Pawlik Timothy M, Nakamura Hideo, Mullen John T, Kasuya Hideki, Yoon Sam S, Chandrasekhar Soundararajalu, Chiocca E Antonio, Tanabe Kenneth K
Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston 02114-2696, USA.
Cancer. 2002 Sep 1;95(5):1171-81. doi: 10.1002/cncr.10776.
Herpes simplex virus 1 (HSV-1) infection of cancer cells results in viral replication with cell destruction and liberation of progeny virion that infect adjacent tumor cells. rRp450 is a novel replication-conditional HSV-1 mutant that expresses both the endogenous herpes viral thymidine kinase gene and the rat p450 CYP2B1 transgene; p450 bioactivates such cancer prodrugs as cyclophosphamide.
Viral cytotoxicity and replication assays were performed in colon carcinoma cells as well as primary human hepatocytes. For in vivo studies, diffuse liver metastases were generated by inoculating MC26 cells into the portal system of BALB/c mice. Mice were treated with control media, rRp450, or rRp450 plus cyclophosphamide.
Cytopathic effects induced by rRp450 replication in colon carcinoma cells were equivalent to those induced by wild type HSV-1 in vitro. Assays developed to separate cytotoxicity mediated by viral replication from cytotoxicity mediated by chemotherapy confirmed that HSV-1 thymidine kinase bioactivates ganciclovir and CYP2B1 bioactivates cyclophosphamide in rRp450-infected cells. rRp450 mediated cytotoxicity in the presence of cyclophosphamide was increased by 21% to 30% above that achieved by viral replication alone. Cyclophosphamide bioactivation produced bystander killing of colon carcinoma cells but not hepatocytes. In contrast to these effects of cyclophosphamide, rRp450 mediated cytotoxicity was reduced in the presence of ganciclovir. These findings are explained by further experiments showing that bioactivation of cyclophosphamide only minimally affected HSV-1 replication in colon carcinoma cells, whereas bioactivation of ganciclovir markedly attenuated HSV-1 replication. In vivo studies revealed a substantial decrease in hepatic tumor burden in all rRp450-treated animals compared to controls. The addition of cyclophosphamide augmented rRp450's in vivo anti-neoplastic effect.
The rRp450 mutant HSV-1 is highly oncolytic against colon carcinoma cells both in vitro and in vivo. rRp450 displays preferential replication in colon carcinoma cells compared to normal hepatocytes. Activation of cyclophosphamide by the p450 transgene augmented the anti-neoplastic effects of rRp450 without simultaneously decreasing viral replication. Oncolysis induced by HSV-1 replication combined with cyclophosphamide prodrug activation warrants further investigation as a potential therapy for colon carcinoma liver metastases.
癌细胞感染单纯疱疹病毒1型（HSV-1）会导致病毒复制，伴随细胞破坏以及子代病毒体释放，进而感染邻近肿瘤细胞。rRp450是一种新型的复制条件性HSV-1突变体，它同时表达内源性疱疹病毒胸苷激酶基因和大鼠p450 CYP2B1转基因；p450可使环磷酰胺等癌症前体药物发生生物活化。
在结肠癌细胞以及原代人肝细胞中进行病毒细胞毒性和复制试验。对于体内研究，通过将MC26细胞接种到BALB/c小鼠的门静脉系统来产生弥漫性肝转移。小鼠分别用对照培养基、rRp450或rRp450加环磷酰胺进行治疗。
rRp450在结肠癌细胞中复制所诱导的细胞病变效应在体外与野生型HSV-1诱导的效应相当。为区分病毒复制介导的细胞毒性和化疗介导的细胞毒性而开发的试验证实，在rRp450感染的细胞中，HSV-1胸苷激酶可使更昔洛韦发生生物活化，CYP2B1可使环磷酰胺发生生物活化。在存在环磷酰胺的情况下，rRp450介导的细胞毒性比单独由病毒复制所达到的水平增加了21%至30%。环磷酰胺的生物活化导致对结肠癌细胞的旁观者杀伤，但对肝细胞无此作用。与环磷酰胺的这些作用相反，在存在更昔洛韦的情况下，rRp450介导的细胞毒性降低。进一步的实验解释了这些发现，即环磷酰胺的生物活化仅对结肠癌细胞中HSV-1的复制产生最小影响，而更昔洛韦的生物活化则显著减弱HSV-1的复制。体内研究显示，与对照组相比，所有接受rRp450治疗的动物肝脏肿瘤负担均大幅降低。添加环磷酰胺增强了rRp450的体内抗肿瘤作用。
rRp450突变体HSV-1在体外和体内对结肠癌细胞均具有高度溶瘤性。与正常肝细胞相比，rRp450在结肠癌细胞中表现出优先复制。p450转基因对环磷酰胺的活化增强了rRp450的抗肿瘤作用，同时并未降低病毒复制。HSV-1复制诱导的溶瘤作用与环磷酰胺前体药物活化相结合，作为结肠癌肝转移的一种潜在治疗方法值得进一步研究。
