Enhancement of anticancer effects by HSV-TK potentiators and evaluation of navel liposome vectors in MSV-TK/prodding gene therapy

在癌症基因治疗试验中，单纯疱疹病毒特异性胸苷激酶(HSV-TK)在更昔洛韦(GCV)或阿昔洛韦(ACV)存在的情况下被用作自杀基因。两种二萜类化合物SDC和PND选择性地刺激HSV-TK的酶活性。因此，我们研究了这些化合物在表达HSV-TK的癌细胞中对GCV/ACV毒性的增强作用。结果如下：1)转染体：将含单纯疱疹病毒L tk基因的真核表达载体导入人癌细胞。克隆后获得稳定表达HSV-TK的(TK^+)细胞克隆。2)体内抗癌效应：SDC/PND与ACV或SDC/PND与GCV联合给药对TK^+细胞的生长有明显的抑制作用，但剂量较低。3)旁观者效应：体外和体内实验检测SDC和PND对TK^+细胞增强旁观者效应的能力。体外联合应用SDC/PND。与前药相比，ACV/GCV使肿瘤细胞对前药更敏感，只有1%~20%的TK^+细胞对前药敏感。在裸鼠体内实验中，注射3%或10%TK^+细胞后，联合用药组小鼠的肿瘤体积较单纯用药组显著缩小。4)毒性和疗效：即使SDC和PND的剂量比体内实验中使用的剂量高10倍，SDC和PND在小鼠身上也没有看到毒性。SDC具有比PND更强的作用，并且有可能从植物的器官培养中稳定地供应。5)脂质体载体效率的评估：在大豆衍生的甾基葡萄糖苷存在的情况下，阳离子脂质体可以提高基因的转染率。该脂质体还提高了肝脏靶向性。

In cancer gene therapy trials, herpes simplex virus-specific thymidine kinase (HSV-TK) has been used as a suicide gene in the presence of ganciclovir (GCV) or acyclovir (ACV). Two diterpenoids, scopadulciol (SDC) and ponicidin (PND), stimulated selectively the HSV-TK enzymatic activity. Thus, we studied the potentiation of GCV/ACV toxicity by these compounds in HSV-TK-expressing cancer cells. The results obtained were as follows:1) Transfectants: Human cancer cells were transfected with the plasmid containing HSV-l tk gene. After cloning, stably HSV-TK-expressing (TK^+) cell clones were obtained. These transfectants showed the ability of forming tumors in nude mice.2) In vivo anticancer effects: The in vivo growth of TK^+ cells was significantly inhibited by coadministration of SDC/PND and ACV, or of SDC/PND and GCV, compared with single administration of ACV or GCV in spite of lower doses.3) Bystander effects: The abilities of SDC and PND to potentiate the bystander effects of the products were determined in both in vitro and in vivo systems. In vitro combined use of SDC/PND. With ACV/GCV rendered tumor cells more sensitive to the prodrugs as compared with the prodrug only in 1 to 20% of TK^+ cells. In the in vivo experiments using nude mice injected with 3 or 10% TK^+ cells, significant reduction in tumor volume was observed in mice treated with drug combinations as compared with those treated with the produg only.4) Toxicity and efficacy: No toxicity of both SDC and PND was seen in mice even at a dose 10-fold higher than that used in the in vivo experiments. SDC has more potent effects than PND, and the possibility for stable supply from organ cultures of the plant.5) Evaluation of the efficiency of liposome vectors: Transfection efficiency of the gene by cationic liposome was increased in the presence of soybean-derived sterylglucoside. The liver targeting was also improved by this liposome.