The Oncolytic Role of Parapoxvirus on Nasopharyngeal Carcinoma Cells and Canine Mammary Tumor Cells

溶瘤病毒為癌症的替代治療方式；在不影響正常細胞的狀況下，此類病毒具有選擇性感染、複製並溶解腫瘤細胞之特質。除此之外，溶瘤病毒也可以刺激免疫反應進而調節腫瘤微環境並改變腫瘤的免疫耐受性。ORFV為感染山羊以及綿羊的一種副痘病毒(Parapoxovis ovis)，對於人亦具有感受性，但僅造成局部輕微病灶，近來已被視為新興的溶瘤療法。ORFV在人之肺癌和乳癌異種小鼠模式中可使腫瘤團塊消退，推測病毒感染可能活化自然殺手細胞而促進腫瘤毒殺。由於此病毒嗜侵犯上皮，本研究遂以上皮來源之人類鼻咽癌(Nasopharyngeal carcinoma，NPC)細胞為模式探究ORFV之溶瘤效果，並同步比較ORFV感染本實驗室培養的犬乳腺腫瘤細胞(DMGT)，藉此探究ORFV可能之作用機制。本研究使用兩種重組ORFV，分別為ORFV-eGFP WT及VEGF△-vvTK-eGFP，前者基因體為野生型，後者缺損血管內皮基因，但均帶有eGFP螢光基因。從螢光訊號顯示，在測試的四種細胞中，NPC有最顯著的感染率，A549次之，MDCK和DMGT則相對不顯著。此結果與西方墨點法之定量分析相符；NPC感染後比起其他細胞株有顯著較高的病毒蛋白 (F1L)。此外，NPC細胞中ORFV子代病毒產量顯著高於其他測試細胞。由於ORFV在不同細胞中的感染率具顯著差異，有鑒於溶瘤病毒特異性的感染腫瘤細胞可能是因為病毒感染對於細胞訊息之依賴性；因此本實驗另針對常見的癌症訊息傳遞路徑(例如pAkt/mTOR以及ERK)活化情形是否與ORFV感染率具關連性。結果顯示，DMGT細胞之Akt活化程度顯著高於其他細胞，而且經Akt抑制劑MK2006 (500nM)處理之後，ORFV-eGFP WT對於犬乳腺腫瘤細胞株的感染力顯著提升，因此推測pAkt/mTOR訊息傳遞路徑可能影響ORFV的感染。綜上所述，本研究首先發現ORFV有效率的感染人鼻咽癌細胞株，並於感染12-24小時之後即造成多數NPC細胞失去貼附性，型態圓型化以及懸浮的現象；這些結果強烈支持ORFV極有可能因感染而毒殺NPC細胞，然而溶瘤效果以及機制需進一步探究。此外，ORFV對於腫瘤生長之影響需進一步於體內模式中測試。ORFV對於DMGT感受性差，而抑低Akt/mTOR訊息路徑之活化顯著增強ORFV的感染，故未來針對pAkt訊息傳遞路徑為標的基因工程重組ORFV或許可增強溶瘤能力。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.致謝 i 摘要 ii Abstract iv Contents vi List of abbreviations viii List of Tables ix List of Figures x Chapter 1. Introduction 1 1.1. Oncolytic therapy 1 1.2. Oncolytic therapy in veterinary medicine 3 1.3. The role of poxvirus in oncolytic therapy 4 1.4. Canine mammary gland tumor 5 1.5. Nasopharyngeal cell carcinoma 6 Chapter 2. Materials and methods 8 2.1. Cell culture 8 2.2. Viruses 8 2.3. Experiment design 9 2.4. Western blotting 9 2.5. Cell viability test 11 2.6. Plaque assay 12 2.7. Inhibitors toward PI3K/Akt pathway in DMGT cells 12 2.7.1. Inhibitor effect in DMGT 12 2.7.2. ORFV infectivity after using inhibitor in DMGT 13 2.8. Statistical analysis 13 Chapter 3. Results 14 3.1. Infectivity of ORFV between cell lines 14 3.2. Oncolytic effect of ORFV on NPC 17 3.3. Factors affecting ORFV infection 20 3.3.1. Cell survival signaling pathway 20 3.3.2. DMGT Akt expression with different Akt pathway inhibitor 22 3.3.3. ORFV infectivity of DMGT under Akt pathway inhibitor 24 3.3.4. Cell density influenced the infectivity of ORFV 29 Chapter 4. Discussions 31 4.1. Oncolytic effect of ORFV 31 4.1.1. Susceptibility of ORFV in the four cell types 31 4.1.2. Infectivity between two recombinant ORFVs 32 4.2. Akt expression 35 4.3. Erk expression 38 4.4. Cell density 40 Reference 4