Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo.
Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo.
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分子化疗联合放射治疗可增强体外和体内对胆管癌细胞的杀伤作用。
作者：
L. Pederson;D. Buchsbaum;S. Vickers;S. Kancharla;Matthew S. Mayo;D. Curiel;M. Stackhouse
胆管癌是一种几乎无法治愈的肿瘤，对目前的手术、化疗和放疗干预具有抗性。我们将无毒前体药物转化为化疗药物的基因治疗策略与放射治疗相结合应用于胆管癌的治疗。在这方面，5-氟尿嘧啶（5-FU）是目前用于癌症治疗的公认的放射增敏剂和化学治疗剂。大肠杆菌酶胞嘧啶脱氨酶（CD）将前药5-氟胞嘧啶（5-FC）转化为5-FU。因此，我们的目标是在人胆管癌细胞系SK-ChA-1中表达CD基因，评估细胞内产生的5-FU的细胞毒性，并确定通过加入外部束辐射增强的细胞杀伤。SK-ChA-1细胞对重组腺病毒感染的敏感性通过荧光激活细胞分选分析来确定。我们使用重组腺病毒载体AdCMVLacZ，编码E.在人巨细胞病毒（CMV）启动子控制下的大肠杆菌β-半乳糖苷酶报告基因，以10和100空斑形成单位（pfu）/细胞感染SK-ChA-1和HeLa细胞，随后进行FACS分析。为了评价CD介导的5-FC向5-FU的转化以及随后的细胞毒性，用编码CD的重组腺病毒AdCMVCD感染SK-ChA-1细胞。然后将细胞接种在96孔微量滴定板中，并暴露于不同浓度的5-FC。在接种后2-8天开始进行细胞增殖测定（四唑盐转化为甲瓒比色测定）。我们评价了体外照射的影响，使用单剂量8戈伊60 Co的AdCMVCD感染的细胞，与5-FC预先暴露2-3天。在放射处理后进行MTS测定。通过克隆形成试验进行的辐射剂量反应分析被用作更灵敏的试验，以确认处理条件的相互作用。S.C.在无胸腺裸鼠中建立SK-ChA-1肿瘤，然后接受三次瘤内注射1 × 10（9）pfu AdCMVCD。从初始AdCMVCD注射当天（第-2天）开始，小鼠接受400 mg/kg 5-FC腹腔注射，每天两次，持续7天。放射治疗组在第0天接受10戈伊的60 Co照射。SK-ChA-1细胞分别被10和100 pfu/细胞的AdCMVLacZ有效地转导（48.7和99.2%）。用10 pfu/cell AdCMVCD感染SK-ChA-1细胞并暴露于不同浓度的5-FC（5、10、30、50和100 μ g/ml）8天后，37.9%至84.4%的SK-ChA-1细胞被杀死。较高的5-FC浓度和较长的暴露持续时间导致更大的细胞杀伤。当与10或20 μ g/ml的5-FC组合时，辐射处理（8戈伊）增强细胞杀伤大于70%。放射剂量反应分析与克隆形成试验证实增强SK-ChA-1细胞的细胞毒性作为一个结果，辐射治疗后的AdCMVCD感染和5-FC暴露，放射生物学参数α = 0.44和D 0 = 0.96。与非放射治疗组相比，SK-ChA-1肿瘤与AdCMVCD、5-FC和放射联合治疗动物的存活率、肿瘤再生长时间和倍增时间显著延长（分别为P = 0.03、0.015和0.002）。与未接受放射治疗组相比，在放射治疗组中观察到肿瘤大小的变化显著更大，最终肿瘤大小与原始肿瘤大小的比值更小，最终肿瘤大小更小（分别为P = 0.02，0.03和0.03）。人胆管癌细胞转染重组腺病毒在体外高效率和CD介导的细胞内5-FU的生产是敏感的。放射生物学存活曲线参数证实了病毒感染和前药治疗与外部束辐射暴露相结合时的相互作用的细胞毒性效应。（摘要截断）
Cholangiocarcinoma is a virtually incurable tumor, resistant to current surgical, chemotherapy, and radiotherapy interventions. We applied the gene therapy strategy of toxin gene conversion of nontoxic prodrug to chemotherapeutic drug in combination with radiation therapy to the treatment of cholangiocarcinoma. In this regard, 5-fluorouracil (5-FU) is an accepted radiosensitizing and chemotherapeutic agent presently used in cancer therapy. The Escherichia coli enzyme cytosine deaminase (CD) converts the prodrug 5-fluorocytosine (5-FC) to 5-FU. Therefore, our goal was to express the CD gene in the human cholangiocarcinoma cell line, SK-ChA-1, assess the cytotoxicity of intracellular production of 5-FU, and determine any enhanced cell killing by the addition of external beam radiation. The susceptibility of SK-ChA-1 cells to recombinant adenoviral infection was determined by fluorescence-activated cell sorting analysis. We used the recombinant adenoviral vector AdCMVLacZ, encoding the E. coli beta-galactosidase reporter gene under control of the human cytomegalovirus (CMV) promoter, to infect SK-ChA-1 and HeLa cells at 10 and 100 plaque forming units (pfu)/cell, followed by FACS analysis. To evaluate CD-mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes CD. Cells were then plated in 96-well microtiter plates and exposed to varying concentrations of 5-FC. Cell proliferation assays (tetrazolium salt conversion to formazan colorimetric assay) were performed beginning 2-8 days after plating. We evaluated the effects of external beam radiation using a single 8 Gy 60Co dose to AdCMVCD infected cells, with prior exposure to 5-FC for 2-3 days. MTS assays were performed following radiation treatment. Radiation dose-response analysis, via clonogenic assay, was used as a more sensitive assay to confirm the interaction of the treatment conditions. s.c. SK-ChA-1 tumors in athymic nude mice were established, which then received three intratumoral injections of 1 x 10(9) pfu AdCMVCD. Mice received i.p. injections of 400 mg/kg of 5-FC twice daily for 7 days beginning the day of initial AdCMVCD injection (day -2). The radiation treatment group received 10 Gy of 60Co exposure to their tumor on day 0. SK-ChA-1 cells were efficiently transduced (48.7 and 99.2%) by 10 and 100 pfu/cell of AdCMVLacZ, respectively. From 37.9 to 84.4% of SK-ChA-1 cells were killed following infection with 10 pfu/cell AdCMVCD and 8 days of exposure to various concentrations of 5-FC (5, 10, 30, 50, and 100 microg/ml). Higher 5-FC concentrations and longer duration of exposure resulted in greater cell killing. Radiation treatment (8 Gy) enhanced cell killing by greater than 70% when combined with 10 or 20 microg/ml of 5-FC. Radiation dose-response analysis with clonogenic assay confirmed enhanced SK-ChA-1 cell cytotoxicity as a result of radiation treatment following AdCMVCD infection and 5-FC exposure, with radiobiological parameters alpha = 0.44 and D0 = 0.96. Combined treatment of SK-ChA-1 tumors with AdCMVCD, 5-FC, and radiation in animals resulted in significantly greater survival, time to tumor regrowth, and doubling time compared to the nonradiation treatment group (P = 0.03, 0.015, and 0.002, respectively). Significantly greater change in tumor size, smaller ratio of final tumor size to original tumor size, and smaller final tumor size were observed in the radiation treatment group compared to the no radiation treatment group (P = 0.02, 0.03, and 0.03, respectively). Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency and were susceptible to CD-mediated intracellular 5-FU production. Radiobiological survival curve parameters confirmed an interactive cytotoxic effect when viral infection and prodrug therapy were combined with external beam radiation exposure. (ABSTRACT TRUNCATED)
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DOI：
10.1016/0360-3016(93)90423-s
发表时间：
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期刊：
International journal of radiation oncology, biology, physics
影响因子：
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DOI：
10.1093/jnci/88.17.1193
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