Transfection of siRNA targeted against caspase 8 mRNA prevents FasL-mediated apoptosis of HepG2 hepatoma cells. HepG2 cells were transfected with either a nonsense siRNA or a specific siRNA targeted against caspase 8. After 48 h cells were infected with AdFasL or AdGFP. (a) Caspase 8 assay (caspase 8 sequence 1). Cells pretreated with caspase 8 siRNA show ≈2.5-fold reduced caspase 8 activity compared with cells pretreated with scrambled siRNA 8 h after adenoviral infection. (b) Histone ELISA (caspase 8 sequence 1). Caspase 8 siRNA treatment inhibits Fas (CD95)-mediated apoptosis as shown by photometric histone ELISA 12 h after infection. The figure shows representative data obtained from three independent experiments.

Caspase 8 siRNA inhibits caspase 8 gene expression in the liver and prevents Jo2-mediated apoptosis of hepatocytes. (a) Mice were treated with 0.45 nmol of siRNA per g of body weight into the tail vein. After 48 h livers were harvested for mRNA isolation and Northern blot analysis. Compared with animals treated with scrambled (scr) siRNA or control animals without prior siRNA treatment, caspase 8 siRNA-treated animals show >3-fold-reduced mRNA expression in Northern blot analysis. Each lane contains mRNA extracted from pooled tissues of three animals. (b) For analysis of caspase 8 activity, mice were treated with Fas (CD95)-activating antibody Jo2 48 h after siRNA application. Six hours after Jo2 application, livers were harvested for measurement of caspase 8 activity. Extracts of pooled liver tissue from three animals in each group were prepared. Pretreatment with caspase 8 siRNA significantly inhibits up-regulation of caspase 8 activity after Jo2 application. (c) Mice treated systemically with caspase 8 siRNA show heterogenous and overall reduced apoptosis in TUNEL staining 6 h after Jo2 application compared with mice treated with scrambled-siRNA. Inhibition of Jo2-mediated liver cell apoptosis can be improved by portal-vein application compared with tail-vein application. (Magnification, ×100 and ×200.) Shown are representative data from three independent experiments. (d) Hematoxylin/eosin-stained tissue sections were investigated for histopathology to show the morphological effects of caspase 8 siRNA treatment. Unprotected livers were totally destroyed, whereas 10–20% of hepatocytes from mice pretreated with caspase 8 siRNA were still alive 10 h after Jo2 treatment. (Magnification, ×200.)

Caspase 8 siRNA prevents liver damage in a model of AdFasL-induced liver failure. Delivery of caspase 8 siRNA 48 h before application of AdFasL is capable of preventing liver damage as shown by significantly reduced serum transaminases (a) and distinctly improved histopathologies (b; magnification, ×200) in caspase 8 siRNA-protected mice compared with mice treated with scrambled siRNA 32 and 40 h after adenoviral application. The difference between the siRNA scrambled- and caspase 8 siRNA-treated animals for serum alanine aminotransferase and serum aspartate transaminase at each time point was highly significant (P < 0.001). Furthermore, the increase for siRNA scrambled-treated animals and decrease for caspase 8 siRNA-treated animals in time course was also significant (P < 0.001 and 0.05, respectively).

Application of caspase 8 siRNA leads to significantly improved survival in different models of ALF. Prevention of Fas (CD95)-mediated apoptosis by caspase 8 siRNA was investigated by application of caspase 8 siRNA or scrambled siRNA 48 h before administration of Jo2 or AdFasL. Control animals received Jo2 or AdFasL without siRNA treatment. (a) Whereas all animals with unprotected livers died, prevention of apoptosis by caspase 8 siRNA led to 100% survival in AdFasL-mediated liver failure and 45% survival in Jo2-mediated ALF. (b) Therapeutic intervention by RNAi in an ongoing ALF was investigated by application of caspase 8 siRNA after viral liver infection with AdFasL or Adwt. Increase of transaminases (serum alanine aminotransferase and serum aspartate transaminase) demonstrates liver injury already 4 and 8 h after application of Adwt. Treatment with caspase 8 siRNA 8 h after application of AdFasL or Adwt significantly improves survival of animals. Control animals received AdFasL or Adwt without siRNA treatment.