TKI refractoriness and tumor recurrence. In this review, we summarize the evidence for dysregulated IGF/IGF-1R signaling especially in hepatitis B virus (HBV)-associated HCC. The regulation of cancer stemness expression and drug resistance will be highlighted. Current clinical treatments and potential therapies targeting IGF/IGF-1R signaling for the treatment of HCC will be discussed.
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http://dx.doi.org/10.3390/ijms22041931DOI Listing
February 2021

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells.

ACS Chem Biol 2021 02 3;16(2):414-428. Epub 2021 Feb 3.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein-protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT-KD3 and cR10-KD3, able to activate the p53 pathway in cancer cells. cTAT-KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1-12 μM. cR10-KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 μM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.
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http://dx.doi.org/10.1021/acschembio.0c00988DOI Listing
February 2021

Immuno-Metabolism: The Role of Cancer Niche in Immune Checkpoint Inhibitor Resistance.

Int J Mol Sci 2021 Jan 27;22(3). Epub 2021 Jan 27.

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

The use of immune checkpoint inhibitors (ICI) in treating cancer has revolutionized the approach to eradicate cancer cells by reactivating immune responses. However, only a subset of patients benefits from this treatment; the majority remains unresponsive or develops resistance to ICI therapy. Increasing evidence suggests that metabolic machinery in the tumor microenvironment (TME) plays a role in the development of ICI resistance. Within the TME, nutrients and oxygen are scarce, forcing immune cells to undergo metabolic reprogramming to adapt to harsh conditions. Cancer-induced metabolic deregulation in immune cells can attenuate their anti-cancer properties, but can also increase their immunosuppressive properties. Therefore, targeting metabolic pathways of immune cells in the TME may strengthen the efficacy of ICIs and prevent ICI resistance. In this review, we discuss the interactions of immune cells and metabolic alterations in the TME. We also discuss current therapies targeting cellular metabolism in combination with ICIs for the treatment of cancer, and provide possible mechanisms behind the cellular metabolic rewiring that may improve clinical outcomes.
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http://dx.doi.org/10.3390/ijms22031258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865434PMC
January 2021

Niche Modulation of IGF-1R Signaling: Its Role in Stem Cell Pluripotency, Cancer Reprogramming, and Therapeutic Applications.

Front Cell Dev Biol 2020 12;8:625943. Epub 2021 Jan 12.

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

Stem cells work with their niches harmoniously during development. This concept has been extended to cancer pathology for cancer stem cells (CSCs) or cancer reprogramming. IGF-1R, a classical survival signaling, has been shown to regulate stem cell pluripotency, CSCs, or cancer reprogramming. The mechanism underlying such cell fate determination is unclear. We propose the determination is due to different niches in embryo development and tumor malignancy which modulate the consequences of IGF-1R signaling. Here we highlight the modulations of these niche parameters (hypoxia, inflammation, extracellular matrix), and the targeted stem cells (embryonic stem cells, germline stem cells, and mesenchymal stem cells) and CSCs, with relevance to cancer reprogramming. We organize known interaction between IGF-1R signaling and distinct niches in the double-sided cell fate with emerging trends highlighted. Based on these new insights, we propose that, through targeting IGF-1R signaling modulation, stem cell therapy and cancer stemness treatment can be further explored.
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http://dx.doi.org/10.3389/fcell.2020.625943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835526PMC
January 2021

Evaluation of the Aphrodisiac Activity of a Cyclotide Extract from .

J Nat Prod 2020 12 9;83(12):3736-3743. Epub 2020 Dec 9.

Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia.

is an Indian folk medicinal herb that has been widely used as a libido enhancer. This plant belongs to the Violaceae plant family, which ubiquitously contains disulfide-rich cyclic peptides named cyclotides. Cyclotides are an expanding plant-derived peptide family with numerous interesting bioactivities, and their unusual stability against proteolysis has attracted much attention in drug design applications. Recently, has been reported to be a rich source of cyclotides, and hence, it was of interest to investigate whether cyclotides contribute to its aphrodisiac activity. In this study, we evaluated the aphrodisiac activity of the herbal powder, extract, and the most abundant cyclotide, hyen D, extracted from on rats in a single dose regimen. After dosing, the sexual behaviors of male rats were observed, recorded, analyzed, and compared with those of the vehicle group. The results show that the extract and hyen D significantly decreased the intromission latency of sexually naïve male rats and the extract improved a range of other measured sexual parameters. The results suggest that the extract could enhance libido as well as facilitate erectile function in male rats and that the cyclotide hyen D could contribute to the libido-enhancing activity of this ethnomedicinal herb.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01045DOI Listing
December 2020

Cyclic gomesin, a stable redesigned spider peptide able to enter cancer cells.

Biochim Biophys Acta Biomembr 2021 Jan 24;1863(1):183480. Epub 2020 Sep 24.

Queensland University of Technology, School of Biomedical Sciences, Institute of Health & Biomedical Innovation and Translational Research Institute, Brisbane, QLD 4102, Australia; Institute for Molecular Bioscience, and Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address:

Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.
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http://dx.doi.org/10.1016/j.bbamem.2020.183480DOI Listing
January 2021

Whole genome sequencing identifies genetic variants associated with co-trimoxazole hypersensitivity in Asians.

J Allergy Clin Immunol 2020 Aug 10. Epub 2020 Aug 10.

Department of Dermatology, Drug Hypersensitivity Clinical and Research Center, Chang Gung Memorial Hospital, Linkou, Taipei, and Keelung, Taiwan; Cancer Vaccine and Immune Cell Therapy Core Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital and Chang Gung University, Linkou, Taiwan; Department of Dermatology, Xiamen Chang Gung Hospital, Xiamen, China; College of Medicine, Chang Gung University, Taoyuan, Taiwan; Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial Hospital, Keelung, Taiwan; Immune-Oncology Center of Excellence, Chang Gung Memorial Hospital, Linkou, Taiwan; Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Genomic Medicine Core Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan; Department of Dermatology, Beijing Tsinghua Chang Gung Hospital, School of Clinical Medicine, Tsinghua University, Beijing; School of Medicine, Shanghai Jiao Tong University, Shanghai. Electronic address:

Background: Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear.

Objective: We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR.

Methods: We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia.

Results: The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P = 8.2 × 10; odds ratio [OR] = 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). A replication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P = 7.2 × 10; OR = 8.7]). A strong HLA association was also observed in the case patients from Thailand (P = 3.2 × 10; OR = 3.6) and Malaysia (P = .002; OR = 12.8), respectively. A meta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P = 4.2 × 10; OR = 40.1).

Conclusion: This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians.
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http://dx.doi.org/10.1016/j.jaci.2020.08.003DOI Listing
August 2020

Drivers of community turnover differ between avian hemoparasite genera along a North American latitudinal gradient.

Ecol Evol 2020 Jun 9;10(12):5402-5415. Epub 2020 Jun 9.

Department of Biological Sciences University at Albany State University of New York Albany NY USA.

