This is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. According to molecular docking analysis, 7HF preferred to bind to β-tubulin over α-tubulin. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. 7HF reduced polymerization of α-, β-tubulin in dose-dependent manner. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. We observed that 7HF was able to enter the MDA-MB-468 cells. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting.
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Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. Polymerization of α- and β-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Then, the effect of 7HF treatment (6, 12, and 24 µM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. MethodsħHF uptake by MDA-MB-468 cells was assessed using spectrophotometry. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Triple-negative breast cancer (TNBC) responds poorly to the available drugs thus, the mortality rate associated with TNBC remains high. Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells. Cite this article Chimplee S, Smythe C, Tipmanee V, Sukrong S, Kanokwiroon K. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. Licence This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. 3 Center of Excellence in DNA Barcoding of Thai Medicinal Plants, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand DOI 10.7717/peerj.13508 Published Accepted Received Academic Editor Vladimir Uversky Subject Areas Biochemistry, Cell Biology, Computational Biology, Molecular Biology Keywords 7-α-Hydroxyfrullanolide, Sesquiterpene lactones, Natural product, Triple-negative breast cancer, Microtubules, G2/M arrest, G1 arrest, DNA damage response, Molecular docking Copyright © 2022 Chimplee et al.
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