1. ¹Department of Medicine, Dartmouth Medical School, Hanover, NH, USA
2. ²Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH, USA

Correspondence: R P Perez, Section of Hematology/Oncology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA; Fax: 603-650-7791

Received 8 September 2001; Accepted 10 June 2002.

TO THE EDITOR

The HMG-CoA reductase family of drugs ('statins'), which includes drugs such as mevastatin, lovastatin and atorvastatin, is widely used to treat patients with hypercholesterolemia. Statins inhibit HMG-CoA reductase, blocking the synthesis of mevalonate, a precursor of cholesterol, ubiquinone, and other intracellular moieties. In vitro, lovastatin causes apoptosis in a number of malignant cell lines and acute myeloid leukemia (AML) cell lines are particularly sensitive to this effect.^1,2 Serum concentrations of ubiquinone, a coenzyme involved in mitochondrial respiratory metabolism, are decreased in patients taking statin drugs.³ Oral supplementation with ubiquinone may allow patients to tolerate higher doses of statins as may be required for the treatment of AML and other malignancies.⁴ Therefore, prior to considering a therapeutic trial of statins for the treatment of leukemia, we wished to determine whether ubiquinone would rescue AML cells from the cytotoxic effects of statins.

In preliminary experiments, ubiquinone did not rescue AML cells from the inhibitory effects of statins at either physiologic concentrations (5 M) or at supraphysiologic concentrations (50 M), nor did it stimulate cellular proliferation (data not shown). Ubiquinone, 50 M, did not rescue leukemic cell proliferation from the inhibitory effects of mevastatin in any cell line (see Figure 1).

Figure 1.

Ubiquinone does not restore cell proliferation in AML cell lines exposed to mevastatin. Five AML cell lines (HL60, KG1, MEL, ML-1, U937) and one chronic myeloid leukemia (CML) cell line (K562) were grown in 96-well microtiter plates, in triplicate, in the presence of serial dilutions of mevastatin (MST) using 1:2 serial dilutions from 200 M to 0.01 M. Viable cell numbers were determined at day 6 of culture using a tetrazolium-based colorimetric assay (Promega, Madison, WI, USA; catalog No. G3581) and compared to vehicle-treated controls. Ubiquinone (50 M) was added to cell cultures containing serial dilutions of MST as above. Results are shown for HL60, KG1 and U937 cells. Two to three independent assays of each drug for each cell line were performed in these cell lines. The lines are nearly superimposed indicating that ubiquinone does not rescue cells from statin-induced inhibition of cellular proliferation. Similar results were seen in single experiments using K562, MEL, or ML-1 cells (not shown).

Full figure and legend (30K)

We also examined the effects of ubiquinone on colony formation in bone marrow cultures of cells obtained from patients with AML and myelodysplasia (MDS). As shown in Figure 2a, the number of colonies was decreased in the presence of mevastatin. Ubiquinone did not rescue primary leukemic cells from the inhibitory effects of mevastatin on colony formation. Also, there was no evidence that ubiquinone alone stimulated leukemic cells as it did not increase the numbers of colonies present in cultures without mevastatin.

Figure 2.

(a) Colony formation in primary AML cells is inhibited by mevastatin in the presence or absence of ubiquinone. Bone marrow cells from patients with AML were cultured in colony forming assays with growth factors in control conditions (DMSO alone), mevastatin (MST, 30 M + DMSO), ubiquinone (Ubi, 5 M), or mevastatin + ubiquinone (MST + Ubi). Patients A, B and C were newly diagnosed and untreated AML patients with 51 to 85% blasts in their bone marrows. Patient D had MDS with excess blasts (6%). Samples were obtained with written informed consent from patients undergoing bone marrow biopsies for clinical purposes under protocols approved by the Committee for Protection of Human Subjects. Cells obtained after ammonium chloride lysis (Stem Cell Technologies, SCT, Vancouver, BC, Canada, catalog No. 07850) were plated in methylcellulose media with recombinant growth factors (SCT, Methocult GF, catalog No. H4434). The number of myeloid colonies present on day 16 of culture in untreated cultures was compared to the number of colonies formed in the presence of mevastatin (30 M), with or without ubiquinone at physiological concentrations (5 M). Mevastatin decreased colony formation and ubiquinone did not rescue cells from this effect. (b) Mevastatin decreases membrane localization of Ras and this is not affected by ubiquinone. Cells were cultured in the presence or absence of 50 M mevastatin 50 M ubiquinone (Ubi). Prenylated Ras was assayed by fractionating cell lysates into lipophilic (membrane, MB) and aqueous (cytosol, CS) components using Triton X114. The samples were separated by SDS gel electrophoresis and immunoblotted using a pan-Ras antibody. Mevastatin inhibited prenylation of Ras in both K562 cells and in MEL cells. This effect was not abrogated by ubiquinone.

Full figure and legend (40K)

The mechanism of the anti-proliferative effect of statins is not known. One hypothesis is that cell viability is affected by inhibition of prenylation, a post-translational modification of proteins important for activity of intracellular signaling proteins such as Ras. All statins inhibit the production of mevalonate by HMG-CoA reductase, thereby decreasing the production of all synthetic products downstream from mevalonate. In addition to cholesterol and ubiquinone, these include the prenylation precursors, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These lipophilic moieties are added to Ras and other intracellular proteins during post-translational processing and are required for activity. Xia et al⁵ reported that, of the mevalonate metabolites, only geranylgeranyl pyrophosphate rescued AML cells from statin-induced apoptosis. Therefore, AML cell viability may be critically sensitive to the prenylation status of one of the intracellular proteins that can be activated by geranylgeranylation. Among these are the Rho, Rac, and Ras-related proteins.

As an assay for the prenylation status of these proteins, the prenylation state of Ras was determined using X-114⁶ to separate the lipophilic (membrane) and aqueous (cytosolic) fractions of cell extracts from K562 cells and MEL cells. Ras was detected by immunoblotting with a pan-Ras antibody (Ras Clone 10; Upstate Biotechnologies, Lake Placid, NY, USA) and detected by chemiluminescence. As shown in Figure 2c, Ras from the untreated cells is found primarily in the membrane fraction, suggesting that prenylation was inhibited. Mevastatin caused an increase in the unprenylated fraction as shown by the increase in the proportion of Ras in the cytosolic fraction. Ubiquinone did not affect Ras localization in either the presence or absence of mevastatin, suggesting that ubiquinone does not alter prenylation.

Ubiquinone did not rescue AML cells from statin-induced in vitro alterations on cellular proliferation, colony formation, or prenylation suggesting that ubiquinone will not abrogate the potential therapeutic effects of statins in AML. Ubiquinone may allow some patients to tolerate the relatively high doses of statins that may be required to treat AML. Appropriately designed clinical trials will be needed to address the potential utility of statins, with or without ubiquinone, in the treatment of patients with AML.