Antiviral protection against enterovirus 71 mediated by autophagy induction following FLICE-inhibitory protein inactivation.

Even with the recent awareness of enterovirus 71 (EV71) as a major public health issue, there are no preventive or therapeutic agents that are effective against EV71 infection. Although FLICE-like inhibitory protein (FLIP) has been identified as a factor that modulates virus pathogenesis, there are no reports regarding its effects on EV71 infection. The aim of the present study was to identify whether FLIP influences EV71 pathogenesis and to understand the underlying mechanisms. Virus replication was markedly reduced in MRC5 cells preincubated with anti-FLIP peptides, and infected cells were rescued from the cytopathic effects of the virus. The anti-FLIP peptides induced autophagy by disrupting intrinsic FLIP functions. The antiviral activity of these peptides was reduced when autophagy was inhibited by treatment with siRNA targeted to beclin-1. Thus, the present study provides evidence that anti-FLIP peptides induce autophagy by inactivating cFLIP, and that this is associated with antiviral effects against EV71.

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin.

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.

Persistent anti-tumor effects via recombinant adeno-associated virus encoding herpes thymidine kinase gene monitored by PET-imaging.

Despite the well-documented advantages of the recombinant adeno-associated virus (rAAV) as a gene delivery vehicle, including its non-pathogenic and long-term therapeutic gene expression, there have been very limited studies on its potential for producing persistent anti-tumor effects, particularly in vivo. To address this issue, we constructed rAAV vectors encoding herpes simplex virus 1-thymidine kinase (HSV-TK) or its mutant form (sc39TK) as therapeutic genes, and GFP as a control gene. Effective rAAV-mediated gene delivery was readily observed in human cancer cells using immunocytochemistry and Western blotting. Cell survival analysis following prodrug ganciclovir treatment implied that both preferential and superior cytotoxicity was achieved by rAAV-sc39TK introduction. Persistent anti-tumor effects in vivo were investigated in Balb/c nude mice bearing human cancer cells treated with either rAAV-sc39TK or -GFP. Severe tumor growth inhibition was clearly observed only in the case of sc39TK with ganciclovir treatment. Non-invasive micro-PET imaging using 18F-FHBG directly correlated with persistent anti-tumor effects by sc39TK. Therefore, the present study provides evidence that rAAV-mediated persistent therapeutic gene expression can occur, resulting in long-term anti-tumor activities and that these events can be readily monitored using micro-PET imaging.