Enhanced cytotoxicity of RIPTK gene therapy of pancreatic cancer via PDX-1 co-delivery
Enhanced cytotoxicity of RIPTK gene therapy of pancreatic cancer via PDX-1 co-delivery
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DOI：
10.1016/j.jss.2006.04.039
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发表时间：
2007-01-01
影响因子：
2.2
通讯作者：
Brunicardi, F. Charles
中科院分区：
文献类型：
--
作者：
Liu, Shihe;Wang, Xiao-Ping;Brunicardi, F. Charles
背景资料。利用小鼠体内模型，我们证明了胰岛素启动子驱动的自杀基因治疗(RIPTK)可以用于治疗小鼠胰岛素瘤和人胰腺癌细胞。然而，这种疗法的局限性包括肿瘤细胞缺乏足够的PDX-1蛋白，以及脂质体介导的转基因表达水平较低。本研究的目的是确定1)瞬时转导PDX-1是否会导致RIPTK细胞毒性增加；2)腺病毒递送系统是否会在体外增加RIPTK基因的整体表达。RIPlacZ和RSVlacZ质粒DNA以及AdCMVlacZ和AdRIPlacZ分别用于人胰腺癌细胞株Panc-1和MIA PaCa2(n=8)的转染实验。还使用了含有小鼠PDX-1基因的表达载体DNA。进行LacZ报告分析。RIPTK基因构建于载体或腺病毒载体中，用于细胞毒实验。用RT-PCR方法检测PDX-1的表达水平。人胰腺癌PANC-1细胞表达PDX-1蛋白，MIA、PaCa2细胞表达少量PDX-1蛋白。脂质体(L)介导的RSVlacZ和RIPlacZ在PANC-1细胞中的转基因表达分别为10.1%和9.3%。联合应用PDX-1对RSVlacZ基因表达无显著影响(9.3%，P=NS)，但显著增加RIPlacZ基因表达(14.9%P&t；0.05)。腺病毒(Ad)介导的RIPlacZ转基因在PANC-1细胞中高表达(66.1%)，共转导PDX-1后报告基因活性进一步增强(70.2%，P<0.05)。用RSVlacZ和RIPlacZ报告基因脂质体转染MIA PaCa2细胞，分别获得9.3%和1.0%的基因表达。PDX-1共转染组RIPlacZ基因表达显著激活(14.5%，P<0.05)，而RSVlacZ组(9.8%)无明显变化。ADCM-VlacZ和AdRIPlacZ可显著提高MIA PaCa2细胞的报告活性(分别为63.0%和9.8%)。PDX-1基因的转染组AdRIPlacZ活性显著升高(46.0%，P<；0.05)，而AdCMVlacZ组的AdRIPlacZ活性(68.2%)无明显变化。脂质体-RIPTK/更昔洛韦(GCV)对PANC-1细胞的细胞毒作用为18.6%，与PDX-1共转染组为22.8%(P=NS)。单独用RIPTK处理Mia PaCa2细胞后，细胞死亡率为4.9%，与外源性PDX-1联合作用时细胞死亡率增加至18.2%(P<；0.05)。经GCV处理后，Ad-RIPTK基因对PANC-1(29.3%)和MIA PaCa2(12.4%)细胞产生明显的细胞毒作用。当PDX-1与PdX-1共转染时，PANC-1和MIA PaCa2细胞的细胞毒作用分别增加到43.4%和29.4%(P<；0.05)。这些数据表明，与脂质体相比，腺病毒介导的基因传递导致转基因表达显著增加。RIPTK介导的细胞毒性也通过外源性PDX-1在这些细胞中的共传递而显著增强。因此，这些结果也表明PDX-1在胰岛素启动子激活中起关键作用，并证明PDX-1的产生在胰岛素启动子导向的基因治疗中是必不可少的。(C)2007 Elsevier Inc.保留所有权利。
Background. Using in vivo mouse models, we have demonstrated that the insulin promoter-driven suicidal gene therapy (RIPTK) could be used in the treatment of mouse insulinoma and human pancreatic cancer cells. However, limitations of this therapy include tumor cells lack of sufficient PDX-1 protein and low levels of transgene expression mediated by liposome delivery system. The purpose of this study was to determine 1) whether transient transfection of PDX-1 into selected pancreatic cancer cells would lead to increased RIPTK cytotoxicity, and 2) whether an adenoviral delivery system would increase the overall RIPTK gene expression in vitro.Material and methods. RIPlacZ and RSVlacZ plasmid DNA as well as AdCMVlacZ and AdRIPlacZ were used in transfection assays in human pancreatic cancer cell lines PANC-1 and MIA PaCa2 (n = 8). An expression plasmid DNA containing the mouse PDX-1 cDNA was also used. LacZ reporter assays were performed. RIPTK genes constructed either in plasmid or in adenoviral vectors were used in cytotoxic assays. RT-PCR assays were used to determine PDX-1 expression levels.Results. PDX-1 protein was detected in the human pancreatic ductal carcinoma cell line PANC-1, a little in MIA, PaCa2 cells. Liposome mediated (L) RSVlacZ and RIPlacZ transfection in PANC-1 cells resulted in 10.1% and 9.3% transgene expression, respectively. Co-delivery of PDX-1 had no significant effect on RSVlacZ expression (9.3%, P = NS) but significantly increased RIPlacZ gene expression (14.9% P < 0.05). Adenoviral mediated (Ad) RIPlacZ transgene was highly expressed in PANC-1 cells (66.1%) and the reporter activity was further enhanced when PDX-1 was co-delivered (70.2%, P < 0.05). Liposomal transfection of MIA PaCa2 cells using RSVlacZ and RIPlacZ reporter genes resulted in 9.3% and 1.0% gene expression, respectively. Co-transfection of PDX-1 in these cells resulted in a significant activation of RIPlacZ gene expression (14.5%, P < 0.05) with no effects on RSVlacZ treated cells (9.8%). AdCM- VlacZ and AdRIPlacZ significantly increased reporter activities in MIA PaCa2 cells (63.0% and 9.8%, respectively). Transfection of PDX-1 also significantly enhanced the AdRIPlacZ activities (46.0%, P < 0.05), with no significant effect in AdCMVlacZ treated cells (68.2%). The cytotoxic effect of liposome-RIPTK/ganciclovir (GCV) in PANC-1 cells was 18.6% and increased to 22.8% when PDX-1 was co-transfected into the cells (P = NS). MIA PaCa2 cells treated with RIPTK alone resulted in 4.9% cell death and increased to 18.2% when exogenous PDX-1 was co-delivered (P < 0.05). The Ad-RIPTK gene delivery with GCV treatment caused significant cytotoxic effect in PANC-1 (29.3%) and MIA PaCa2 (12.4%) compared with untreated cells. The cytotoxic effects were further increased to 43.4% and 29.4% in PANC-1 and MIA PaCa2 cells, respectively, when PDX-1 was co-transfected (P < 0.05 for both).Conclusions. These data demonstrated that adenoviral mediated gene delivery resulted in a significant increase of transgene expression compared with liposomal delivery systems. RIPTK mediated cytotoxicity was also significantly enhanced via co-delivery of exogenous PDX-1 in these cells. Thus, these results also indicated that PDX-1 plays critical roles in insulin promoter activation and demonstrated that PDX-1 production is essential for insulin promoter-directed gene therapy. (c) 2007 Elsevier Inc. All rights reserved.