Chiorazzo Michael G, Bloch Noah B, Popov Anatoliy V, Delikatny Edward J
Department of Pharmacology, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
Department of Radiology, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
Bioconjug Chem. 2015 Dec 16;26(12):2360-70. doi: 10.1021/acs.bioconjchem.5b00417. Epub 2015 Oct 28.
Activatable fluorophores selective to cytosolic phospholipase A2 (cPLA2) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA2 was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA2 when compared to esterification with palmitic acid. In vitro analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA2 group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally in vitro and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA2. In contrast to the in vitro analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA2 enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA2 resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA2 selective activatable fluorophore capable of detecting cPLA2 in triple negative breast cancer cells.
合成了对胞质磷脂酶A2（cPLA2）具有选择性的可激活荧光团，并评估了它们对三阴性乳腺癌细胞成像的能力。通过将小分子荧光团与脂肪酸进行酯化反应来合成可激活构建体，从而消除荧光。通过选择荧光团和脂肪酸来实现对cPLA2的选择性。与用棕榈酸酯化相比，用花生四烯酸酯化足以赋予对cPLA2的特异性。对掺入磷脂酰胆碱脂质体中的探针进行的体外分析表明，一种非选择性磷脂酶（sPLA2组IB）能够水解花生四烯酸和棕榈酸偶联的荧光团，从而产生荧光。在所测试的四种荧光团中，观察到DDAO（7-羟基-9H-（1,3-二氯-9,9-二甲基吖啶-2-酮））在体外表现最佳，并在4175-Luc+细胞中进行了进一步分析，该细胞系是一种表达高水平cPLA2的转移性三阴性人乳腺癌细胞系。与体外分析相反，通过荧光显微镜测量并通过荧光光谱法定量，与对照DDAO棕榈酸酯相比，DDAO花生四烯酸酯在4175-Luc+细胞中显示出选择性激活。添加两种已知可激活cPLA2的试剂可增强DDAO花生四烯酸酯的荧光，而不会引起DDAO棕榈酸酯的任何变化。抑制cPLA2会导致DDAO花生四烯酸酯的荧光降低，但不会导致DDAO棕榈酸酯的荧光降低。我们共同报道了一种能够在三阴性乳腺癌细胞中检测cPLA2的cPLA2选择性可激活荧光团的合成。
