Li Rongxiu, Boehm Amanda L, Miranda Michelle B, Shangary Sanjeev, Grandis Jennifer R, Johnson Daniel E
Department of Medicine, University of Pittsburgh School of Medicine, and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213-1863, USA.
Neoplasia. 2007 Oct;9(10):801-11. doi: 10.1593/neo.07394.
Head and neck squamous cell carcinomas (HNSCC) are frequently characterized by chemotherapy and radiation resistance, and by overexpression of Bcl-XL, an antiapoptotic member of the Bcl-2 protein family. In this report we examined whether cell-permeable peptides derived from the BH3 domains of proapoptotic Bax, Bad, or Bak could be used to target Bcl-XL and/or Bcl-2 in HNSCC cells, and induce apoptotic death in these cells. To render the peptides cell permeable, Antennapedia (Ant) or polyarginine (R8) peptide transduction domains were fused to the amino termini. Fluorescence microscopy of peptide-treated HNSCC cells revealed that the BH3 peptides colocalized with mitochondria, the site of Bcl-XL and Bcl-2 expression. By contrast, a mutant peptide (BaxE BH3) which cannot bind Bcl-XL or Bcl-2 was diffusely localized throughout the cytoplasm. Treatment of three HNSCC cell lines (1483, UM-22A, UM-22B) with the wild-type BH3 peptides resulted in loss of viability and induction of apoptosis, as assessed by MTS assays and annexin V staining. In general, Ant-conjugated peptides were more potent than R8-conjugated peptides, and Bad BH3 peptide was typically more potent than Bax BH3 or Bak BH3. Treatment of purified HNSCC mitochondria with BH3 peptides resulted in robust release of cytochrome c. Thus, the relative apoptosis resistance of HNSCC cells is not due to a deficit in this step of the intrinsic, mitochondrial-mediated apoptosis pathway. We conclude that cell-permeable BH3 peptides can be used to target Bcl-XL and/or Bcl-2 in HNSCC, and targeting of these proteins may have therapeutic value in the treatment of this disease.
头颈部鳞状细胞癌（HNSCC）的特征通常是具有化疗和放疗抗性，以及抗凋亡的Bcl-2蛋白家族成员Bcl-XL的过表达。在本报告中，我们研究了源自促凋亡蛋白Bax、Bad或Bak的BH3结构域的细胞穿透肽是否可用于靶向HNSCC细胞中的Bcl-XL和/或Bcl-2，并诱导这些细胞发生凋亡性死亡。为使肽具有细胞穿透性，将触角足蛋白（Ant）或聚精氨酸（R8）肽转导结构域融合至氨基末端。对经肽处理的HNSCC细胞进行荧光显微镜观察发现，BH3肽与线粒体共定位，而线粒体是Bcl-XL和Bcl-2的表达位点。相比之下，一种不能结合Bcl-XL或Bcl-2的突变肽（BaxE BH3）则弥漫性地定位在整个细胞质中。用野生型BH3肽处理三种HNSCC细胞系（1483、UM-22A、UM-22B），通过MTS分析和膜联蛋白V染色评估，结果显示细胞活力丧失并诱导了凋亡。一般来说，与Ant偶联的肽比与R8偶联的肽更有效，并且Bad BH3肽通常比Bax BH3或Bak BH3更有效。用BH3肽处理纯化的HNSCC线粒体导致细胞色素c大量释放。因此，HNSCC细胞相对的凋亡抗性并非由于内源性线粒体介导的凋亡途径这一步骤存在缺陷。我们得出结论，细胞穿透性BH3肽可用于靶向HNSCC中的Bcl-XL和/或Bcl-2，并且靶向这些蛋白可能对该疾病的治疗具有治疗价值。
