Orzechowska Emilia Joanna, Kozlowska Ewa, Czubaty Alicja, Kozlowski Piotr, Staron Krzysztof, Trzcinska-Danielewicz Joanna
Department of Molecular Biology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
BMC Cancer. 2014 Oct 18;14:771. doi: 10.1186/1471-2407-14-771.
Low cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study.
Uptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay.
TAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID.
TAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.
BID的低细胞水平对众多癌细胞的存活至关重要。通过腺病毒或pcDNA载体过表达BID使细胞对抗癌药物敏感是一种提出的癌症治疗策略；然而，它并未对BID的细胞水平提供任何严格的控制。这项工作的目的是研究BID与TAT细胞穿透肽（TAT-BID）的融合是否可用于使癌细胞对通过死亡受体（TRAIL）或DNA损伤（喜树碱）起作用的抗癌药物进行可控性敏感化。研究中使用了前列腺癌PC3和LNCaP细胞、非小细胞肺癌A549细胞以及子宫颈癌HeLa细胞。
通过对细胞提取物进行定量蛋白质免疫印迹分析来研究细胞对TAT-BID蛋白的摄取。通过MTT试验监测细胞活力。通过流式细胞术和细胞色素c释放试验检测细胞凋亡。
TAT-BID以取决于时间、剂量和细胞系的量递送至所有癌细胞。重组BID使PC3细胞对TRAIL敏感，或在较小程度上对喜树碱敏感。在其余细胞中，TAT-BID使A549细胞对TRAIL敏感，而仅使HeLa细胞对TRAIL有轻微敏感。后两种细胞系均未对喜树碱敏感。在所有情况下，不能被CK2磷酸化的突变体（TAT-BIDT59AS76A）在敏感化方面与野生型TAT-BID同样有效。
TAT-BID可以可控方式递送至癌细胞，并有效地使PC3和A549细胞对TRAIL敏感。因此，它可被视为一种潜在的治疗剂，可增强TRAIL治疗前列腺癌和非小细胞肺癌的疗效。
