Helge Joa1, Tina Blažević2, Christoph Grojer3, Iris Zeller4, Elke H Heiss5, Atanas G Atanasov6, Ines Feldler5, Päivi Gruzdaitis7, Christa Czaloun8, Peter Proksch9, Barbara Messner10, David Bernhard11, Verena M Dirsch5. 1. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria; vasopharm GmbH, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany. 2. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria. Electronic address: tina.blazevic@univie.ac.at. 3. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria; Pfizer Corp. Austria GmbH, Floridsdorfer Hauptstraße 1, 1210 Wien, Austria. 4. Cardiac Surgery Research Laboratory, Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria; Division of Clinical Microbiology, Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. 5. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria. 6. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria; Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 05-552, Jastrzebiec, Poland. 7. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria; Pharmaceutical Information Centre Ltd., Korkeavuorenkatu 35, 00130 Helsinki, Finland. 8. Department of Pharmacognosy, University of Vienna, Althanstraße 14, Vienna 1090, Austria; Auge Gottes Apotheke, Nussdorfer Straße 79, 1090 Wien, Austria. 9. Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Universitätsstraße 1, Düsseldorf 40225, Germany. 10. Cardiac Surgery Research Laboratory, Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria. 11. Cardiac Surgery Research Laboratory, Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria; Center for Medical Research, Johannes Kepler University Linz, Krankenhausstr. 7a, 4020 Linz, Austria.
Abstract
BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/ PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.
BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/ PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.
Authors: Ehab M Mostafa; Hamdoon A Mohammed; Arafa Musa; Mohamed A Abdelgawad; Mohammad M Al-Sanea; Suliman A Almahmoud; Mohammed M Ghoneim; Hesham A M Gomaa; Fatema El-Zahraa S Abdel Rahman; Khaled Shalaby; Samy Selim; Riaz A Khan Journal: Plants (Basel) Date: 2022-05-12