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Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27^Kip1/Cyclin E Expression

Department of Pharmacological Sciences, University of Milan, Milan, Italy (N.F., A.G., C.P., F.T., A.C.); Novartis-Pharma AG, Basel, Switzerland (P.J.P.); and Department of Cardiology, Tenon Hospital, Assistance Publique, Hopitaux de Paris, Paris, France (R.D.)

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G₀ and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 x 10^-7 M), lowering the IC₅₀ value from 2.5 x 10^-9 to 1.0 x 10^-9 M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [³H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G₁ phase. In particular, the drug combination significantly up-regulated p27^Kip1 levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [³H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27^kip1, which affects Rb phosphorylation and leads to G₁ phase arrest.

Address correspondence to: Dr. Nicola Ferri, Department of Pharmacological Sciences, Via Balzaretti 9, 20133, Milan, Italy. E-mail: nicola.ferri{at}unimi.it