BiochimiePub Date : 2025-04-04DOI: 10.1016/j.biochi.2025.04.001
Linda Saxe Einbond, Kunhui Huang, Michael Balick, Hongbao Ma, Rajendra Gharbaran, Stephen Redenti, Hsan-Au Wu
{"title":"Transcriptomic analysis of digitoxin: Synergy with doxorubicin in Her2-overexpressing MDA-MB-453 breast cancer cells.","authors":"Linda Saxe Einbond, Kunhui Huang, Michael Balick, Hongbao Ma, Rajendra Gharbaran, Stephen Redenti, Hsan-Au Wu","doi":"10.1016/j.biochi.2025.04.001","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.04.001","url":null,"abstract":"<p><p>The aim of this research is to further elucidate the mechanism of action of digitoxin and explore its potential synergistic effects with doxorubicin. MDA-MB-453 breast cancer cells, characterized by Her2 overexpression and low ER levels, were exposed to digitoxin at three doses (0.1 (0.13 μM), 0.2, and 1.0 μg/ml). RNA was extracted over 6 and 24-hour periods to subject to transcriptomic analysis, using IPA software. To validate the findings, cell growth inhibitory, Western blot, and enzymatic assays were performed. In addition, molecular docking was carried out to assess the interaction of digitoxin and doxorubicin with the Na+/K+- ATPase. IPA analysis indicates that the effects of digitoxin are dose and time-dependent; at the highest dose, digitoxin activates the transcription of cholesterol biosynthetic genes at early times, and the stress response gene ATF3 at later times. Key genes at the central point of the pathways altered by digitoxin include: (activated) TP53, CREB1, and TGFB1 at the highest dose at 6 and 24 h and (repressed) MYCN at the middle dose at 24 h. ATF3 also plays a role in the action of doxorubicin, and digitoxin exhibits synergy with doxorubicin in MDA-MB-453 cells. Molecular docking studies demonstrated binding potential of both digitoxin and doxorubicin to Na+/K+-ATPase, with doxorubicin showing a stronger binding affinity. Our results highlight the role of bioelectric signaling through ion channel proteins, like Na+/K+-ATPase, in cancer development. Our findings suggest it is worthwhile to study the use of digitoxin, alone or combined with doxorubicin, for treating estrogen receptor-negative breast cancer, but caution of possible risks to patients who take both drugs in combination.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143797259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Master of disguise: Ribosomal Protein L5 Beyond Translation.","authors":"Guglielmo Rambaldelli, Lorenza Bacci, Daniela Pollutri, Kamil Filipek, Marianna Penzo","doi":"10.1016/j.biochi.2025.03.009","DOIUrl":"https://doi.org/10.1016/j.biochi.2025.03.009","url":null,"abstract":"<p><p>Ribosomal proteins (RPs), key components of ribosomes, are traditionally associated with protein synthesis. However, emerging evidence suggests their involvement in diverse cellular functions beyond ribosomal biogenesis and translation, including transcriptional regulation. This study aimed at investigating the potential of RPs as transcriptional regulators by analyzing their interacting protein network. A subset of RP interactors exhibiting transcriptional regulatory functions was subjected to Gene Ontology analysis to identify enriched functional pathways. The results indicated that these interactions may play a role in different cellular pathways relevant to a number of biological processes, including cancer. To further explore this hypothesis, a virtual knockdown of RPL5 was performed in ovarian and breast cancer public data. As proof of concept the same experiments were conducted in vitro to validate the computational findings, confirming the potential of RPL5 in transcriptional regulation in cancer. This seminal study provides a foundation for future investigations into the multifaceted roles of RPs in the regulation of gene expression in physiological and pathological contexts.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BiochimiePub Date : 2025-02-20DOI: 10.1016/j.biochi.2025.02.002
Jean-Philippe Robin, Vincent Mocquet
{"title":"Hijacking a real time detection thermocycler for enzymology: Improvement of a fluorescent bulk assay monitoring helicase activity.","authors":"Jean-Philippe Robin, Vincent Mocquet","doi":"10.1016/j.biochi.2025.02.002","DOIUrl":"10.1016/j.biochi.2025.02.002","url":null,"abstract":"<p><p>Helicases are enzymes involved in all aspects of nucleic acid synthesis, regulation and degradation. As a consequence, several methods were developed to monitor their enzymatic activity. In this report, we described an improvement of bulk fluorescent helicase assays to overcome their specific limitations (cost, health and safety regulations, etc.). Using a real time detection thermocycler to monitor the fluorescence in real-time, we managed to precisely control the initiation of the helicase reaction through temperature tuning. Therefore, we were able to demonstrate that this setup could provide a qualitative and a quantitative evaluation of the helicase domain of the UPF1 helicase (UPF1-HD) and that several fluorophores could be used in parallel during the same run.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BiochimiePub Date : 2024-05-15DOI: 10.1016/j.biochi.2024.05.014
Ivan Petushkov, Andrey Feklistov, Andrey Kulbachinskiy
{"title":"Highly specific aptamer trap for extremophilic RNA polymerases.","authors":"Ivan Petushkov, Andrey Feklistov, Andrey Kulbachinskiy","doi":"10.1016/j.biochi.2024.05.014","DOIUrl":"https://doi.org/10.1016/j.biochi.2024.05.014","url":null,"abstract":"<p><p>During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.</p>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140960867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BiochimiePub Date : 2023-01-24DOI: 10.1016/j.biochi.2023.01.009
Zoltán Szeltner , Györgyi Ferenc , Tünde Juhász , Zoltán Kupihár , Zoltán Váradi , Dávid Szüts , Lajos Kovács
{"title":"Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions","authors":"Zoltán Szeltner , Györgyi Ferenc , Tünde Juhász , Zoltán Kupihár , Zoltán Váradi , Dávid Szüts , Lajos Kovács","doi":"10.1016/j.biochi.2023.01.009","DOIUrl":"https://doi.org/10.1016/j.biochi.2023.01.009","url":null,"abstract":"<div><p><span><span>Guanine<span> quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the </span></span>nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model </span>oligonucleotides<span><span><span><span>. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the </span>cytosine </span>deamination product 5-hydroxyuracil and its nucleoside counterpart in </span>RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.</span></p></div>","PeriodicalId":93898,"journal":{"name":"Biochimie","volume":"214 ","pages":"Pages 33-44"},"PeriodicalIF":0.0,"publicationDate":"2023-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49670757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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