Zangemeister-Wittke U, Leech S H, Olie R A, Simões-Wüst A P, Gautschi O, Luedke G H, Natt F, Häner R, Martin P, Hall J, Nalin C M, Stahel R A
Department of Internal Medicine, University Hospital Zürich, Switzerland.
Clin Cancer Res. 2000 Jun;6(6):2547-55.
Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in solid tumors. The bcl-2 and bcl-xL mRNAs share a region of homology comprising nucleotides 605-624 and 687-706, respectively, which differs by only three nucleotides. This sequence does not occur in the proapoptotic splice variant bcl-xS. To test the possibility that oligonucleotides targeting this region have the potential to down-regulate bcl-2 and bcl-xL expression simultaneously, three 2'-O-methoxy-ethoxy-modified phosphorothioate oligonucleotides were designed. These oligonucleotides differed in the number of mismatches to bcl-2 and bcl-xL and in the number of nucleotides to which the modifications were made. The effects of these oligonucleotides on bcl-2 and bcl-xL expression, as well as their abilities to induce apoptosis, were assessed in small cell and non-small cell lung cancer cell lines expressing different basal levels of bcl-2 and bcl-xL. Although all oligonucleotides down-regulated bcl-2 and bcl-xL expression, oligonucleotide 4625, which has no mismatching nucleotides to bcl-2 but three to bcl-xL, two of which were modified by 2'-O-methoxy-ethoxy residues, showed the strongest bispecific activity on the transcript and protein level. In all cell lines this bispecific activity induced apoptotic cell death, as demonstrated by increased uptake of propidium iodide, a 10-100-fold increase in caspase-3-like protease activity, and nuclear condensation and fragmentation. This is the first report of a bcl-2/bcl-xL bispecific antisense oligonucleotide that deserves attention as a therapeutic compound in lung cancer and other malignancies in which bcl-2 and/or bcl-xL are overexpressed.
Bcl-2和Bcl-xL是凋亡抑制因子，在实体瘤中经常过度表达。bcl-2和bcl-xL mRNA分别共享一个同源区域，包含核苷酸605 - 624和687 - 706，两者仅相差三个核苷酸。该序列不存在于促凋亡剪接变体bcl-xS中。为了测试靶向该区域的寡核苷酸是否有可能同时下调bcl-2和bcl-xL的表达，设计了三种2'-O-甲氧基-乙氧基修饰的硫代磷酸酯寡核苷酸。这些寡核苷酸与bcl-2和bcl-xL的错配数以及进行修饰的核苷酸数不同。在表达不同基础水平bcl-2和bcl-xL的小细胞和非小细胞肺癌细胞系中评估了这些寡核苷酸对bcl-2和bcl-xL表达的影响及其诱导凋亡的能力。尽管所有寡核苷酸都下调了bcl-2和bcl-xL的表达，但寡核苷酸4625对bcl-2没有错配核苷酸，对bcl-xL有三个错配核苷酸，其中两个被2'-O-甲氧基-乙氧基残基修饰，在转录本和蛋白质水平上显示出最强的双特异性活性。在所有细胞系中，这种双特异性活性诱导了凋亡细胞死亡，表现为碘化丙啶摄取增加、caspase-3样蛋白酶活性增加10 - 100倍以及核浓缩和碎片化。这是关于bcl-2/bcl-xL双特异性反义寡核苷酸的首次报道，作为一种治疗化合物，在bcl-2和/或bcl-xL过度表达的肺癌和其他恶性肿瘤中值得关注。
