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1.
J Nat Prod ; 83(6): 1817-1828, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32437150

RESUMO

Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many Viola 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 Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three Viola 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 V. arcuata 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.

2.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~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. Hybanthus enneaspermus (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 one known cyclotide (cycloviolacin O2) from H. enneaspermus 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, 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 H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

3.
ACS Chem Biol ; 15(6): 1650-1661, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32315152

RESUMO

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.

4.
J Chin Med Assoc ; 83(5): 446-453, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32243271

RESUMO

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.

5.
Biochimie ; 171-172: 124-135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32147511

RESUMO

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 IC50 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 Kd 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.

6.
ACS Chem Biol ; 15(4): 962-969, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32203656

RESUMO

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, Oldenlandia affinis, and can be produced recombinantly for use in vitro 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.

7.
PLoS One ; 15(2): e0229101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084186

RESUMO

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.


Assuntos
Antibioticoprofilaxia/métodos , Hemorragia Gastrointestinal/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Idoso , Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Feminino , Hemorragia Gastrointestinal/microbiologia , Mortalidade Hospitalar , Humanos , Cirrose Hepática/microbiologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
8.
J Med Chem ; 63(7): 3475-3484, 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32003561

RESUMO

Tachyplesin I (TPI) is a cationic ß-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.

9.
J Exp Clin Cancer Res ; 39(1): 10, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931847

RESUMO

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'.

10.
BMC Med Genomics ; 12(Suppl 8): 169, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31856824

RESUMO

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.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sistema Imunitário/citologia , Simulação por Computador , Humanos , Leucócitos Mononucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Máquina de Vetores de Suporte
11.
Chromosome Res ; 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712937

RESUMO

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.

12.
Toxicon ; 172: 33-44, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31682883

RESUMO

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.


Assuntos
Ciclotídeos/química , Magnoliopsida/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ciclotídeos/farmacologia , Ciclotídeos/toxicidade , Insetos/efeitos dos fármacos , Toxinas Biológicas/química , Toxinas Biológicas/toxicidade
13.
J Exp Clin Cancer Res ; 38(1): 474, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31771617

RESUMO

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.

14.
Biochem Biophys Res Commun ; 520(2): 353-358, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31604524

RESUMO

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 Šresolution (PDB entry 6JDG) that revealed a new binding mode, namely, (SSB)3:1. In the (SSB)65 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)3:1 mode. The bound ssDNA in the PaSSB-ssDNA complex adopts an Ω-shaped conformation rather than a χ-shaped conformation in the (SSB)65 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)3:1 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)3:1.

15.
Cancers (Basel) ; 11(10)2019 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-31614906

RESUMO

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.

16.
Int J Mol Sci ; 20(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540435

RESUMO

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.


Assuntos
Neoplasias Gastrointestinais/terapia , Imunoterapia/métodos , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/imunologia , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/imunologia , Humanos , Imunidade , Prognóstico , Microambiente Tumoral
17.
Cancers (Basel) ; 11(9)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505803

RESUMO

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.

18.
Biochem Biophys Res Commun ; 519(1): 160-165, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31481233

RESUMO

Dihydropyrimidinase (DHPase) catalyzes the reversible cyclization of dihydrouracil to N-carbamoyl-ß-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 Šresolution (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.

19.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31455019

RESUMO

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic ß-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.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Ciclização , Estabilidade de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Estrutura Molecular , Espectrometria de Fluorescência , Relação Estrutura-Atividade
20.
ACS Chem Biol ; 14(9): 2071-2087, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390185

RESUMO

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 in vitro 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.


Assuntos
Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Cíclicos/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Sequência de Aminoácidos , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/toxicidade , Sequências Hélice-Alça-Hélice , Humanos , Bicamadas Lipídicas/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/toxicidade , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/síntese química , Proteínas Supressoras de Tumor/toxicidade
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