The latitudinal diversity gradient (LDG) is an established macroecological pattern, but is poorly studied in microbial organisms, particularly parasites. In this study, we tested whether latitude, elevation, and host species predicted patterns of prevalence, alpha diversity, and community turnover of hemosporidian parasites. We expected parasite diversity to decrease with latitude, alongside the diversity of their hosts and vectors. Similarly, we expected infection prevalence to decrease with latitude as vector abundances decrease. Lastly, we expected parasite community turnover to increase with latitudinal distance and to be higher between rather than within host species. We tested these hypotheses by screening blood and tissue samples of three closely related avian species in a clade of North American songbirds (Turdidae: ,  = 466) across 17.5° of latitude. We used a nested PCR approach to identify parasites in hemosporidian genera that are transmitted by different dipteran vectors. Then, we implemented linear-mixed effects and generalized dissimilarity models to evaluate the effects of latitude, elevation, and host species on parasite metrics. We found high diversity of hemosporidian parasites in thrushes ( = 44 lineages) but no evidence of latitudinal gradients in alpha diversity or prevalence. Parasites in the genus were most prevalent and lineage rich in this study system; however, there was limited turnover with latitude and host species. Contrastingly, parasites were less prevalent and diverse than parasites, yet communities turned over at a higher rate with latitude and host species. communities were skewed by the dominance of one or two highly prevalent lineages with broad latitudinal distributions. The few studies that evaluate the hemosporidian LDG do not find consistent patterns of prevalence and diversity, which makes it challenging to predict how they will respond to global climate change.
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http://dx.doi.org/10.1002/ece3.6283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319150PMC
June 2020

Exploring the Sequence Diversity of Cyclotides from Vietnamese Species.

J Nat Prod 2020 06 21;83(6):1817-1828. Epub 2020 May 21.

The Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of cyclotides from species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three species (, , and ) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the cyclotides isolated from was assessed. All tested cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.
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http://dx.doi.org/10.1021/acs.jnatprod.9b01218DOI Listing
June 2020

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb .

J Biol Chem 2020 08 15;295(32):10911-10925. Epub 2020 May 15.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb and have uncovered cyclotides with potential anticancer activity.
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http://dx.doi.org/10.1074/jbc.RA120.012627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415973PMC
August 2020

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold.

ACS Chem Biol 2020 06 6;15(6):1650-1661. Epub 2020 May 6.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.
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http://dx.doi.org/10.1021/acschembio.0c00297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831380PMC
June 2020

Identification of key genes and pathways associated with topotecan treatment using multiple bioinformatics tools.

J Chin Med Assoc 2020 May;83(5):446-453

Division of Radiation Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan, ROC.

Background: The goal of this study is to determine critical genes and pathways associated with topotecan using publicly accessible bioinformatics tools.

Methods: Topotecan signatures were downloaded from the Library of Integrated Network-Based Cellular Signatures (LINCS) database (http://www.ilincs.org/ilincs/). Differentially expressed genes (DEGs) were defined as genes that appeared at least three times with p values <0.05 and a fold change of ≥50% (|log2FC| ≥ 0.58). Hub genes were identified by evaluating the following parameters using a protein-protein interaction network: node degrees, betweenness, and eigenfactor scores. Hub genes and the top-40 DEGs by |log2FC| were used to generate a Venn diagram, and key genes were identified. Functional and pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Information on ovarian cancer patients derived from The Cancer Genome Atlas (TCGA) database was analyzed, and the effect of topotecan on the protein expression was examined by Western blotting.

Results: Eleven topotecan signatures were downloaded, and 65 upregulated and 87 downregulated DEGs were identified. Twenty-one hub genes were identified. We identified eight key genes as upregulated genes, including NFKBIA, IKBKB, GADD45A, CDKN1A, and HIST2H2BE, while EZH2, CDC20, and CDK7 were identified as downregulated genes, which play critical roles in the cell cycle and carcinogenesis in KEGG analysis. In the TCGA analysis, the CDKN1A+/EZH2- group had the longest median survival, while the CDKN1A-/EZH2+ group had the shortest median survival. Topotecan-treated murine ovarian (MOSEC), colorectal (CT26), and lung (LLC) cancer cell lines displayed upregulated CDKN1A encoding p21 and downregulated Ezh2.

Conclusion: Using publicly accessible bioinformatics tools, we evaluated key genes and pathways related to topotecan and examined the key genes using the TCGA database and in vitro studies.
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http://dx.doi.org/10.1097/JCMA.0000000000000313DOI Listing
May 2020

Circular Permutation of the Native Enzyme-Mediated Cyclization Position in Cyclotides.

