Molecular and Cellular Pharmacology Hydrophobic statins induce autophagy and cell death in human rhabdomyosarcoma cells by depleting geranylgeranyl diphosphate Makoto Araki a, b , Masatomo Maeda b , Kiyoto Motojima a, ⁎ a Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588, Japan b Department of Molecular Biology, School of Pharmacy, Iwate Medical University, Yahaba, Shiwa, Iwate 028-3694, Japan abstract article info Article history: Received 21 August 2011 Received in revised form 11 October 2011 Accepted 27 October 2011 Available online 9 November 2011 Keywords: Statin Autophagy Rhabdomyolysis Geranylgeranyl diphosphate mTORC1 Statins are the most common type of medicine used to treat hypercholesterolemia; however, they are asso- ciated with a low incidence of myotoxicity such as myopathy and rhabdomyolysis. The mechanisms for the adverse effects remain to be fully elucidated for safer chronic use and drug development. The results of our earlier work suggested that hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma A204 cells. In this study, we first confirmed the statin-induced autophagy by assessing other criteria, includ- ing induced expression of the autophagy-related genes, enhanced protein degradation of autophagy marker protein p62 and electron microscopic observation of induced formation of autophagosome. We next demon- strated that the extent of inhibition of HMG-CoA reductase in the cell is parallel with the ability of a statin to induce autophagy. Thus, the primary activity of statins causes autophagy in A204 cells. Considering the mech- anism for the induction, we showed that statins induce autophagy by depleting cellular levels of geranylger- anyl diphosphate (GGPP) mostly through an unknown pathway that does not involve two major small G proteins, Rheb and Ras. Finally, we demonstrated that the ability of statins to induce autophagy parallels their toxicity to A204 cells and that both can be suppressed by GGPP. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Statins block de novo synthesis of cholesterol by inhibiting the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and are widely used for the treatment of hy- percholesterolemia. Since the discovery of mevastatin by Endo et al. in 1973, a variety of statins have been discovered as natural products or developed by drug design (Endo et al., 1977). Although statins are widely prescribed as supposedly safe for many patients, there is a risk of myotoxicity, such as muscle pain or even severe muscle disorder rhabdomyolysis (Arora et al., 2006; Omar and Wilson, 2002). The hy- drophilic statins are thought to be actively transported into hepato- cytes by expressing the organic anion transporter (OATP), whereas hydrophobic statins diffuse non-selectively into extra-hepatic tissues such as muscle and cause the adverse effects (Neuvonen et al., 2006). However, the mechanism underlying statin-induced myotoxicity has not been elucidated. Importantly, it has not been clarified whether in- hibition of HMG-CoA reductase itself or other targets causes myotoxicity. In our earlier work, we reported that hydrophobic statins induced autophagy in cultured human rhabdomyosarcoma A204 cells based on both the biochemical observation of the enhanced processing of the autophagy marker protein LC3 and the morphological observation of accumulation of the green fluorescent protein-labeled LC3 (GFP- LC3) on autophagosomes (Araki and Motojima, 2008). These changes were induced only by hydrophobic statins in A204 cells but not HEK293, HepG2 and HuH7 cells and were blocked by mevalonate but not by cholesterol. Autophagy, found in all mammalian cells, is a system for the non- selective degradation of proteins and organelles to promote protein turnover for cell survival and protection against a variety of stresses (Mizushima, 2007). Both insufficient and excess autophagy can be harmful for the cell and the process is stringently regulated by several signaling pathways (Mizushima et al., 2008). The mammalian target of the rapamycin complex 1 (mTORC1) plays a major regulatory role in autophagy in collaboration with the autophagy-related pro- teins (ATG proteins). The small G protein Rheb, when modified by farnesylation, activates mTORC1, which inhibits the induction of autophagy by inhibiting the kinase activity of ULK (the mammalian Atg1 homolog) (Hosokawa et al., 2009; Jung et al., 2009). Under star- vation conditions, Rheb is inactivated by the Rheb GTPase-activating protein TSC1–TSC2 and mTORC1 is inactivated, leading to the induc- tion of autophagy (Huang and Manning, 2008). In addition, the Ras/ PI3K/Akt pathway regulates autophagy negatively. In this pathway, inactivated Ras inhibits polyisoprenylation and induces autophagy by inactivating Akt (He and Klionsky, 2009). Other signaling path- ways are known to control autophagy but the highly complicated reg- ulatory processes remain to be fully elucidated. European Journal of Pharmacology 674 (2012) 95–103 ⁎ Corresponding author at: Department of Biochemistry, Meiji Pharmaceutical Uni- versity, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. Tel./fax: + 81 42 495 8474. E-mail address: motojima@my-pharm.ac.jp (K. Motojima). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.10.044 Contents lists available at SciVerse ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar