Abstract
Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis, and are widely used to treat hypercholesterolaemia, a risk factor for cardiovascular disease. Myopathy (muscle toxicity) is an adverse effect that is thought to occur due to the secondary effects of inhibiting HMGCR; however the mechanism of this myopathy is not fully understood. Therefore, the aim of this study was to use two human cell lines as an in vitro model for comparing the effects of a variety of statins on both cholesterol synthesis and protein prenylation between the liver, the intended target tissue for therapeutic benefit, and skeletal muscle, the major site of toxicity. Initially, the differentiation of RD rhabdomyosarcoma muscle cells was characterised in terms of morphological and molecular changes that occur, using microscopy and TaqMan analysis respectively, and a 5-day cell differentiation period was chosen for use in later experiments. The initial hypothesis to then be tested was that the process of protein prenylation exhibits circadian variation, as has already been shown for cholesterol synthesis, and this might enable temporal separation between the cholesterol-lowering efficacy and the toxicity of statins. However, the ability of the RD cell line to establish and maintain circadian rhythmicity in vitro could not be verified and so the circadian contributions to cholesterol metabolism and protein prenylation in liver and muscle in this model could not be investigated. Using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, significant, dose-dependent reductions in cell viability were observed in both liver and muscle cells on treatment with each of the statins (P = 0. 0041, atorvastatin; P < 0. 0001, all other statins, two-way analysis of variance (ANOVA)). However, greater cell toxicity was observed with the more lipophilic statins, simvastatin (acid), lovastatin (acid) and cerivastatin, and also in RD muscle cells compared to Huh7 liver cells. Taken together, these data represent the first comprehensive comparative analysis of statin toxicity in human liver and muscle cell lines. Cholesterol metabolism was then examined in these cells: Although cholesterol was present in all cell types, the levels in RD cells were somewhat lower than in Huh7 cells, probably due to much lower levels of HMGCR as measured at both the transcript and protein level; and 48 hour treatment with 10μM simvastatin and pravastatin, and 1μM cerivastatin, seemed to lead to a decrease in total cellular cholesterol. Subsequently, protein was extracted from Huh7 and RD cells treated with simvastatin, cerivastatin or pravastatin, and then subjected to analysis by western blot to detect any changes in the prenylation status of selected small GTPases. Inhibition of prenylation was found by directly analysing changes in the unprenylated form of Rap1A, using a specific antibody, and by examining the reduction in membrane association of H-Ras, M-Ras and Rab1A. Treatment with simvastatin and cerivastatin inhibited the prenylation of Rap1A, and seemed to also affect membrane association of the small GTPases H-Ras, M-Ras and Rab1A. In both muscle and liver cells the inhibition of prenylation of Rap1A, a Ras-related GTPase, was 10-fold more sensitive to treatment with cerivastatin than simvastatin. Inhibition of Rap1A prenylation in RD muscle cells was also 10-fold more sensitive to simvastatin and 3-fold more sensitive to cerivastatin treatment than in liver cells. Hydrophilic pravastatin, which showed the lowest toxicity in MTT assays, did not affect the prenylation of any of the small GTPases examined, as far as was detectable using western blot analysis. In conclusion, this work has shown that RD muscle cells show both greater sensitivity to the toxic effects of statins and preferential disruption of protein prenylation in response to statin treatment when compared to Huh7 liver cells. This further supports a mechanism whereby disrupting production of intermediates in the cholesterol synthesis pathway, including the isoprenoids used in protein prenylation, is a significant cause of statin-induced muscle toxicity. This study also highlights the importance of examining the effects of drugs at both the target site of action and at the site of toxicity, rather than only studying one site in isolation, in order to gain insight into the mechanism of adverse drug effects.