ACS Chem Biol 2020 04 30;15(4):962-969. Epub 2020 Mar 30.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, , and can be produced recombinantly for use as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.
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http://dx.doi.org/10.1021/acschembio.9b00996DOI Listing
April 2020

Identification and characterization of dihydropyrimidinase inhibited by plumbagin isolated from Nepenthes miranda extract.

Biochimie 2020 Apr - May;171-172:124-135. Epub 2020 Mar 5.

School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan. Electronic address:

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. This enzyme is important in pyrimidine metabolism, and blocking its activity would be detrimental to cell survival. This study investigated the dihydropyrimidinase inhibition by plumbagin isolated from the extract of carnivorous plant Nepenthes miranda (Nm). Plumbagin inhibited dihydropyrimidinase with IC value of 58 ± 3 μM. Double reciprocal results of Lineweaver-Burk plot indicated that this compound is a competitive inhibitor of dihydropyrimidinase. Fluorescence quenching analysis revealed that plumbagin could form a stable complex with dihydropyrimidinase with the K value of 37.7 ± 1.4 μM. Docking experiments revealed that the dynamic loop crucial for stabilization of the intermediate state in dihydropyrimidinase might be involved in the inhibition effect of plumbagin. Mutation at either Y155 or K156 within the dynamic loop of dihydropyrimidinase caused low plumbagin binding affinity. In addition to their dihydropyrimidinase inhibition, plumbagin and Nm extracts also exhibited cytotoxicity on melanoma cell survival, migration, and proliferation. Further research can directly focus on designing compounds that target the dynamic loop in dihydropyrimidinase during catalysis.
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http://dx.doi.org/10.1016/j.biochi.2020.03.005DOI Listing
December 2020

Limited effects of antibiotic prophylaxis in patients with Child-Pugh class A/B cirrhosis and upper gastrointestinal bleeding.

PLoS One 2020 21;15(2):e0229101. Epub 2020 Feb 21.

Department of Biochemistry and Molecular Cell Biology, College of Medicine, Taipei Medical University, Taipei, Taiwan.

Current guidelines recommend antibiotic prophylaxis for all patients with various degrees of cirrhosis and upper gastrointestinal (UGI) bleeding. This study assessed the need for antibiotic prophylaxis in patients with low Child-Pugh scores. We retrospectively screened all patients with cirrhosis who underwent upper endoscopies for UGI bleeding in a referral hospital in Taiwan between 2003 and 2014, from which 913 patients were enrolled after excluding patients with active bacterial infections, recent antibiotic use, early death, and Child-Pugh class C cirrhosis. Among them, 73 (8%) received prophylactic antibiotics, and 45 (4.9%) exhibited 14-day bacterial infection. Neither Child-Pugh score nor model for end stage liver disease score were optimal for predicting bacterial infection because their areas under the curves were 0.610 (95% confidence interval [CI]: 0.529-0.691) and 0.666 (95% CI: 0.591-0.742), respectively. Antibiotic prophylaxis did not reduce the risks of 14-day bacterial infection (relative risk [RR]: 0.932, 95% CI: 0.300-2.891, P = 0.902), 14-day rebleeding (RR: 0.791, 95% CI: 0.287-2.181, P = 0.650), or 42-day mortality (RR: 2.710, 95% CI: 0.769-9.524, P = 0.121). The results remained similar after propensity score adjustment. On-demand antibiotic treatment might suffice for patients with Child-Pugh class A/B cirrhosis and UGI bleeding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229101PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034903PMC
May 2020

Enhanced Activity against Multidrug-Resistant Bacteria through Coapplication of an Analogue of Tachyplesin I and an Inhibitor of the QseC/B Signaling Pathway.

J Med Chem 2020 04 14;63(7):3475-3484. Epub 2020 Feb 14.

College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China.

Tachyplesin I (TPI) is a cationic β-hairpin antimicrobial peptide with broad-spectrum, potent antimicrobial activity. In this study, the all d-amino acid analogue of TPI (TPAD) was synthesized, and its structure and activity were determined. TPAD has comparable antibacterial activity to TPI on 14 bacterial strains, including four drug-resistant bacteria. Importantly, TPAD has significantly improved stability against enzymatic degradation and decreased hemolytic activity compared to TPI, indicating that it has better therapeutic potential. The induction of bacterial resistance using low concentrations of TPAD resulted in the activation of the QseC/B two-component system. Deletion of this system resulted in at least five-fold improvement of TPAD activity, and the combined use of TPAD with LED209, a QseC/B inhibitor, significantly enhanced the bactericidal effect against three classes of multidrug-resistant bacteria.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01563DOI Listing
April 2020

Correction to: DNMT3b/OCT4 expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through IL-6/STAT3 regulation.

J Exp Clin Cancer Res 2020 Jan 13;39(1):10. Epub 2020 Jan 13.

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.

In the original publication of this article [1], labelling within Fig. 7a was incorrect. The updated figure is shown below, with 'DMT1' now corrected to read 'DNMT1'.
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http://dx.doi.org/10.1186/s13046-019-1518-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956484PMC
January 2020

Improved cell composition deconvolution method of bulk gene expression profiles to quantify subsets of immune cells.

BMC Med Genomics 2019 12 20;12(Suppl 8):169. Epub 2019 Dec 20.

Institute of Biomedical Informatics, National Yang-Ming University, No.155, Sec. 2, Li-Nong St., Beitou Dist, Taipei, 11221, Taiwan.

Background: To facilitate the investigation of the pathogenic roles played by various immune cells in complex tissues such as tumors, a few computational methods for deconvoluting bulk gene expression profiles to predict cell composition have been created. However, available methods were usually developed along with a set of reference gene expression profiles consisting of imbalanced replicates across different cell types. Therefore, the objective of this study was to create a new deconvolution method equipped with a new set of reference gene expression profiles that incorporate more microarray replicates of the immune cells that have been frequently implicated in the poor prognosis of cancers, such as T helper cells, regulatory T cells and macrophage M1/M2 cells.

Methods: Our deconvolution method was developed by choosing ε-support vector regression (ε-SVR) as the core algorithm assigned with a loss function subject to the L1-norm penalty. To construct the reference gene expression signature matrix for regression, a subset of differentially expressed genes were chosen from 148 microarray-based gene expression profiles for 9 types of immune cells by using ANOVA and minimizing condition number. Agreement analyses including mean absolute percentage errors and Bland-Altman plots were carried out to compare the performances of our method and CIBERSORT.

Results: In silico cell mixtures, simulated bulk tissues, and real human samples with known immune-cell fractions were used as the test datasets for benchmarking. Our method outperformed CIBERSORT in the benchmarks using in silico breast tissue-immune cell mixtures in the proportions of 30:70 and 50:50, and in the benchmark using 164 human PBMC samples. Our results suggest that the performance of our method was at least comparable to that of a state-of-the-art tool, CIBERSORT.

Conclusions: We developed a new cell composition deconvolution method and the implementation was entirely based on the publicly available R and Python packages. In addition, we compiled a new set of reference gene expression profiles, which might allow for a more robust prediction of the immune cell fractions from the expression profiles of cell mixtures. The source code of our method could be downloaded from https://github.com/holiday01/deconvolution-to-estimate-immune-cell-subsets.
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http://dx.doi.org/10.1186/s12920-019-0613-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923925PMC
December 2019

DNMT3b/OCT4 expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through IL-6/STAT3 regulation.

J Exp Clin Cancer Res 2019 Nov 26;38(1):474. Epub 2019 Nov 26.

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.

Background: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear.

Methods: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo.

Results: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells.

Conclusions: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b-mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.
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http://dx.doi.org/10.1186/s13046-019-1442-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878666PMC
November 2019

The maize B chromosome is capable of expressing microRNAs and altering the expression of microRNAs derived from A chromosomes.

Chromosome Res 2020 06 11;28(2):129-138. Epub 2019 Nov 11.

Department of Agronomy, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan.

Supernumerary B chromosomes (Bs) are nonessential chromosomes that are considered genetically inert. However, the maize B carries control elements that direct its behavior, such as that of nondisjunction, during the second pollen mitosis, and affects normal A chromosomes during cell division. Recently, the maize B has been found to contain transcriptionally active sequences and to affect the transcription of genes on A chromosomes. To better understand the regulatory mechanisms underlying the maize B, we constructed two small RNA libraries from maize B73 inbred lines with and without Bs. The sequencing results revealed that 18 known microRNAs (miRNAs) were significantly differentially expressed in response to the presence of the B, and most target mRNAs were characterized as transcription factors. Moreover, three novel B-derived miRNAs were identified via stem-loop reverse transcriptase-polymerase chain reaction (RT-PCR)-based analysis, and all showed consistent B-specific expression in almost all analyzed inbred lines and in all tissue types, including leaves, roots, and pollen grains. By the use of B-10L translocations, the three B-derived miRNAs were mapped to specific B regions. The results from this study suggest that the maize B can express miRNAs and affect the expression of A-derived miRNAs, which could regulate the expression of A-located genes.
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http://dx.doi.org/10.1007/s10577-019-09620-2DOI Listing
June 2020

Cyclotides: Disulfide-rich peptide toxins in plants.

Toxicon 2019 Oct 1;172:33-44. Epub 2019 Nov 1.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia. Electronic address:

Cyclotides are a plant-derived family of peptides that comprise approximately 30 amino acid residues, a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action mechanisms. We focus on their toxicities to insect pests and their medical and agricultural applications.
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http://dx.doi.org/10.1016/j.toxicon.2019.10.244DOI Listing
October 2019

High Expression of MicroRNA-196a is Associated with Progression of Hepatocellular Carcinoma in Younger Patients.

Cancers (Basel) 2019 Oct 13;11(10). Epub 2019 Oct 13.

Institute of Clinical Medicine, National Yang-Ming University, Taipei 11221, Taiwan.

MicroRNAs are small RNAs involved in various biological processes and cancer metastasis. miR-196a was associated with aggressive behaviors in several cancers. The role of miR-196a in hepatocellular carcinoma (HCC) metastasis remains unknown. This study aimed to examine the role of miR-196a in HCC progression. Expression of miR-196a was measured in 83 human HCC samples. The HCC patients with high miR-196a expression had younger ages, lower albumin levels, higher frequency with alpha-fetoprotein (AFP) levels ≥20 ng/mL, more macrovascular invasion, and non-early stages. Kaplan-Meier analysis showed that high miR-196a expression was associated with lower recurrence-free survival. Knockdown of miR-196a decreased transwell invasiveness, sphere formation, transendothelial invasion, and Slug, Twist, Oct4, and Sox2 expression, suppressed angiogenesis, and reduced sizes of xenotransplants and number of pulmonary metastasis. Down-regulation of miR-196a decreased Runx2 and osteopontin (OPN) levels. Knockdown of Runx2 in vitro resulted in comparable phenotypes with miR-196a down-regulation. Restoration of Runx2 in miR-196a-knockdown HCC reverted tumor phenotypes. This study showed that high expression of miR-196a is associated with HCC progression in a subset of younger patients. miR-196a mediates HCC progression via upregulation of Runx2, OPN, epithelial-mesenchymal transition (EMT) regulators, and stemness genes. We proposed that miR-196a can be used as a prognostic marker and a potential therapeutic target.
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http://dx.doi.org/10.3390/cancers11101549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826650PMC
October 2019

Complexed crystal structure of SSB reveals a novel single-stranded DNA binding mode (SSB): Phe60 is not crucial for defining binding paths.

