Adjuvant gene therapy for pancreatic cancer using hybrid type liposome vector

使用混合型脂质体载体进行胰腺癌的辅助基因治疗

• 批准号: 10470251
• 负责人: SUNAMURA Makoto
• 金额: $ 7.87万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470251/
• 关键词: pancreatic cancer; gene therapy; uracil phosphoribosyl transferase; 5FU; angiogenesis; soluble VEGF receptor; flt 1; ハイブリッド型リポソームベクター; 癌; P53遺伝子; アデイウイルス; UPRT; UPRT,CD,; 増殖型アテツウイルス; 変異型アデノウィルスベクター; UPRT遺伝子

E. coli uracil phosphoribosyltransferase (UPRT) catalyzes the synthesis of FUMP from 5-FU, resulting in increased production of FdUMP, which induces cytotoxic effect by inhibiting the function of thymidylate synthesis. We constructeded UPRT expressing mutant adenovirus vector (AxElAdB-UPRT) that replicates preferentially in tumor cells without p53 function. Pancreatic cancer cells lacking p53 function were transfected with either UPRT or lacZ gene using adenovirus vectors (Ax-UPRT and Ax-lacZ) and cultured in medium containing various concentration of 5-FU. Therapeutic advantage of AxE1AdB-UPRT/5-FU treatment was evaluated using intraperitoneal disseminated ASPC-1 tumor mice model. The mice were treated with intraperitoneal injection of vectors 10 days after tumor inoculation, followed by the administration of 5-FU(10 mg/Kg) for 7days. Mice were sacrificed and tumor weight was measured 4 weeks later. The sensitivity against 5-FU of 5 pancreatic cancer cells increased significantly (7.1 … More -72 fold) after the transfection of UPRT gene. Tumor weight in Ax-UPRT/5-FU group (0.24 g) was significantly reduced as compared to Ax-lacZ/5-FU group (0.56 g) (P<0.001). Furthermore, that of AxElAdB-UPRT/5-FU group was significantly decreased (0.15 g).(P=0.042 vs Ax-UPRT/5-FU group). Furthermore, the adverse effect of FdUMP on alimentary tract was diminished because of the decreasd dosage of 5-FU.The growth of a tumor and its ability to metastasize is angiogenesis-dependent. Angiogenesis is one of the crucial steps in tumor's transition from a small, harmless cluster of mutated cells to a large, malignant growth, capable of spreading to other organs throughout the body soluble. We transfected Panc-1 cells with soluble VEGF receptor (flt1) and as control with the lacZ gene using the adenovirus vectors Adsflt and AdlacZ, respectively. Adsflt or AdlacZ (3.5X10^7 PFU) or 100 μl of PBS were injected directly into tumors daily from day 8 through day 12 after the implantation. Furthermore, we implanted in dorsal skinfold chamber the wild-type Panc-1 and its transfectants and monitored the tumor angipgenesis using in vivo microscope system. VEGF levels in conditioned media were higher in all cell lines (mean, 1969 pg/ml ; Panc-1, 980 pg/ml), compared to the level in media of HUVEC (8 pg/ml). Expression of VEGF mRNA was detected in the all cell lines by RT-PCR and Northern blot analysis and it was confirmed by DNA sequencing. Tumor growth rate of Adsflt-infected cells was lower than those of AdlacZ group and no treatment group, and the difference was statistically significant. Wild type Panc-1 and the LacZ transfectant implantation prompted strong tumor angiogenesis as observed in skinfold chamber, whereas soluble flt-1 transfectants failed to exert such an effect. Less

