Uchida Hiroaki, Shinoura Nobusada, Kitayama Joji, Watanabe Toshiaki, Nagawa Hirokazu, Hamada Hirofumi
Department of Molecular Medicine, Sapporo Medical University, S1 W17 Chuo-ku, Sapporo 060-8556, Japan.
J Gene Med. 2003 Apr;5(4):287-99. doi: 10.1002/jgm.339.
In order to develop a safe and effective gene therapy for cancer, more powerful therapeutic genes must be selected and a gene transduction methodology needs to be devised that minimizes the total dose of vector required. We investigated the combination effect of 5-fluorouracil (5-FU), a first-choice drug for the treatment of colorectal cancer and adenovirus-mediated transfer of caspase-8 in DLD-1 colon cancer cells.
The degree of cell death was assessed by determining the percentage of cells which had died, and the degree of DNA fragmentation. The protein expression levels and degree of activation of caspase-8 were analyzed by Western blot analysis. The degree of transgene expression was assessed using adenoviral vectors expressing lacZ and GFP.
Combination treatment led to a significant induction of apoptosis, whereas treatment with either approach alone resulted in only minimal cytotoxicity. Caspase-8 was only activated in cells that received the combined treatment. Exposure to 5-FU increased the quantity of transgene expression per cell, 48 h post-infection. A potentiating effect of adenoviral treatment was also seen when 5-FU treatment was substituted by the overexpression of cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p27(KIP1), suggesting that the cytostatic effect of 5-FU augmented apoptosis induced by caspase-8 gene transduction by inhibiting the dilution of gene products associated with cell division.
This combination strategy may be very useful in the treatment of 5-FU-resistant colorectal cancers and may also be more generally helpful in minimizing the dose of therapeutic vectors used in cancer gene therapy.
为了开发一种安全有效的癌症基因治疗方法，必须选择更强大的治疗基因，并设计一种基因转导方法，以尽量减少所需载体的总剂量。我们研究了5-氟尿嘧啶（5-FU）（一种治疗结直肠癌的首选药物）与腺病毒介导的半胱天冬酶-8在DLD-1结肠癌细胞中的转移的联合作用。
通过确定死亡细胞的百分比和DNA片段化程度来评估细胞死亡程度。通过蛋白质印迹分析来分析半胱天冬酶-8的蛋白质表达水平和激活程度。使用表达lacZ和GFP的腺病毒载体评估转基因表达程度。
联合治疗导致显著的细胞凋亡诱导，而单独使用任何一种方法治疗仅导致最小的细胞毒性。半胱天冬酶-8仅在接受联合治疗的细胞中被激活。感染后48小时，暴露于5-FU增加了每个细胞的转基因表达量。当用细胞周期蛋白依赖性激酶抑制剂p21（WAF1/CIP1）和p27（KIP1）的过表达替代5-FU治疗时，也观察到腺病毒治疗的增强作用，这表明5-FU的细胞生长抑制作用通过抑制与细胞分裂相关的基因产物的稀释来增强半胱天冬酶-8基因转导诱导的细胞凋亡。
这种联合策略可能对治疗5-FU耐药的结直肠癌非常有用，并且在尽量减少癌症基因治疗中使用的治疗载体剂量方面可能也更普遍地有帮助。
