scholarly journals Callyspongiolide kills cells by inducing mitochondrial dysfunction via cellular iron depletion

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jaeyoung Ha ◽  
Seung Bum Park
Keyword(s):  
Mitochondrial Dysfunction ◽  
Target Identification ◽  
Cancer Therapeutics ◽  
Marine Natural Product ◽  
Great Promise ◽  
Label Free ◽  
Antibody Drug Conjugate ◽  
Iron Depletion ◽  
Cellular Iron ◽  
Lysosomal Dysfunction

AbstractThe highly cytotoxic marine natural product callyspongiolide holds great promise as a warhead of antibody-drug conjugate in cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of protein-spot separation on 2-D gels upon treatment with callyspongiolide at increasing temperatures. During our exploration of what proteins were associated with this phenomenon as well as why it happens, we found that callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that callyspongiolide causes lysosomal dysfunction, which induces cellular iron depletion and leads to mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through iron supplementation. Although our approach was unable to reveal the direct protein targets of callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of callyspongiolide and specifically its cytotoxic activity via induction of mitochondrial dysfunction through cellular iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.

scholarly journals Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells

2006 ◽  
Vol 50 (11) ◽  
pp. 3597-3606 ◽  
Author(s):  
Tulika Prasad ◽  
Aparna Chandra ◽  
Chinmay K. Mukhopadhyay ◽  
Rajendra Prasad
Keyword(s):  
Multidrug Resistance ◽  
Membrane Fluidity ◽  
Iron Uptake ◽  
Efflux Pump ◽  
Disulfonic Acid ◽  
Candida Spp ◽  
Copper Transporter ◽  
Iron Depletion ◽  
Cellular Iron ◽  
Increased Sensitivity

ABSTRACT Inthis study, we show that iron depletion in Candida albicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely,Δ ftr1 and Δftr2, as well as the copper transporter mutation Δccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

scholarly journals Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 845
Author(s):  
Jesús Maldonado ◽  
Ana Belén González-Guerrero ◽  
Adrián Fernández-Gavela ◽  
Juan José González-López ◽  
Laura M. Lechuga
Keyword(s):  
Antimicrobial Resistance ◽  
Negative Impact ◽  
Multidrug Resistant ◽  
Therapeutic Interventions ◽  
Great Promise ◽  
Resistant Bacteria ◽  
Label Free ◽  
Multidrug Resistant Bacteria ◽  
Label Free Detection ◽  
Encoding Gene

Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.

scholarly journals MicroRNAs as Emerging Regulators of Signaling in the Tumor Microenvironment

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 911 ◽  
Author(s):  
Shahzad Nawaz Syed ◽  
Bernhard Brüne
Keyword(s):  
Tumor Microenvironment ◽  
Signaling Pathways ◽  
Target Identification ◽  
Cancer Therapeutics ◽  
Protein Translation ◽  
Transcriptional Gene Silencing ◽  
Post Transcriptional Gene Silencing ◽  
Cancer Initiation ◽  
Regulate Protein

A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME.

scholarly journals Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 508 ◽  
Author(s):  
Yonghyo Kim ◽  
Yutaka Sugihara ◽  
Tae Young Kim ◽  
Sung Min Cho ◽  
Jin Young Kim ◽  
...  
Keyword(s):  
Natural Products ◽  
Natural Compound ◽  
Target Identification ◽  
Growth Factor Receptor ◽  
Docking Simulation ◽  
Kinase Domain ◽  
Label Free ◽  
Downstream Signaling ◽  
Systematic Strategy ◽  
Specific Localization

Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.

scholarly journals Phosphotyrosine-based Phosphoproteomics for Target Identification and Drug Response Prediction in AML Cell Lines

2020 ◽  
Vol 19 (5) ◽  
pp. 884-899 ◽  
Author(s):  
Carolien van Alphen ◽  
Jacqueline Cloos ◽  
Robin Beekhof ◽  
David G. J. Cucchi ◽  
Sander R. Piersma ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Target Identification ◽  
Response Prediction ◽  
Clinical Samples ◽  
Label Free ◽  
Poor Treatment ◽  
Poor Treatment Outcome ◽  
The Individual

Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal.In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested.Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation.Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

scholarly journals Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI

2020 ◽  
Author(s):  
Yonghyo Kim ◽  
Yutaka Sugihara ◽  
Tae Young Kim ◽  
Sung Min Cho ◽  
Jin Young Kim ◽  
...  
Keyword(s):  
Natural Products ◽  
Natural Compound ◽  
Target Identification ◽  
Growth Factor Receptor ◽  
Docking Simulation ◽  
Kinase Domain ◽  
Label Free ◽  
Downstream Signaling ◽  
Systematic Strategy ◽  
Specific Localization

Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.

scholarly journals OrBITS: A High-throughput, time-lapse, and label-free drug screening platform for patient-derived 3D organoids

2021 ◽  
Author(s):  
Christophe Deben ◽  
Edgar Cardenas De La Hoz ◽  
Maxim Le Compte ◽  
Paul Van Schil ◽  
Jeroen M. Hendriks ◽  
...  
Keyword(s):  
Personalized Medicine ◽  
High Throughput ◽  
Basic Research ◽  
Automated Analysis ◽  
Time Lapse ◽  
Industrial Applications ◽  
Great Promise ◽  
Label Free ◽  
Current Standard ◽  
Convolutional Network

AbstractPatient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications. Here, we report the development of a high-throughput automated organoid analysis platform that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS). The combination of computer vision with a convolutional network machine learning approach allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D®-96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. We used OrBITS to screen chemotherapeutics and targeted therapies, and incorporation of a fluorescent cell death marker, revealed further insight into the mechanistic action of the drug, a feature not achievable with the current gold standard ATP-assay. This manuscript describes the validation of the OrBITS deep learning analysis approach against current standard assays for kinetic imaging and automated analysis of organoids. OrBITS, as a scalable, high-throughput technology, would facilitate the use of patient-derived organoids for drug development, therapy screening, and guided clinical decisions for personalized medicine. The developed platform also provides a launching point for further brightfield-based assay development to be used for fundamental research.

scholarly journals Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-β–dependent mechanisms

2016 ◽  
Vol 113 (31) ◽  
pp. E4558-E4566 ◽  
Author(s):  
Xiarong Shi ◽  
Valia T. Mihaylova ◽  
Leena Kuruvilla ◽  
Fang Chen ◽  
Stephen Viviano ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Acquired Resistance ◽  
Cancer Therapeutics ◽  
Receptor Expression ◽  
Great Promise ◽  
Therapeutic Benefits ◽  
Colorectal Cancer Cells ◽  
Tripartite Motif ◽  
Domain Protein ◽  
Β Receptor

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-β receptor expression and signaling, and blocking TGF-β receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-β signaling with BET bromodomain inhibition may offer new therapeutic benefits.

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