diamide and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Anion Transport Proteins; Apoptosis; Calixarenes; Caspases; Cell Line; Chlorides; Cytochromes c; Diamide; HCT116 Cells; HeLa Cells; Humans; Ion Transport; Liposomes; Mitochondria; Porphyrins; Pyridines; Rats; Reactive Oxygen Species; Small Molecule Libraries

The dithiol-reducing thioredoxin/thioredoxin reductase system normally maintains the reduced state of key enzymes responsible for the cell's anti-oxidant defences. We therefore addressed the question of whether AW 464--a novel thioredoxin inhibitor--as well as broad spectrum dithiol ligands diamide and phenylarsine oxide are able to induce and execute a regular apoptotic sequence of events without overwhelming the cell's ability to detoxify reactive oxygen species. All three agents were found to target the thioredoxin system in a cell-free assay. In HL-60 leukaemia cells, they were also found to induce Bak activation, cytochrome c release from mitochondria, decreasing Delta Psi m, chromatin condensation, phosphatidyl serine exposure and Tdt-sensitive DNA nicks. At the onset of apoptosis there was no evidence of increases in oxygen free radicals or peroxide in cells treated with AW 464 or diamide. Phenylarsine oxide induced both free radicals and hydrogen peroxide, but this did not appear to interfere with apoptosis. We conclude that pharmacological targeting of thioredoxin can induce a well-orchestrated apoptotic programme.

Topics: Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Arsenicals; bcl-2 Homologous Antagonist-Killer Protein; Caspases; Diamide; Enzyme Inhibitors; HL-60 Cells; Humans; Membrane Proteins; Mitochondria; Oxidation-Reduction; Sulfhydryl Reagents; Thioredoxins

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antigens, Surface; Apoptosis; Autoantigens; Autoimmune Diseases; Benzimidazoles; Blotting, Western; Caspase Inhibitors; Cells, Cultured; Diamide; Enzyme Inhibitors; Fluorescent Dyes; Free Radical Scavengers; Humans; In Situ Nick-End Labeling; Infant, Newborn; Keratinocytes; Oxidative Stress; Ribonucleoproteins; RNA, Small Cytoplasmic; Ultraviolet Rays