E.大肠杆菌尿嘧啶磷酸核糖基转移酶（UPRT）催化5-FU合成FUMP，导致FdUMP的产生增加，其通过抑制胸苷酸合成的功能而诱导细胞毒性作用。我们构建了表达UPRT的突变型腺病毒载体（AxElAdB-UPRT），该载体在无p53功能的肿瘤细胞中优先复制。使用腺病毒载体（Ax-UPRT和Ax-lacZ）将UPRT或lacZ基因转染缺乏p53功能的胰腺癌细胞，并在含有不同浓度的5-FU的培养基中培养。使用腹膜内播散的ASPC-1肿瘤小鼠模型评价AxE 1AdB-UPRT/5-FU治疗的治疗优势。在肿瘤接种后10天通过腹膜内注射载体治疗小鼠，随后给予5-FU（10 mg/Kg）7天。4周后处死小鼠并测量肿瘤重量。5株胰腺癌细胞对5-FU的敏感性显著提高（7.1 ...更多信息 -72倍）。Ax-UPRT/5-FU组瘤重（0.24 g）较Ax-lacZ/5-FU组（0.56 g）显著降低（P<0.001）。AxE 1AdB-UPRT/5-FU组细胞凋亡率明显降低（0.15g）。（P=0.042 vs Ax-UPRT/5-FU组）。FdUMP对消化道的不良反应也因5-FU剂量的减少而减轻。肿瘤的生长和转移能力依赖于血管生成。血管生成是肿瘤从一个小的，无害的突变细胞簇转变为一个大的，恶性生长的关键步骤之一，能够扩散到整个身体的其他器官。我们转染Panc-1细胞与可溶性VEGF受体（flt 1）和作为对照与lacZ基因使用腺病毒载体Adsflt和AdlacZ，分别。从植入后第8天到第12天，每天将Adsflt或AdlacZ（3.5 × 10^7 PFU）或100 μl PBS直接注射到肿瘤中。此外，我们将野生型Panc-1及其转染子植入背侧皮褶室中，并使用体内显微镜系统监测肿瘤血管发生。与HUVEC培养基中的水平（8 pg/ml）相比，在所有细胞系中条件培养基中的VEGF水平（平均值，1969 pg/ml ; Panc-1，980 pg/ml）更高。RT-PCR和北方印迹分析检测VEGF mRNA在所有细胞系中的表达，并通过DNA测序证实。Adsflt感染细胞的肿瘤生长率低于AdlacZ组和未治疗组，差异有统计学意义。野生型Panc-1和LacZ转染子植入促进了如在皮褶腔室中观察到的强肿瘤血管生成，而可溶性flt-1转染子未能发挥这种作用。少

项目成果

Sunamura M: "The anti-angiogenesis effect of IL-12 during early growth of human pancreatic cancer in SCID mice"Pan Creas. 20・3. 227-233 (2000)

Sunamura M：“IL-12 在 SCID 小鼠早期生长过程中的抗血管生成作用”Pan Creas 20・3（2000）。

砂村眞琴: "膵癌に対する遺伝子治療法の開発"日本臨床59:98-103,2001. 59・1. 98-103 (2001)

Makoto Sunamura：“胰腺癌基因治疗的发展”日本临床研究59：98-103，2001 59・1（2001）。

Sunamura M., Motoi F., Matsuno S.: "Restricted Replication-Competent Adenovirus : a Novel Strategy for Cancer Gene Therapy"Int J Clin Oncol. 5. 147-152 (2000)

Sunamura M.、Motoi F.、Matsuno S.：“具有限制性复制能力的腺病毒：癌症基因治疗的新策略”Int J Clin Oncol。

Sunamura M: "Restricted Replication-Competent Adenovirus : a Novel Strategy for Cancer Gene Therapy"Int J Clin Oncol. 5・7. 147-152 (2000)

Sunamura M：“限制性复制腺病毒：癌症基因治疗的新策略”Int J Clin Oncol 5・7（2000）。

Sunamura M, Motoi F, Onuma M, Abe H, Matsuno M: "Development of gene therapy for pancreatic cancer"Nippon Rinsho. 59. 98-103 (2001)

Sunamura M、Motoi F、Onuma M、Abe H、Matsuno M：“胰腺癌基因治疗的开发”Nippon Rinsho。