FIG. 4.

Cis-bixin–induced cytotoxicity can be overcome by the antioxidants NAC and GSH, but not by inhibitors of DNA, RNA, or protein synthesis, and is associated with the potent induction of oxidative stress in cells. (A) Inhibitors of DNA (aphidicolin), RNA, (5,6-dichloro-β-d-ribofuranosyl-benzimidazole; DRB), and protein (cycloheximide) synthesis do not alter cis-bixin–associated cytotoxicity, as assessed with colony-forming assays (24-h cis-bixin exposures in A549 cells, indicated inhibitors added 30 min before cis-bixin addition). (B) N-acetyl cysteine (NAC) and reduced glutathione (GSH) rescue cis-bixin–treated cells (colony-forming assays, 24-h cis-bixin exposures in A549 cells; indicated inhibitors added 30 min before cis-bixin addition). (C) Representative FACS data showing that cis-bixin shifts the fluorescence intensity of an ROS-indicator (CMH₂DCFDA) in A549 cells, inducing high levels of ROS, starting at very short exposure times (2 min, cis-bixin concentration 200 μM). (D) Quantitated oxidative stress levels induced in A549 cells by cis-bixin, cis-norbixin, crocin, acitretin, hydrogen peroxide, or tert-butyl hydroperoxide (24-h treatments, unless otherwise noted; all drug concentrations were 200 μM). All values were statistically significant from DMSO (p < 0.001) except for t-BHP, which was not statistically different. Levels of oxidative stress induced by other agents were also significantly different from cis-bixin, as follows: *p < 0.01 and **p < 0.001. Error bars indicate mean ± 1 sample standard deviation of triplicate samples. (E) Effects of cis-bixin on intracellular reduced glutathione levels in A549 cells (24-h treatments; indicated statistical significances are in comparison to DMSO control). (F) Levels of oxidative stress in CD138^+/- patient myeloma cells treated with cis-bixin for 24 h as assessed by dihydroethidium staining for superoxide by using FACS (results expressed as percentage of total cells; results from one myeloma patient of three examined are shown).