Bcl-XL antisense oligonucleotides coupled with antennapedia enhances radiation-induced apoptosis in pancreatic cancer
Bcl-XL antisense oligonucleotides coupled with antennapedia enhances radiation-induced apoptosis in pancreatic cancer
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DOI：
10.1016/j.surg.2006.03.014
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发表时间：
2006-08-01
期刊：
影响因子：
3.8
通讯作者：
Doi, Ryuichiro
中科院分区：
文献类型：
--
作者：
Masui, Toshihiko;Hosotani, Ryo;Doi, Ryuichiro
背景。胰腺癌对放化疗具有高度的耐药性，其耐药性反映了包括Bcl-2家族在内的凋亡抑制基因的增强。Antennapedia （pAnt）能够通过脂质双分子层几乎100%内化到细胞中，而没有任何细胞毒性作用。本研究的目的是检验Bcl-XL的作用。利用反义寡核苷酸与天线环偶联的方法研究胰腺癌的体外和体内放射敏感性。体外实验检测5种胰腺癌细胞系中Bcl-XL蛋白的表达。在AsPC-1细胞中，证实了寡核素的内化，并检测了触角反义（pAnt-AS）或触角乱序（pAnt-Scr）对Bcl-XL蛋白表达的影响。用pAnt-AS、pAnt-Scr或硫代反义（S-AS）处理细胞3 d，观察辐照对细胞存活、caspase-3活性和凋亡指数的影响。在AsPC-1异种移植小鼠中，注射pAnt-AS、pAnt-Scr或S-AS，并加5或10 Gy辐照。辐照前检测Bcl-Xl蛋白表达。照射后48小时观察细胞凋亡情况。在10-Gy照射后第14天测量肿瘤湿重，并在5周内观察肿瘤生长情况。在体外实验中，所有胰腺癌细胞系均表达Bcl-XL蛋白。pAnt-AS在2小时内内化到AsPC-1细胞中。10 μ mol/L的pAnt-AS可降低AsPC-1细胞中90%以上的Bcl-XL蛋白，而相同浓度的pAnt-Scr或S-AS可降低10%以上的Bcl-XL蛋白。与pAnt-Scr或S-AS相比，pAnt-AS处理后辐照显著降低了细胞活力。pant - as处理组Caspase-3活性显著上调（P = 0.033）。pAnt-AS组细胞核碎裂率显著高于对照组（P = 0.013）。在体内实验中，经pant - as处理的小鼠体内Bcl-XL蛋白减少约40%。10-Gy辐照可延长pant - as处理小鼠的肿瘤倍增时间。与pAnt-Scr和10-Gy辐照组相比，pAnt-AS和10-Gy辐照组小鼠肿瘤湿重显著降低（P = 0.046）。照射后48 h， panas处理小鼠的细胞凋亡指数显著升高（P < 0.01）。结果表明，当与天线基偶联时，针对Bcl-XL的反义寡核苷酸可能成为胰腺癌放射增敏的良好治疗工具。
Background. Pancreatic cancer is highly resistant to radiation and chemotherapy, and its resistance,reflects the enhancement of apoptosis inhibitory genes, including Bcl-2 family. Antennapedia (pAnt) is capable of almost 100% internalization into cells through the lipid bilayer without any cytotoxic effect. The aim of this study was to examine the effects of the Bcl-XL. antisense oligonucleotide for radiosensitivity of in vitro and in vivo pancreatic cancer using oligonucleotide conjugated with antennapedia.Methods. In in vitro experiments, expression of Bcl-XL protein was examined in 5 pancreatic cancer cell lines. In AsPC-1 cells, internalization of the oligonucteolide was confirmed, and the effects of antennopedia-antisense (pAnt-AS) or antennapedia-scramble (pAnt-Scr) on Bcl-XL protein expression were examined. Cells were treated with pAnt-AS, pAnt-Scr or phosphorothioate antisense (S-AS) for 3 days, then the effects of irradiation on the cell survival, caspase-3 activity, and apoptotic index were evaluated. In AsPC-1 xenograft mice, pAnt-AS, pAnt-Scr, or S-AS was injected, and 5 or 10 Gy irradiation was added. Bcl-Xl protein, expression was measured before irradiation. Apoptosis was evaluated at 48 hours after irradiation. On the 14th day after 10-Gy irradiation, tumor wet weight was measured, and tumor growth was estimated over 5 weeks.Results. In in vitro experiments, all pancreatic cancer cell lines expressed Bcl-XL protein. pAnt-AS was internalized into AsPC-1 cells within 2 hours. pAnt-AS at 10 mu mol/L reduced more than 90% of the Bcl-XL protein in AsPC-1 cells, whereas pAnt-Scr or S-AS treatment at the same concentration reduced as much as 10% of the Bcl-XL protein. Treatment with pAnt-AS followed by irradiation significantly reduced cell viability when compared with that of pAnt-Scr or S-AS. Caspase-3 activity was significantly upregulated in the pAnt-AS-treated group (P = .033). The rate of nuclear fragmentation was significantly higher in the pAnt-AS group (P = .013). In in vivo experiments, Bcl-XL protein was,reduced about 40% in the pAnt-AS-treated mice. Tumor doubling time of the pAnt-AS-treated mice was elongated by 10-Gy irradiation. The tumor wet weight of mice treated with pAnt-AS and 10-Gy irradiation was significantly reduced when compared with mice treated with pAnt-Scr and 10-Gy irradiation (P = .046). The opoptosis index at 48 hours after irradiation was significantly increased in pAnt AS-treated mice (P < .01).Conclusions. The results suggest that, when coupled with antennapedia, the antisense oligonucleotide against Bcl-XL could, be a good therapeutic tool for radiosensitization of pancreatic cancer.