The Bcl-2 antagonist HA14-1 forms a fluorescent albumin complex that can be mistaken for several oxidized ROS probes.
The proapoptotic effects of the Bcl-2 antagonist HA14-1 are believed to derive from its affinity for the hydrophobic groove on Bcl-2 and Bcl-x(L), thereby displacing proapoptotic factors, e.g. Bax and Bak. We have reported that HA14-1 promotes the efficacy of low-dose photodynamic therapy (PDT). A recent report proposed that the proapoptotic activity of HA14-1 reflects its ability to generate reactive oxygen species (ROS) when incubated in an aqueous environment. This later study, like several other HA14-1 investigations, relied on the use of fluorescent probes for ROS detection. We found that HA14-1 reacts with the albumin in serum to yield a fluorescent product. After correcting for this effect, the putative formation of ROS by HA14-1 could not be demonstrated with the fluorescent probes H(2)DCFDA, dihydroethidium or dihydrorhodamine. Indeed, the fluorescence excitation/emission spectra of HA14-1 encompassed the excitation/emission wavelengths used to detect these ROS probes. Cells cultured in a medium supplemented with ovalbumin, instead of serum, underwent apoptosis following HA14-1 addition, but did not exhibit fluorescence. Hence, HA14-1 fluorescence was unrelated to its proapoptotic activity. We conclude that the enhancement of PDT by HA14-1 reflects a pharmacologic effect, rather than its direct contribution of ROS.
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA. dhkessel@med.wayne.edu
David
D
Kessel
Michael
M
Price
John J
JJ
Reiners
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001578:Benzopyrans / Q000737:chemistry* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D009570:Nitriles / Q000737:chemistry* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010778:Photochemotherapy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D001578:Benzopyrans; D005456:Fluorescent Dyes; D009570:Nitriles; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D012709:Serum Albumin; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate