A Novel Roscovitine Derivative Potently Induces G₁-Phase Arrest in Platelet-Derived Growth Factor-BB-Activated Vascular Smooth Muscle Cells

Abstract

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize, a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC. Cell proliferation was quantified measuring DNA synthesis via 5-bromo-2Ј-deoxyuridine incorporation. Analysis of cell cycle distribution was done by flow cytometry using propidium iodide-stained nuclei. Key regulators of the cell cycle and relevant signaling pathways were dissected by Western blot analyses. In addition, in vitro kinase assays and in silico studies regarding the pharmacokinetic profile of both compounds were performed. LGR1406 shows a stronger (IC 50 ϭ 3.0 M) antiproliferative activity than ROSC (IC 50 ϭ 16.9 M), halting VSMCs in G 0 /G 1 phase of the cell cycle, whereas ROSC does not arrest but rather delays cell cycle progression. Neither of the compounds interferes with early PDGF-BB-induced signaling pathways (p38, extracellular signal-regulated kinase 1/2, c-Jun NH 2 -terminal kinase, Akt, signal transducer and activator of transcription 3), and both inhibit CDKs, with LGR1406 exerting a slightly higher potency against CDK1/2 and 4 than ROSC. Expression of cyclins A and E as well as hyperphosphorylation of the pocket proteins retinoblastoma protein and p107 are negatively affected by both compounds, although to a different extent. In silico calculations predicted a much higher metabolic stability for LGR1406 compared with ROSC. Altogether, ROSC derivatives, such as LGR1406 seem to be promising compounds for further development in antirestenotic therapy.Developing strategies against restenosis, the renarrowing of an artery after angioplastic interventions, remains an important goal of vascular biology and pharmacological research. Currently, drug-eluting stents have become the treatment of choice for patients undergoing percutaneous coronary revascularization (Kukreja et al., 2008). Current marketed first-generation drug eluting stents use drugs such as rapamycin and paclitaxel targeting mammalian target of rapamycin and tubulin, respectively (Windecker and Jü ni, 2008). Unfortunately, some concerns have been raised recently due to a potential increased risk of late-stent thrombosis (Steffel et al., 2008). Therefore, the identification of new drug candidates interfering with vascular smooth muscle cell (VSMC) proliferation using mechanisms other than rapamycin and paclitaxel is an important pharmacological topic.Roscovitine (ROSC) has been characterized as a selective inhibitor of cyclin dependent kinases (CDK) 1, 2, and 5 in enzyme-based assays (Meijer et al., 1997), inhibiting the proliferation of various cell types ranging from numerous cancer cell lines to keratinocytes and fibroblasts. The ob-

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“…Unless stated otherwise, the experiments presented in this study were performed in VSMCs isolated from thoracic aortas of Sprague-Dawley rats using a digestion method, as described previously (24). Cells were passaged twice a week and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 m m l -glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 10% newborn calf serum (all from Lonza, Basel, Switzerland) at 37 °C and 5% CO 2 .…”

Indirubin-3′-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration

“…15 Murine WT and Nrf2 −/− embryonic fibroblast were kindly provided by Thomas Kensler, University of Pittsburgh. 16 Rats and mice were kept and handled in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health.…”

Cyclin-Dependent Kinase 9 Activity Regulates Neutrophil Spontaneous Apoptosis

“…A novel roscovitine analogue LGR1406 [11] was a gift from Libor Havlíček (Institute of Experimental Botany ASCR, Prague) and NU6102 was kindly provided by D.R.Newell (University of Newcastle, UK).…”