The Study Of Mechanisms Of Cellular Uptake Of L-K6, An Anticancer Peptide With α- Helical Structure

Anticancer peptide L-K6 is an analogue of temporin-1CEb which is isolated and purified from the skin secretions of Rana chensinensis. In our previous study, L-K6 exhibited significant anticancer activities against various breast cancer cells and could inhibit the proliferation of cancer cells. But not like other anticancer peptides with α- helical structure, L-K6 didn’t exhibit the depolarization of cell membrane and affect the permeability of membrane, suggesting that the target of L-K6 was not cell membrane. In this study, the pathway of L-K6 entering into MCF-7 cells, and intracellular mechanisms of action of anticancer peptides L-K6 were investigated.We first evaluated the impacts of L-K6 on morphologies of cell membrane, cytoskeleton, nucleus of MCF-7 human breast cancer cells using confocal microscopy and ultra-high resolution microscopy. The morphological observation showed that one-hour treatment of MCF-7 cell with L-K6 has no impact on cell membrane and cytoskeleton morphology, whereas the nucleus fluorescence was diminished in a dose-dependent manner, which indicating a nucleus damage. Secondly, by using flow cytometry, fluorescence microplate reader and ultra-high resolution microscopy, the internalizing manner of L-K6 was further determined. Results showed that endocytosis inhibitors methyl-β-cyclodextrin(M-β-CD), amiloride(EIPA), chlorpromazine(CPZ) and cytochalasin D(CyD) inhibited FITC-labeled L-K6 to internalize into cells, with CyD the most potent, suggesting that L-K6 enter into MCF-7 cells through a endocytosis-related mechanism. In order to explore energy-dependent property of L-K6 internalization process, we co-incubated cells with L-K6 under a low temperature at 4oC. The results showed a significant reduction of L-K6 internalization compared to 37 o C, indicating that the internalization of L-K6 was energy-dependent.As a cationic antimicrobial peptide, L-K6 may first interact through electrostatic attraction with negatively charged phosphatidylserine(PS) on cell surface membrane, then aggregate on cell surface to trigger endocytosis. Isothermal Titration Calorimetry(ITC) experiments supported our hypothesis that L-K6 bind with PS with the ratio of 1: 1; CD chromatography experiments further proved PS can promote L-K6 to form α- helix structure in a dose-dependent manner. In contrst, other negatively charged components of MCF-7 cell surface, HS, CS, and gangliosides, does not participate in the internalization of L-K6.