Abstract
Phenoxazine derivatives such as Nile Blue analogues are assumed to be increasingly relevant in cell biology due to their fluorescence staining capabilities and antifungal and anticancer activities. However, the mechanisms underlying their effects remain poorly elucidated. Using S. cerevisiae as a eukaryotic model, we found that BaP1, a novel 5- and 9-N-substituted benzo[a]phenoxazine synthesized in our laboratory, when used in low concentrations, accumulates and stains the vacuolar membrane and the endoplasmic reticulum. In contrast, at higher concentrations, BaP1 stains lipid droplets and induces a regulated cell death process mediated by vacuolar membrane permeabilization. BaP1 also induced mitochondrial fragmentation and depolarization but did not lead to ROS accumulation, changes in intracellular Ca2+, or loss of plasma membrane integrity. Additionally, our results show that the cell death process is dependent on the vacuolar protease Pep4p and that the vacuole permeabilization results in its translocation from the vacuole to the cytosol. In addition, although nucleic acids are commonly described as targets of benzo[a]phenoxazines, we did not find any alterations at the DNA level. Our observations highlight BaP1 as a promising molecule for pharmacological application, using vacuole membrane permeabilization as a targeted approach.
Highlights
To assess whether BaP1 could induce cell death, S. cerevisiae cells were incubated with this compound and its effects on cell viability were determined by colony forming units (CFUs) counting over time
To understand whether the toxic effect of BaP1 could be reduced by its efflux out of the cell, we evaluated the effect of the compound on cell viability of a PDRefflux deleted mutant, AD1-7, in contrast to US50-18C, a strain with the activating mutation pdr1–3 in the gene encoding the transcription factor
In previous work in our laboratory, we found that several newly synthetized fluorescent compounds derived from benzo[a]phenoxazine displayed antiproliferative activity against S. cerevisiae, used as a model eukaryotic organism, and that minor changes in substituents of the benzo[a]phenoxazine nucleus drastically influenced their activity, with
Summary
Phenoxazine derivatives are long wavelength fluorescent dyes of great importance as probes since they absorb and emit fluorescence in the 600–900 nm region of the spectrum where there is minimum interference with the biological molecules’ auto-fluorescence [1,2,3,4,5,6].They have been used in the covalent labeling of amino acids and proteins and in the non-covalent labeling of nucleic acids in various contexts such as blotting experiments, gel electrophoresis, and living cell assays [7,8,9,10,11].In addition to their use as fluorophores, oxazine heterocycles such as phenoxazine and benzo[a]phenoxazine derivatives have assumed significance in life sciences due to their antiproliferative proprieties, which has prompted their study in pharmacological applications such as antimicrobial [12,13,14] and antitumor agents [15,16]. Phenoxazine derivatives are long wavelength fluorescent dyes of great importance as probes since they absorb and emit fluorescence in the 600–900 nm region of the spectrum where there is minimum interference with the biological molecules’ auto-fluorescence [1,2,3,4,5,6] They have been used in the covalent labeling of amino acids and proteins and in the non-covalent labeling of nucleic acids in various contexts such as blotting experiments, gel electrophoresis, and living cell assays [7,8,9,10,11].
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