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）已被用作更昔洛韦（GCV）或阿昔洛韦（ACV）存在下的自杀基因。两种二萜类化合物，东莨菪碱（SDC）和ponicidin（PND），选择性地刺激HSV-TK酶活性。因此，我们研究了这些化合物在表达HSV-TK的癌细胞中增强GCV/ACV毒性。结果如下：1）转染细胞：用含HSV-1 tk基因的质粒转染人癌细胞。克隆后，获得稳定表达HSV-TK（TK^+）的细胞克隆。2）体内抗肿瘤作用：与单用ACV或GCV相比，SDC/PND与ACV或GCV联合应用可显著抑制TK^+细胞的体内生长，尽管剂量较低。3）旁观者效应：在体外和体内系统中测定SDC和PND增强产物的旁观者效应的能力。体外联合使用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。在使用注射3或10%TK ^+细胞的裸鼠的体内实验中，与仅用前药治疗的小鼠相比，在用药物组合治疗的小鼠中观察到肿瘤体积的显著减小。4）毒性和功效：即使在比体内实验中使用的剂量高10倍的剂量下，也没有在小鼠中观察到SDC和PND两者的毒性。SDC比PND具有更强的效果，并且有可能从植物的器官培养物中稳定供应。5）脂质体载体效率的评估：在大豆衍生的甾醇葡萄糖苷的存在下，阳离子脂质体对基因的转染效率增加。该脂质体还提高了肝靶向性。

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.