Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate

Abstract

Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G1, S, and G2/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G1/S transition than in the G1 and S/G2/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates

“…This conjugate can mediate the photoinduction of apoptosis. Recently, this construct has been successfully used for the analysis of cell-cycle-dependent protein/peptide function via PCI-mediated peptide transduction [33].…”

“…A number of studies have shown that the use of CPP-photosensitizer (CPP-PS) conjugates offers a promising way to deliver macromolecules into cells [30][31][32]. PCI mediated by CPP-PS conjugates is an efficient tool for transducing transient high-cytosolic concentrations of nucleic acids and proteins/peptides in a short duration [31,33]. Optimal use of CPP-PS conjugates for therapeutic delivery depends on efficient membrane destabilization induced by photogenerated ROS and minimal photoinduced damage to cells, except for target endosomes.…”

Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer

“…We developed a PCI-based apoptosis-inducing molecule, TatBim-Alexa. This molecule, which is a conjugate of the Tat cell-penetrating peptide, the BH3 domain from the apoptosis-inducing protein Bim, and the photosensitizer Alexa, can spatially control apoptosis, depending on photo-activation 9 , 10 .…”