Abstract
RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.
Highlights
RL2 is a recombinant analogue of a natural peptide, lactaptin, with a molecular weight of 8.6 kDa, that has been isolated from human milk
We found that all the experimental spectra of the A549 cells incubated with spectra of A549 cells incubated with RL2 (sRL2) can be well reproduced by a superposition of easier two or three electron paramagnetic resonance (EPR) spectral components (Figure 4b) with different contributions
It is shown that stable spin probe 2 cannot penetrate into the cell, whereas the attachment of this spin label to a human kappa casein fragment, RL2, capable of penetrating the cell allows us to study its stability in human lung adenocarcinoma A549 cells and to follow its transformation inside the cells by EPR spectroscopy
Summary
RL2 is a recombinant analogue of a natural peptide, lactaptin, with a molecular weight of 8.6 kDa, that has been isolated from human milk. It has been shown that this peptide is a proteolytic fragment of human k-casein that induces apoptotic death of human breast adenocarcinoma MCF-7 cells in culture [1]. RL2 (14 kDa) induces apoptosis of MCF-7 cells as well as other cancer cell types, including A549 cells and is not toxic to nonmalignant human adipose-tissue mesenchymal stem cells (MSCs). It has been reported that RL2 can penetrate the cytoplasm of both MCF-7 cancer cells and nonmalignant adipose-tissue MSCs [2,3,4].
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