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J Pharmacol Exp Ther. 2009 Jul;330(1):276-82. doi: 10.1124/jpet.109.151860. Epub 2009 Mar 27.

Heat shock protein 90 inhibitor induces apoptosis and attenuates activation of hepatic stellate cells.

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1
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 28 Yungun-dong, Chongno-gu, Seoul 110-744, Korea.

Abstract

Activated hepatic stellate cells (HSCs) are major participants in hepatic fibrosis; thus, the induction of HSC apoptosis has been proposed as an antifibrotic treatment strategy. Heat shock protein (Hsp) 90 is a molecular chaperone that stabilizes major signal transduction proteins, and its inhibitors have antitumor activity. In this study, the susceptibility of HSCs to an Hsp90 inhibitor was evaluated. LX-2 cells, an immortalized human HSC line, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, and monensin, an acidic sphingomyelinase inhibitor, were used in this study. Cellular apoptosis was quantified by 4',6-diamidino-2-phenylindole dihydrochloride staining, and signaling cascades were explored using immunoblotting and immunoprecipitation techniques. Nuclear factor (NF) kappaB activities were evaluated by immunofluorescent microscopy and enzyme-linked immunosorbent assay. Collagen alpha1 and alpha-smooth muscle actin expressions were determined by real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. It was found that 17AAG induced HSC apoptosis and that caspase 8 cleavage preceded the downstream activation of apoptotic signaling cascades. Furthermore, this caspase 8 activation was dependent on ceramide generation by acidic sphingomyelinase. In addition, 17AAG prevented NFkappaB nuclear translocation and activation, specifically by inducing complex formation between NFkappaB and the glucocorticoid receptor. In accordance, NFkappaB-dependent cellular FLICE-like inhibitory protein expression level was found to be reduced by 17AAG. Finally, 17AAG down-regulated collagen alpha1 and alpha-smooth muscle actin expression levels in HSCs before inducing apoptosis. These results demonstrate that the Hsp90 inhibitor induces HSC apoptosis via a sphingomyelinase- and NFkappaB-dependent mechanism. Because this inhibitor also reduces HSC activation before apoptosis, Hsp90 inhibitor treatment might be therapeutically useful as an antifibrotic strategy in a variety of liver diseases.

PMID:
19329756
DOI:
10.1124/jpet.109.151860
[Indexed for MEDLINE]
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