Inhibition of SOS Response by Nitric Oxide Donors in Escherichia coli Blocks Toxin Production and Hypermutation

BackgroundPrevious reports have differed as to whether nitric oxide inhibits or stimulates the SOS response, a bacterial stress response that is often triggered by DNA damage. The SOS response is an important regulator of production of Shiga toxins (Stx) in Shiga-toxigenic E. coli (STEC). In addition, the SOS response is accompanied by hypermutation, which can lead to de novo emergence of antibiotic resistance. We studied these effects in vitro as well as in vivo.ResultsNitric oxide donors inhibited induction of the SOS response by classical inducers such as mitomycin C, ciprofloxacin, and zidovudine, as measured by assays for E. coli RecA. Nitric oxide donors also inhibited Stx toxin protein production as well as stx2 RNA in vitro and in vivo. In vivo experiments were performed with ligated ileal segments in the rabbit using a 20 h infection. The NO donor S-nitroso-acetylpenicillamine (SNAP) reduced hypermutation in vitro and in vivo, as measured by emergence of rifampin resistance. SNAP blocked the ability of the RecA protein to bind to single-stranded DNA in an electrophoretic mobility shift assay (EMSA) in vitro, an early event in the SOS response. The inhibitory effects of SNAP were additive with those of zinc acetate.ConclusionsNitric oxide donors blocked the initiation step of the SOS response. Downstream effects of this blockade included inhibition of Stx production and of hypermutation. Infection of rabbit loops with STEC resulted in a downregulation, rather than stimulation, of nitric oxide host defenses at 20 h of infection.

The Application of Nitric Oxide for Ocular Hypertension Treatment

Despite of various therapeutic methods for treating ocular hypertension and glaucoma, it still remains the leading cause of irreversible blindness. Intraocular pressure (IOP) lowering is the most effective way to slow disease progression and prevent blindness. Among the ocular hypotensive drugs currently in use, only a couple act on the conventional outflow system, which is the main pathway for aqueous humor outflow and the major lesion site resulting in ocular hypertension. Nitric oxide (NO) is a commendable new class of glaucoma drugs that acts on the conventional outflow pathway. An increasing number of nitric oxide donors have been developed for glaucoma and ocular hypertension treatment. Here, we will review how NO lowers IOP and the types of nitric oxide donors that have been developed. And a brief analysis of the advantages and challenges associated with the application will be made. The literature used in this review is based on Pubmed database search using ‘nitric oxide’ and ‘glaucoma’ as key words.

NCX-4040, a Unique Nitric Oxide Donor, Induces Reversal of Drug-Resistance in Both ABCB1- and ABCG2-Expressing Multidrug Human Cancer Cells

The emergence of multidrug resistance (MDR) in the clinic is a significant problem for a successful treatment of human cancers. Overexpression of various ABC transporters (P-gp, BCRP and MRP’s), which remove anticancer drugs in an ATP-dependent manner, is linked to the emergence of MDR. Attempts to modulate MDR have not been very successful in the clinic. Furthermore, no single agent has been found to significantly inhibit their functions to overcome clinical drug resistance. We have previously shown that nitric oxide (●NO) inhibits ATPase functions of ABC transporters, causing reversal of resistance to clinically active anticancer drugs. In this study, we have used cytotoxicity and molecular docking studies to show that NCX4040, a nitric oxide donor related to aspirin, inhibited the functions of ATPase which resulted in significant reversal of resistance to both adriamycin and topotecan in P-gp- and BCRP-expressing human cancer cell lines, respectively. We also used several other cytotoxic nitric oxide donors, e.g., molsidomine and S-nitroso glutathione; however, both P-gp- and BCRP-expressing cells were found to be highly resistant to these NO-donors. Molecular docking studies showed that NCX4040 binds to the nucleotide binding domains of the ATPase and interferes with further binding of ATP, resulting in decreased activities of these transporters. Our results are extremely promising and suggest that nitric oxide and other reactive species delivered to drug resistant tumor cells by well-designed nitric oxide donors could be useful in sensitizing anticancer drugs in multidrug resistant tumors expressing various ABC transporters.

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.