Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase

Abstract

In this paper we present the finding that lovastatin arrests cells by inhibiting the proteasome, which results in the accumulation of p21 and p27, leading to G1 arrest. Lovastatin is an inhibitor of hydroxymethyl glutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Previously, we reported that lovastatin can be used to arrest cultured cells in the G1 phase of the cell cycle, resulting in the stabilization of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27. In this report we show that this stabilization of p21 and p27 may be the result of a previously unknown function of the pro-drug, beta-lactone ring form of lovastatin to inhibit the proteasome degradation of these CKIs. The lovastatin mixture used in this study is 80% open-ring form and 20% pro-drug, beta-lactone form. We show that while the lovastatin open-ring form and pravastatin (a lovastatin analogue, 100% open ring) inhibit the HMG-CoA reductase enzyme, lovastatin pro-drug inhibits the proteasome but does not inhibit HMG-CoA reductase. In addition, many of the properties of proteasome inhibition by the pro-drug are the same as the specific proteasome inhibitor lactacystin. Lastly, mevalonate (used to rescue cells from lovastatin arrest) unexpectedly abrogates the lactacystin and lovastatin pro-drug inhibition of the proteasome. Mevalonate increases the activity of the proteasome, which results in degradation of the CKIs, allowing lovastatin- and lactacystin-arrested cells to resume cell division. The lovastatin-mediated inhibition of the proteasome suggests a unique mechanism for the chemopreventative effects of this agent seen in human cancer.

Figure 1

Induction of CKIs by the β-lactone form of lovastatin. (A) Chromatographic separation of lovastatin mixture (Upper) and closed-ring form (Lower) by HPLC analysis as described (15, 25). (B) Fractions corresponding to each HPLC peak were collected and subjected to mass determination by electrospray ionization quadrupole mass spectrometry analysis as described (26). The lovastatin mixture and closed-ring forms also were subjected to mass spectrometry analysis to determine the components of each reagent. (C) MDA-MB-157 tumor cells were treated with the indicated concentrations of lovastatin pro-drug or pravastatin for 36 hr. Cells were harvested and subjected to either flow cytometry (Right) or Western blot analysis with the indicated antibodies (Left).

Figure 2

Inactivation of HMG-CoA reductase enzyme by lovastatin. Microsomes (100 μg) prepared from subconfluent cultures of MDA-MB-157 were incubated at 37°C in the presence of the indicated concentrations of lovastatin, pro-drug, or pravastatin followed by the addition of ¹⁴C-HMG-CoA. After the lactonization of the reaction with HCL, the samples were resolved by TLC and analyzed by PhosphorImaging. The activity of HMG-CoA reductase is a measure of the percent conversion of ¹⁴C-HMG-CoA into mevalolactone, the end product of the HMG-CoA reductase. ●, lovastatin mixture; ○, pro-drug; ■, pravastatin.

Figure 3

Induction of p21 and p27 by proteasome inhibitors. MDA-MB-157 tumor cells were treated with (A) 40 μM of either lovastatin mixture (open and closed rings), lovastatin closed β-lactone ring (pro-drug), or pravastatin for 36 hr, (C) 10 μM LLnL for 0–36 hr, or (D) indicated concentrations of lactacystin for 24 hr and subjected (50 μg/lane) to Western blot analysis with the indicated antibodies. Brackets in A indicate the high molecular weight laddering of p21 and p27, diagnostic for poly-ubiquitination. (B) For turnover studies, cells were treated with 40 μM pro-drug (or no drug) for 36 hr at which point the cells were incubated for 4 hr with [³⁵S]methionine and [³⁵S]cysteine (pulse) and subsequently incubated in the presence of an excess of nonradioactive methionine and cysteine for the additional times indicated (chase). p21 and p27 were precipitated from nondenatured protein extracts with polyclonal antibodies (p27-C19 and p21-C19-G, Santa Cruz Biotechnology), separated by SDS/PAGE, and detected by autofluorography.

Figure 4

Inhibition of the proteasome activity by the pro-drug form of lovastatin and lactacystin. Cell extracts were prepared from either MDA-MB-157 cells (A) without drug treatment or (B) treated with either lovastatin (lov) (40 μM), pro-drug (40 μM), or lactacystin (lact) (1 μM) in the presence or absence of mevalonate (mev) (5 mM) for 36 hr and assayed for proteasome enzyme activity. The extracts in A were incubated in the presence of the indicated concentrations of the drugs for 20 min at 37°C at which point the fluorogenic peptide substrate (100 μM final concentration) for the chymotrypsin-like activity of the proteasome (i.e., Suc-LLVY-AMC) was added to extracts. The fluorescence assays (excitation/emission 380/440 nm) were conducted at 37°C for 750 sec, and n = 3. The pretreated extracts in B were not further incubated in the presence of the inhibitors and were directly assayed for the proteasome activity. ■, lactacystin; ○, pro-drug; ●, lovastatin; □, pravastatin.

Figure 5

Mevalonate reversal of lovastatin, lovastatin pro-drug, and lactacystin. (A) MDA-MB-157 cells were treated with the indicated concentration of lovastatin (lov) or the pro-drug in the presence or absence of 4 mM mevalonate (Mev) for 36 hr and subjected to flow cytometric measurements of DNA content. (B) MDA-MB-157 cells were treated with 10 μM lactacystin (lact) in the presence or absence of 10 mM or 50 mM mevalonate for 36 hr. At the end of treatment cells were subjected to flow cytometric measurement of DNA content. Percent apoptosis reflects the accumulation of cells with sub-G₁ DNA content. (Apoptosis also was measured by chromosome condensation/4′,6-diamidino-2-phenylindole staining; data not shown.) (C) Induction of the proteasome activity by mevalonate. MDA-MB-436 (closed symbols) and MDA-MB-157 (open symbols) were treated with the indicated concentrations of mevalonate for 36 hr. After treatment, crude cell extracts were prepared and assayed for proteasome enzyme activity by measuring the chymotrypsin-like activity of the proteasome (described for Fig. 4B). Values are expressed as fold increase over no treatment controls. n = 3, and the averages of the values are indicated.

Figure 6

Cell cycle regulation by inhibitors of HMG-CoA reductase and proteasome.