RESUMO
The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.
Assuntos
Colagenases/biossíntese , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Metaloproteinase 3 da Matriz/biossíntese , Quinazolinas/farmacologia , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colagenases/genética , Indução Enzimática/efeitos dos fármacos , Hepatócitos/citologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinase 13 da Matriz , Metaloproteinase 3 da Matriz/genética , NF-kappa B/metabolismo , Piperidinas , Quinazolinonas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
EBV infection is associated with virtually all cases of undifferentiated NPC, and the EBV-encoded LMP1 is expressed in a proportion of cases. LMP1 has transforming functions similar to members of the TNF receptor family and activates intracellular signaling cascades through interaction with TRAFs. In B cells, expression of TRAF1 is in turn upregulated by LMP1. LMP1 signaling in epithelial cells may be affected by the presence or absence of TRAF1. By immunohistochemistry, we detected TRAF1 expression in 17 of 42 (40%) EBV+ undifferentiated NPCs. All 7 LMP1+ NPC biopsies were also TRAF1+. Using an RNAse protection assay, high-level TRAF1 expression was detected in an LMP1-expressing NPC-derived cell line (C15) and expression was weaker in 2 LMP1- cell lines (C17, C19). Finally, LMP1 upregulated TRAF1 expression in an EBV- keratinocyte cell line. Our results demonstrate that TRAF1 is expressed in NPC tumor cells in vivo and suggest that TRAF1 expression may be upregulated by LMP1 in NPC. An antiapoptotic function has been proposed for TRAF1, and this may be relevant for the pathogenesis of NPC.