Biochem Biophys Res Commun 2019 12 8;520(2):353-358. Epub 2019 Oct 8.

School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan. Electronic address:

Single-stranded DNA-binding protein (SSB) is essential to cells as it participates in DNA metabolic processes, such as DNA replication, repair, and recombination. Escherichia coli SSB (EcSSB) tetramer cooperatively binds and wraps ssDNA in two major binding modes. In this study, we report the complex crystal structure of Pseudomonas aeruginosa SSB (PaSSB) with ssDNA dT20 at 2.39 Å resolution (PDB entry 6JDG) that revealed a new binding mode, namely, (SSB). In the (SSB) mode revealed by the EcSSB-dC35 complex structure, all four subunits fully participate in the binding to ssDNA. However, only three subunits in the PaSSB tetramer can participate in wrapping ssDNA in the (SSB) mode. The bound ssDNA in the PaSSB-ssDNA complex adopts an Ω-shaped conformation rather than a χ-shaped conformation in the (SSB) mode possibly due to the disability of Phe60. Phe60 is known to play a critical role in defining DNA-binding paths and promoting the wrapping of ssDNA around SSB tetramers. However, it is not important in the (SSB) mode. The ssDNA binding path revealed by our structural evidence suggests that ssDNA occupies half of the binding sites of the two subunits and slightly comes into contact with the ssDNA binding sites of the third subunit. Accordingly, we propose and sketch a possible wrapping mechanism of SSB via this novel ssDNA-binding mode, (SSB).
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http://dx.doi.org/10.1016/j.bbrc.2019.10.036DOI Listing
December 2019

Conceptual Development of Immunotherapeutic Approaches to Gastrointestinal Cancer.

Int J Mol Sci 2019 Sep 18;20(18). Epub 2019 Sep 18.

International PhD Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

Gastrointestinal (GI) cancer is one of the common causes of cancer-related death worldwide. Chemotherapy and/or immunotherapy are the current treatments, but some patients do not derive clinical benefits. Recently, studies from cancer molecular subtyping have revealed that tumor molecular biomarkers may predict the immunotherapeutic response of GI cancer patients. However, the therapeutic response of patients selected by the predictive biomarkers is suboptimal. The tumor immune-microenvironment apparently plays a key role in modulating these molecular-determinant predictive biomarkers. Therefore, an understanding of the development and recent advances in immunotherapeutic pharmacological intervention targeting tumor immune-microenvironments and their potential predictive biomarkers will be helpful to strengthen patient immunotherapeutic efficacy. The current review focuses on an understanding of how the host-microenvironment interactions and the predictive biomarkers can determine the efficacy of immune checkpoint inhibitors. The contribution of environmental pathogens and host immunity to GI cancer is summarized. A discussion regarding the clinical evidence of predictive biomarkers for clinical trial therapy design, current immunotherapeutic strategies, and the outcomes to GI cancer patients are highlighted. An understanding of the underlying mechanism can predict the immunotherapeutic efficacy and facilitate the future development of personalized therapeutic strategies targeting GI cancers.
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http://dx.doi.org/10.3390/ijms20184624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769557PMC
September 2019

Triple-Negative Breast Cancer: Current Understanding and Future Therapeutic Breakthrough Targeting Cancer Stemness.

Cancers (Basel) 2019 Sep 9;11(9). Epub 2019 Sep 9.

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.

Triple-negative breast cancer (TNBC) is cancer that tested as negative for estrogen receptors (ER), progesterone receptors (PR), and excess human epidermal growth factor receptor 2 (HER2) protein which accounts for 15%-20% of all breast cancer cases. TNBC is considered to be a poorer prognosis than other types of breast cancer, mainly because it involves more aggressive phenotypes that are similar to stem cell-like cancer cells (cancer stem cell, CSC). Thus, targeted treatment of TNBC remains a major challenge in clinical practice. This review article surveys the latest evidence concerning the role of genomic alteration in current TNBC treatment responses, current clinical trials and potential targeting sites, CSC and drug resistance, and potential strategies targeting CSCs in TNBC. Furthermore, the role of insulin-like growth factor 1 receptor (IGF-1R) and nicotinic acetylcholine receptors (nAChR) in stemness expression, chemoresistance, and metastasis in TNBC and their relevance to potential treatments are also discussed and highlighted.
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http://dx.doi.org/10.3390/cancers11091334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769912PMC
September 2019

Crystal structure of dihydropyrimidinase in complex with anticancer drug 5-fluorouracil.

Biochem Biophys Res Commun 2019 10 31;519(1):160-165. Epub 2019 Aug 31.

School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan. Electronic address:

Dihydropyrimidinase (DHPase) catalyzes the reversible cyclization of dihydrouracil to N-carbamoyl-β-alanine in the second step of the pyrimidine degradation pathway. Whether 5-fluorouracil (5-FU), the best-known fluoropyrimidine that is used to target the enzyme thymidylate synthase for anticancer therapy, can bind to DHPase remains unknown. In this study, we found that 5-FU can form a stable complex with Pseudomonas aeruginosa DHPase (PaDHPase). The crystal structure of PaDHPase complexed with 5-FU was determined at 1.76 Å resolution (PDB entry 6KLK). Various interactions between 5-FU and PaDHPase were examined. Six residues, namely, His61, Tyr155, Asp316, Cys318, Ser289 and Asn337, of PaDHPase were involved in 5-FU binding. Except for Cys318, these residues are also known as the substrate-binding sites of DHPase. 5-FU interacts with the main chains of residues Ser289 (3.0 Å) and Asn337 (3.2 Å) and the side chains of residues Tyr155 (2.8 Å) and Cys318 (2.9 Å). Mutation at either Tyr155 or Cys318 of PaDHPase caused a low 5-FU binding activity of PaDHPase. This structure and the binding mode provided molecular insights into how the dimetal center in DHPase undergoes a conformational change during 5-FU binding. Further research can directly focus on revisiting the role of DHPase in anticancer therapy.
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http://dx.doi.org/10.1016/j.bbrc.2019.08.153DOI Listing
October 2019

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Int J Mol Sci 2019 Aug 26;20(17). Epub 2019 Aug 26.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic β-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.
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http://dx.doi.org/10.3390/ijms20174184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747087PMC
August 2019

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

ACS Chem Biol 2019 09 21;14(9):2071-2087. Epub 2019 Aug 21.

Institute for Molecular Bioscience , the University of Queensland , St. Lucia , QLD 4072 , Australia.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.
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http://dx.doi.org/10.1021/acschembio.9b00593DOI Listing
September 2019

Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor.

FEBS Lett 2019 06 31;593(12):1336-1350. Epub 2019 May 31.

Syngenta, Jealott's Hill Research Centre, Bracknell, Berkshire, UK.

The insecticidal effects of ω-hexatoxin-Hv1a, κ-hexatoxin-Hv1c and ω/κ-hexatoxin-Hv1h are currently attributed to action at calcium and potassium channels. By characterizing the binding of these toxins to neuronal membranes, we show that they have more potent effects as positive allosteric modulators (PAMs) of insect nicotinic acetylcholine receptors (nAChRs), consistent with their neuroexcitatory toxicology. Alanine scanning analysis of ω-hexatoxin-Hv1a reveals a structure-activity relationship for binding that mirrors that for insecticidal activity. Spinosyn A does not compete with ω-hexatoxin-Hv-1a for binding, and we show that these two PAMs have distinct pharmacology of binding indicating that they act at different receptor populations. These toxins represent valuable tools for the characterization of insect nAChRs and for the development of more selective agrochemicals.
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http://dx.doi.org/10.1002/1873-3468.13435DOI Listing
June 2019