Department of Surgery, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.
Cancer Chemother Pharmacol. 2010 Jul;66(2):325-31. doi: 10.1007/s00280-009-1166-7. Epub 2010 Feb 25.
PNC-27, a peptide that contains an HDM-2-binding domain from p53 attached to a membrane-penetrating peptide on its carboxyl terminal end, is cytotoxic to cancer, but not normal, cells. It forms transmembrane pores in the cancer cell membrane. Our purpose is to determine if the whole peptide or critical fragments induce pore formation in cancer cells.
We have prepared PNC-27 with a green fluorescent label on its amino terminus and a red fluorescent label on its carboxyl terminus and treated MCF-7 breast cancer cells and untransformed MCF-10-2A breast epithelial cells with this double-labeled peptide to determine if combined yellow fluorescence occurs in the membrane of the cancer cells during cancer cell killing.
At 30 min, there is significant combined punctate yellow fluorescence, indicative of intact peptide, in the cell membrane of cancer cells that increases during cancer cell lysis. MCF-10-2A cells show initial (30 min) uniform combined yellow membrane fluorescence that subsequently disappears. Unlike the cancer cells, these untransformed cells remain viable.
PNC-27 induces cancer cell membrane lysis by acting as the whole peptide, not fragments. The punctate yellow fluorescence is due to interaction of PNC-27 with intramembrane targets of MCF-7 cells that do not exist in the membrane of the untransformed cell line. This interaction increases the lifetime of PNC-27. Absence of these targets in the membranes of the untransformed MCF-10-2A cells results in initial uniform fluorescence of the double-labeled peptide in their membranes after which the peptide is degraded.
PNC-27 是一种肽，其羧基末端连接有来自 p53 的 HDM-2 结合域和一个穿膜肽，对癌细胞具有细胞毒性，但对正常细胞没有毒性。它在癌细胞膜上形成跨膜孔。我们的目的是确定整个肽或关键片段是否会在癌细胞中诱导孔形成。
我们用氨基末端的绿色荧光标记和羧基末端的红色荧光标记制备了 PNC-27，并将这种双标记肽处理 MCF-7 乳腺癌细胞和未转化的 MCF-10-2A 乳腺上皮细胞，以确定在癌细胞杀伤过程中，癌细胞膜中是否会出现合并的黄色荧光。
在 30 分钟时，在癌细胞的细胞膜中存在明显的点状黄色荧光，表明完整的肽，随着癌细胞的裂解而增加。MCF-10-2A 细胞显示初始（30 分钟）均匀的合并黄色膜荧光，随后消失。与癌细胞不同的是，这些未转化的细胞仍然存活。
PNC-27 通过作为整个肽而不是片段来诱导癌细胞膜裂解。点状黄色荧光是由于 PNC-27 与 MCF-7 细胞的跨膜靶标相互作用引起的，这些靶标不存在于未转化的细胞系的膜中。这种相互作用延长了 PNC-27 的寿命。未转化的 MCF-10-2A 细胞的膜中不存在这些靶标，导致双标记肽在其膜中初始均匀荧光，随后肽被降解。
