Pravastatin

Human renal proximal tubular epithelial cell line (HK-2), which are immortalized human renal proximal tubular epithelial cell, and the hepatocellular carcinoma HepG2 cell line were obtained from ATCC (Rockville, MD). HK-2 cells at passages 10–15 and HepG2 were cultured. The cell lines were cultured in Dulbecco’s modified Eagle’s medium/F12 (1:1) (Gibco, Grand Island, NY, USA) culture medium containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco). Cells were treated with 1, 2, or 4 μM of pravastatin (Cayman, Ann Arbor, MI, USA), and stimulated with 30 μg/ml cholesterol (Sigma, St Louis, MO, USA) plus 1 μg/ml 25-hydroxycholesterol (Sigma). HK-2 and HepG2 cells were treated with 1, 2, or 4 μM pravastatin plus 25-hydroxy cholesterol and cholesterol for either 24 or 48 h. The expression of pyruvate kinase isozymes L/R (PKLR), PFK-1, PEPCK, and G6PC proteins was then examined by western blotting.

Fluvastatin, pravastatin, lovastatin, and simvastatin were all obtained from Cayman Chemicals (Ann Arbor, MI); atorvastatin and rosuvastatin were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY).

A total of six statins were used in this study. The statins were purchased from Cayman Chemicals (Vitro SA, Madrid, Spain) and included atorvastatin, fluvastatin, simvastatin, pravastatin, mevastatin and lovastatin. The stock solutions for the experiments were prepared in dimethyl sulfoxide (DMSO) and were maintained at −20 °C until required for the experiments. As a positive control, amphotericin B was used.

For the treatment of cells with the several drugs under study (5-FU, verapamil, itraconazole, isoniazid, tacrine, aspirin, cimetidine, chloroquine, losartan, all from Sigma-Aldrich (Sigma-Aldrich Quimica, S.L., Sintra, Portugal) and pravastatin, from Cayman Chemical Company, Ann Arbor, MI, USA), all the compounds were dissolved in autoclaved water, except itraconazole, that was dissolved in dimethyl sulfoxide (DMSO), since it did not present solubility in water. A stock solution of each compound was prepared at a concentration of 10 mM and, except for itraconazole, two stock solutions with a concentration of 25 mM and 50 mM were prepared, because this drug was dissolved in DMSO, which has significant toxicity to cells after a percentage of 0.2%, a percentage that was never exceeded in this work. All these stock solutions were conserved on the freezer at −26 °C. Depending on the purpose of the assay, test compounds were used in concentrations that range from 1 μM to 100 μM, dissolved in culture medium right before contact with cells. The respective concentrations used in each assay are presented in the Results section. The test compounds applied to the cells vary with the purpose of the experiment, also specified in the above-mentioned section.