| Summary: | 碩士 === 國立中興大學 === 獸醫學系暨研究所 === 106 === Oncolytic viruses (OVs), serving as an alternative antitumor strategy, could selectively infect, replicate and lyse tumor cells, while leaving healthy cells intact. Furthermore, infection of OVs could trigger immune response that in turns alter tumor microenvironment and change the tumor immunotolerance. Parapoxovis ovis Orf virus (ORFV), a zoonotic etiologic agent, infects natural hosts (goats and sheep) and also humans. Recently, ORFV has been proposed as a novel candidate for oncolytic therapy; it activates natural killer cells and leads to tumor regression on human lung and breast cancer cell xenograft model. As ORFV is epitheliotrophic, the aim of this study is to evaluate the oncolytic effect of ORFV on nasopharyngeal carcinoma cells (NPC), and the ORFV infectivity of ORFV will be compared in parallel with canine mammary gland tumor cells (DMGT). Two recombinant ORFVs expressing eGFP, namely ORFV-eGFP WT and VEGF△-vvTK-eGFP which contains genome of wild type or with deletion of VEGF gene, respectively. Infectivity of ORFVs in two human cancer cell lines NPC and A549 (a positive control cell line), as well as DMGT and Madin-Darby kidney (MDCK) cells of canine origin were infected by two recombinant ORFVs. As indicated by eGFP fluorescence, among the four cell lines, NPC cell line had the most significant high infection rate, followed by A549 and the least were MDCK and DMGT. Consistently, expression level of viral F1L protein in NPC was statistically higher than other cell lines infected with either ORFV-eGFP WT or VEGF△-vvTK-eGFP ORFV. Similarly, the yield of viral progenies in NPC was significantly higher than the positive cell line, A549. As cell signaling pathway in tumors is possibly responsible for selectively infection of OVs, the common survival signaling, i.e pAkt/mTOR was monitored. It appears Akt was strongly activated (as indicated by high level of phosphorylated Akt, pAkt) in DMGT cells, the least susceptible cell line for ORFV, and that led us to suspect Akt pathway might jeopardize ORFV infection. Indeed, when pAkt/ mTOR pathway was suppressed by Akt inhibitor MK2006 (500nM), viral protein F1L expression was significant elevated in DMGT cells under treatment at three hours prior to ORFV-eGFP WT infection as compared with those of mock group. In conclusion, this study revealed for the first time that epitheliotrophic ORFV significantly infects human NPC cell line. Nevertheless, oncolytic effect should be further tested in in vivo models since the underlying mechanism remains unclear. As for DMGT cell line, since MK2006 could enhance ORFV infection, ORFV engineered to target the Akt pathway perhaps could boost the oncolytic potential.
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