TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells

Abstract

Background Lysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem. Methods We investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT. Results TMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death. Conclusions TMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical internalization. General Significance Understanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies.

The Late Endosome and Its Lipid BMP Act as Gateways for Efficient Cytosolic Access of the Delivery Agent dfTAT and Its Macromolecular Cargos

“…This presumably includes pro-apoptotic ions such as calcium and toxic proteins such as proteases. The leakage of such molecules into the cytosol of cells is known to trigger cell death, when endosomal membrane damage is mediated either photochemically or with lysosomotropic agents (Guicciardi et al, 2004; Muthukrishnan et al, 2012). For these reasons, lysosomes are often referred as the ‘suicide bags’ of the cells and similar potential toxic effects are also often attributed to late endosomes.…”

Breast Cancer Targeting through Inhibition of the Endoplasmic Reticulum-Based Apoptosis Regulator Nrh/BCL2L10

“…5B). To prevent the trapping of the internalized TAT-NDP peptide in the endosomes, we used a light source at 488 nm to excite the FITC fluorophore and induce endosomal leakage (32), thus increasing cytosolic availability of the peptide ( Supplementary Fig. S7B).…”

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research

“…Lysosomal rupture by PDT has been shown to induce accumulation of Ca 2+ in the cytosol, most likely released from the lysosomes, with the involvement, however, of extracellular Ca 2+ not being excluded [87]. The increased accumulation of Ca 2+ in the cytosol usually results in rapid sequestration of excess Ca 2+ by mitochondria and post-PDT inhibition of this sequestration has been shown to reduce cell death, likely due to the inhibition of Ca 2+ -induced opening of the pro-apoptotic mitochondrial permeability transition pore [103].…”