FLUVASTATIN SYNERGISTICALLY IMPROVES THE ANTIPROLIFERATIVE EFFECT OF EVEROLIMUS ON RAT SMOOTH MUSCLE CELLS BY ALTERING P27(KIP1)/CYCLIN E EXPRESSION

Objectives: A combination of two drugs with different mechanism of action may represent a suitable approach to control SMC proliferation, a prominent feature of in stent restenosis. Methods: In the present study, we investigated the effect of everolimus, an mTOR inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. Results: The antiproliferative action of everolimus was amplified by 2.5 fold by the addition of fluvastatin (5x10-7 M), lowering the IC50 value from 2.5x10-9 M to 1.0x10-9 M. Cell cycle analysis and [3H]-thymidine incorporation assay demonstrated that the two drugs synergistically interfered in G1 phase. The drug combination significantly upregulated p27Kip1 levels by 47.0%, suppressed cyclin E by 43.0%, and reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared to everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of SMCs to the antiproliferative action of the drug combination, measured by cell counting, [3H]-thymidine incorporation and cell cycle analysis. Conclusions: Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27kip1 which affect Rb phosphorylation and leading to G1 phase arrest. Funding: This research was supported by Novartis-Pharma, Basel, Switzerland.