Novel Nile Blue Analogue Stains Yeast Vacuolar Membrane, Endoplasmic Reticulum, and Lipid Droplets, Inducing Cell Death through Vacuole Membrane Permeabilization

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.

Novel Benzo[A]Phenoxazinium Chlorides Functionalized with Sulfonamide Groups as Nir Fluorescent Probes for Vacuole, Endoplasmic Reticulum and Plasma Membrane Staining

“…This incubation resulted in the accumulation of the compounds in the cells, as NIR fluorescence emission was observed for all compounds upon excitation (Figure 1). Looking closely at Figure 1, it can be observed that these compounds appear to accumulate particularly at the vacuolar membrane and/or at the perinuclear membrane of the endoplasmic reticulum, which is consistent with our previous observations for this class of compounds [23,35,36]. In fact, Compounds 3a and 3b have a high specific labeling for the vacuole membrane and perinuclear membrane of the endoplasmic reticulum.…”

“…To confirm our previous observations and to evaluate the fluorescence pattern of each compound individually, we analyzed the specific intracellular distribution of the compounds using S. cerevisiae cells expressing GFP-tagged proteins located at the vacuole (W303-1A Vba1-GFP-expressing a GFP-tagged form of the vacuole membrane transporter Vba1 [56]), at the endoplasmic reticulum (BY4741 Sec66-GFP-expressing a GFP-tagged non-essential subunit of Sec63 ER complex [57]) and at the plasma membrane (23344c jen1∆ Jen1-GFP-expressing a GFP-tagged functional lactate transporter Jen1 [58]). We also reduced the concentration of the compounds and stained the cells with 2.5 µM of 3a-f, as we had previously observed that this concentration increased the specificity and labeling ability of the compounds of this family [35]. As previously reported, reducing the concentration of the compounds proved to be beneficial for live-cell imaging experiments.…”

“…In addition to their outstanding optical properties, benzo[a]phenoxazine compounds have also been reported for their pharmaceutical properties. These compounds were reported for their validity as antitumor agents [33,34], antifungals [23,[35][36][37], antibacterials [38,39], antimalarials [40,41], antivirals [42], and as photo-sensitizers in photodynamic therapy [43,44]. Their compact structure, and the presence of amines in 5-and 9-positions allows benzo[a]phenoxazinium chlorides to possess different functional groups, resulting in compounds with variable behaviors.…”

Targeting Lysosomes in Colorectal Cancer: Exploring the Anticancer Activity of a New Benzo[a]phenoxazine Derivative

“…In our previous work, we used the yeast Saccharomyces cerevisiae as a eukaryotic cell model and performed a detailed characterization of the effect of one of our most active compounds, BaP1 (Figure 1). We found that BaP1 accumulates in the vacuole, the yeast lysosome equivalent organelle, and induced a regulated cell death process associated with vacuolar membrane permeabilization [23].…”

“…However, this class of compounds has shown to possess antiproliferative activity, which has increased the interest in evaluating them as pharmaceutical drugs. In fact, there are several reports of different activities for these compounds, such as antifungal [22][23][24][25], antimalarial [26,27], antibacterial [28,29], antiviral [30], and antitumor [31,32]. Still, despite the interest, the information available regarding their mechanisms of action is still limited.…”