β-galactosyl-pyrrolidinyl diazeniumdiolate: an efficient tool to investigate nitric oxide functions on promoting cell death

Abstract

Nitric oxide (NO) is an active free radical gas that plays crucial roles in a broad range of biological processes. Extremely short half-life makes it difficult to use NO directly in research. It has been suggested that different concentrations of NO may lead to quite opposite results on cytotoxicity. However, the net effect of intracellular NO on tumor cell death has been controversial, partly because it is hard to precisely control the amount of NO generated exclusively within the target cells. Therefore, we have developed a cell-specific NO donor, β-galactosyl-pyrrolidinyl diazeniumdiolate (β-Gal-NONOate), in hopes of simulating the actual effects of intracellularly derived NO on the patterns of cell death as well as investigating its underlying mechanisms. In this study, by using three different tumor cell models, we showed that β-Gal-NONOate could steadily transport NO into the target cells with similar delivery efficiencies and exerted a determinative effect on cell death. In addition, β-Gal-NONOate-derived intracellular NO could provoke both apoptosis and necrosis in a concentration-dependent manner. While lower NO concentration primarily induced apoptosis, higher NO concentration mainly triggered necrosis. Moreover, the intrinsic apoptotic pathway, characterized by rapid Ca²⁺ overload and subsequent mitochondrial damage, was the collective mechanism responsible for the apoptotic death in all the three tumor cell lines. Taken together, since this cell-specifically derived NO is cheap to use and easy to quantify, β-Gal-NONOate might be used as a novel and ideal tool to standardize intracellular NO generation and evaluate its net effects in different cellular and experimental settings in the coming future.