Marking patterns and potential targets of action of a benzo[a]phenoxazinium chloride with antiproliferative activity

Abstract

Fluorescent markers are widely used in numerous applications. Despite their great variety, in the last years, research has been conducted focusing on the design and synthesis of new fluorophores based on small organic molecules.1 It was recently demonstrated antimicrobial activity of N-(5-((4-ethoxy-4-oxobutyl)amino)-10-methyl-9H-benzo[a]phenoxazin-9-ylidene)ethanaminium chloride, an heterocyclic compound with an extended planar aromatic system and different substituent groups.2 However, from our knowledge, no studies to elucidate the intracellular distribution and the actual mechanism of action of this compound have been performed. In the present work, fluorescent probes or fusion proteins linked to green fluorescent protein (GFP), specific for different intracellular compartments were used to evaluate by fluorescence microscopy the cellular distribution of the above mentioned benzo[a]phenoxazinium chloride. In order to study the efficiency of the staining as a function of concentration over time, cells incubated with this compound were also analyzed by flow cytometry. The results showed that the benzo[a]phenoxazinium chloride in levels close to the minimum inhibitory concentration (MIC), accumulates at the membranes of the endoplasmic reticulum and the vacuole. It was also found that the fluorescence intensity decreases over time and also when cells are incubated with 10 mM HEPES buffer pH 7.4, 5% glucose, pointing to the possible involvement of efflux pumps of the PDR (pleiotropic drug resistance) family. For concentrations above the MIC, there was a marked disruption of the sub-cellular structures and it was possible to observe some apoptotic nuclei by staining with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI). The results suggest that vacuole and endoplasmic reticulum stress/damage may be triggering of the cell death process. Keywords: Benzo[a]phenoxazinium chlorides, Nile Blue derivatives, yeast; cellular distribution.

1. Gonçalves, M.S.T. Chem. Rev. 2009, 109, 190 – 212. 2. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Biorg. Med. Chem. 2008, 16, 3274 – 3282.