Simvastatin (Zocor)

In Vitro

Simvastatin needs to be activated by NaOH in EtOH treatment before use for cell assay. Simvastatin suppresses cholesterol synthesis in mouse L-M cell, rat H4II E cell, and human Hep G2 cell with IC50s of 19.3 nM, 13.3 nM and 15.6 nM, respectively[1]. Simvastatin causes a dose-dependent increase in serine 473 phosphorylation of Akt within 30 minutes, with maximal phosphorylation occurring at 1.0 µM. Simvastatin (1.0 μM) enhances phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibits serum-free media undergo apoptosis and accelerates vascular structure formation[2]. Simvastatin shows anti-inflammatory effects, reduces anti-CD3/anti-CD28 antibody-stimulated proliferation of PB-derived mononuclear cells and synovial fluid cells from rheumatoid arthritis blood, as well as IFN-γ release at 10 μM. Simvastatin (10 μM) also blocks cell-mediated macrophage TNF-γ release induced via cognate interactions by appr 30%[3]. Simvastatin (5 μM) significantly reduces the expression of ABCA1 in astrocytes and neuroblastoma cells, the expression of apolipoprotein E in astrocytes, and increases cyclin-dependent kinase 5 and glycogen synthase kinase 3β expression in SK-N-SH cells[7].

In Vivo

Simvastatin suppresses the conversion of radiolabeled acetate to cholesterol with an IC50 of 0.2 mg/kg via p.o. administration[1]. Simvastatin (4 mg/day, p.o. for 13 weeks) returns the cholesterol-induced increases in total cholesterol, LDL-cholesterol and HDL-cholesterol to normal level in rabbits fed an atherogenci cholesterol-rich diet[4]. Simvastatin (6 mg/kg) increases LDL receptor-dependent binding and the number of hepatic LDL receptors in rabbits fed a diet containing 0.25% cholesterol[5]. Simvastatin affects inflammation independent of its effect on plasma cholesterol level. Simvastatin (20 mg/kg/day) causes a 1.3-fold less macrophage content in lesions, and 2-fold less vascular cell adhesion molecule-1, interleukin-1beta, and tissue factor expression, companied by a 2.1-fold increases in lesional smooth muscle cell and collagen content in cynomolgus monkeys fed an atherogenic diet[6].

Target

Ki: 0.2 nM (HMG-CoA reductase)[1]

Kinase Assay

For assessment of Akt protein kinase activity in vitro, substrate (2 μg histone H2B or 25 μg eNOS peptide) is incubated with Akt immunoprecipitated from cell lysate using goat polyclonal anti-Akt1 antibody. Kinase reactions are initiated following the addition of Simvastatin to a final concentration of ATP (50 μM) containing 10 μCi of 32P-γATP, dithiotreitol (1 mM), HEPES buffer (20 mM, pH 7.4), MnCl2 (10 mM), MgCl2 (10 mM). After incubation for 30 min at 30°C, phosphorylated histone H2B is visualized after SDS-PAGE (15%) and autoradiography. To estimate the extent of 32P incorporation into eNOS peptides, each reaction mixture is measured by spotting onto phosphocellulose disc filter and the amount of phosphate incorporated is measured by Cerenkov counting. The wild-type peptide sequence is 1174-RIRTQSFSLQERHLRGAVPWA-1194, and the mutant eNOS peptide is identical except that serine 1179 is substituted by alanine[3].

Cell Assay

Primary human astrocytes from four different donors are prepared from tissue obtained from legally aborted fetuses. Primary human astrocytes and SK-N-SH neuroblastoma cell line (ATCC) are plated on 6-well plates and grown in DMEM supplemented with 5% or 8% fetal calf serum, respectively, at 37°C, 5% CO2 until 80% confluent. For measurement of gene expression levels at baseline, cells are just washed and RNA is prepared and assayed as outlined below. Baseline gene expression levels in astrocytes are measured in primary human astrocytes obtained from two donors. For experimental purposes, cells are incubated under serum-free conditions. Primary human astrocytes are obtained from four donors. Based on preliminary time-dependent studies, 48-h incubation is used for all of experiments. Based on the dose-response studies, a majority of our experiments are conducted using the following concentrations of active compounds: Simvastatin at 5 μM, pravastatin at 10 μM, mevalonate at 50 μM, and GGPP and FPP at 10 μM. Following incubation, cells are extensively washed to remove dead cells and cell debris and prepared for further analyses[7].

References & source

[1]. Slater, E.E., et al. Mechanism of action and biological profile of HMG CoA reductase inhibitors. A new therapeutic alternative. Drugs, 1988. 36 Suppl 3: p. 72-82.

[2]. Kureishi, Y., et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med, 2000. 6(9): p. 1004-10.

[3]. Leung BP, et al. A novel anti-inflammatory role for simvastatin in inflammatory arthritis. J Immunol. 2003 Feb 1;170(3):1524-30.

[4]. Kobayashi M, et al. Preventive effect of MK-733 (simvastatin), an inhibitor of HMG-CoA reductase, on hypercholesterolemia and atherosclerosis induced by cholesterol feeding in rabbits. Jpn J Pharmacol. 1989 Jan;49(1):125-33.

[5]. Ishida F, et al. Comparative effects of simvastatin (MK-733) and pravastatin (CS-514) on hypercholesterolemia induced by cholesterol feeding in rabbits. Biochim Biophys Acta. 1990 Feb 23;1042(3):365-73.

[6]. Sukhova GK, et al. Statins reduce inflammation in atheroma of nonhuman primates independent of effects on serum cholesterol. Arterioscler Thromb Vasc Biol. 2002 Sep 1;22(9):1452-8.

[7]. Weijiang Dong, et al. Differential effects of simvastatin and pravastatin on expression of Alzheimer’s disease-related genes in human astrocytes and neuronal cells. J Lipid Res. 2009 Oct; 50(10): 2095-2102.

[8]. Liu Z, et al. Pretreatment Donors after Circulatory Death with Simvastatin Alleviates Liver Ischemia Reperfusion Injury through a KLF2-Dependent Mechanism in Rat. Oxid Med Cell Longev. 2017;2017:3861914.

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