Down-regulation of RhoA is involved in the cytotoxic action of lipophilic statins in HepG2 cells.
OBJECTIVE
Hepatotoxicity is one of the major complaints that occur during lipid-lowering therapy with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins. We reported earlier that lipophilic but not hydrophilic statins induce apoptosis through inhibition of mevalonate biosynthesis cascade in Chang liver cells. The present study was designed to determine the role for small G protein RhoA in the hepatocytotoxicity of statins.


METHODS
Statin-induced hepatocytotoxicity in HepG2 cells were assessed by WST-8 cell viability assay, JC-1 mitochondrial membrane potential assay and caspase-3/7 activity assay. Cytosolic RhoA was detected by Western blotting and RhoA activation was measured by ELISA.


RESULTS
The lipophilic atorvastatin but not the hydrophilic pravastatin induced the mitochondrial membrane depolarization and the activation of caspase-3/7, which led to cell injury. Supplementation of mevalonate or geranylgeranyl pyrophosphate (GGPP) but not farnesyl pyrophosphate (FPP) reversed these cellular events and cell death induced by atorvastatin. Atorvastatin induced a translocation of RhoA protein into the cytosol and inhibited the activity of the protein. In addition, atorvastatin reduced mitochondrial membrane potential, which was mimicked by GGTase inhibitor GGTI-2147 or the specific RhoA inhibitor such as toxin B and C3 exoenzyme. However, only a few cells revealed mitochondrial membrane depolarization and a loss of viability after exposure to the Rho-kinase inhibitors such as Y-27632 and hydroxy fasudil.


CONCLUSIONS
RhoA inactivation and to a lesser extent Rho-kinase inhibition after depletion of GGPP is implicated in the etiology of mitochondrial membrane depolarization and subsequent caspase-dependent cell death induced by the lipophilic statin in HepG2 cells.
Maeda
Asuka
A
Department of Pharmacy, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.
Yano
Takahisa
T
Itoh
Yoshinori
Y
Kakumori
Midori
M
Kubota
Toshio
T
Egashira
Nobuaki
N
Oishi
Ryozo
R
D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology; D015536:Down-Regulation / Q000187:drug effects*; D056945:Hep G2 Cells / Q000187:drug effects; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology / Q000633:toxicity*; D020742:rhoA GTP-Binding Protein / Q000187:drug effects* / Q000502:physiology*
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C066737:RHOA protein, human; D020742:rhoA GTP-Binding Protein