The goal of this study is to investigate the mechanisms by which statins inhibit a novel target, the proteasome, and how such inhibition leads to cell cycle arrest. Statins such as Lovastatin are widely used in the treatment of high blood cholesterol by inhibiting HMG-COA Reductase the enzyme responsible for the conversion of HMG-COA to mevalonate, the rate limiting enzyme in cholesterol biosynthesis. We propose a new mechanism of interaction for the non lipid-related effects of Lovastatin whereby it inhibits the proteasome, upregulates cyclin dependent kinase inhibitors (CKI) and leads to cell cycle arrest. In support of this novel mechanism, we provide experimental evidence that drug treated cells undergo changes characteristic of proteasome inhibition and cell arrest. We show that upon treating cells with Lovastatin, they arrest in the G1 phase of the cell cycle, as indicated by FACS analysis. Western blot analysis shows upregulation of p21 and p27 leading to the inhibition of the CDK2 kinase activity. In vitro proteasome activity assay utilizing the prodrug form of Lovastatin inhibits the proteasome activity in a dose dependent fashion reminescent of the effects of the well characterized proteasome inhibitors such as lactacystin and MG-132. Lastly, agents possessing a lactone moiety, including inhibitors of downstream enzymes in the cholesterol biosynthesis pathway such as farnesyl transferase and geranyl geranyl transferase inhibitors (i.e. FTI-277 and GGTI-298), also block the proteasome activity in a concentration dependent manner. In this career development award, I propose to investigate how the drugs Lovastatin, structurally similar to inhibitors such as GGTI-298 and FTI-277 and lactacystin cause CKI induction and whether their paths intercept. I suspect that they do since both lactacystin and Lovastatin like agents inhibit the proteasome; both class of agents induce the CKIs similarly. I have 3 aims: 1) Determine if the accumulation of p21 and/or p27 resulting from Lovastatin treatment is sufficient to lead to G1 cell arrest in cultured cells by developing/using model systems devoid of p21, p27 or both. 2) Characterize the biochemical interaction of Lovastatin with the proteasome and determine how such interaction (s) cause an increase in p21 and p27 in breast tumor cells.3) Define the generality of lactone containing farnesyl, and geranylgeranly transferase inhibitors that account for their dual mode of action in the cholesterol biosynthesis vs the proteasome proteolytic pathway. The proposed project will contribute to the understanding of this novel action of statins, by helping to elucidate the mechanism of interaction of Lovastatin with the proteasome.
|Keyomarsi, Khandan; Efuet, Ekem T; Bui, Tuyen N (2011) Semi-high throughput method of measuring proteasome inhibition in vitro and in cultured cells. Cell Biol Toxicol 27:123-31|