Carpi Sara, Fogli Stefano, Giannetti Ambra, Adinolfi Barbara, Tombelli Sara, Da Pozzo Eleonora, Vanni Alessia, Martinotti Enrica, Martini Claudia, Breschi Maria Cristina, Pellegrino Mario, Nieri Paola, Baldini Francesco
Department of Pharmacy, University of Pisa, Pisa, Italy.
Institute of Applied Physics "Nello Carrara," IFAC-CNR, Sesto Fiorentino, Florence, Italy.
PLoS One. 2014 Dec 11;9(12):e114588. doi: 10.1371/journal.pone.0114588. eCollection 2014.
Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.
生存素是一种凋亡抑制因子，在不同类型肿瘤中过度表达，而在大多数终末分化的正常组织中无法检测到。在本研究中，我们试图评估靶向生存素mRNA的分子信标-寡脱氧核苷酸（MB）的体外诊疗特性。我们使用激光扫描共聚焦显微镜研究MB在活细胞中的递送情况，并通过实时PCR和蛋白质印迹法评估其在人恶性黑色素瘤细胞中对生存素的选择性靶向作用。我们还通过测量核小体间DNA片段化、线粒体膜电位（MMP）的消散以及核形态的变化来评估MB的促凋亡作用。将MB转染到A375和501 Mel细胞中后，细胞质产生了高信号强度，而在细胞外环境和生存素阴性细胞（即人黑素细胞和单核细胞）中未检测到信号。MB可使黑色素瘤细胞中的生存素mRNA和蛋白质表达随时间依赖性降低，在72小时时达到最大效应。用MB处理黑色素瘤细胞可通过MMP的显著变化、细胞质中组蛋白复合DNA片段的积累以及核浓缩诱导细胞凋亡。MB还增强了在临床相关浓度下测试的标准化疗药物的促凋亡作用。本研究中测试的MB结合了成像能力和对人黑色素瘤细胞中生存素mRNA的药理学沉默活性，可能代表了一种癌症诊断和治疗的创新方法。
