Cis-bixin selectively kills freshly collected ex vivo–treated patient myeloma cells and also highly drug-resistant myeloma cell lines. (A) A 24-h cis-bixin treatment kills CD138⁺ cells while largely sparing paired normal CD138⁻ marrow cells (results from three representative patient samples shown). (B, C) Doxorubicin-resistant 8226R cells are killed as readily as doxorubicin-sensitive 8226S cells by cis-bixin, indicating non–cross resistance. (D, E) Dexamethasone-resistant MM1R_L cells are killed equally as well as parental dexamethasone-sensitive MM1S cells by cis-bixin, indicating non–cross resistance.

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).

Cis-bixin inhibits the reduction of thioredoxin by thioredoxin reductase. (A) A gel-based assay was devised by using AMS to trap reduced thioredoxin and induce a gel motility shift in this species relative to the migration of oxidized thioredoxin. Gel bands from enzyme reaction product stopped at time points from 0 to 20 min are shown for each of five concentrations of cis-bixin, with densitometry quantitation of gel bands used to construct (B) showing the fraction of reduced thioredoxin produced over time. (C) The fraction of reduced thioredoxin relative to control is shown as a function of cis-bixin concentration at three time points, to allow the determination of IC₅₀ values for the inhibition of reduction of Trx by TrxR1. Data shown are representative of three separate experiments.

Cis-bixin has antineoplastic effects in multiple cancer cell lines. (A) Photo of seed pods of the rain forest plant Bixa orellena, or annatto (Alan J. Wolf, Ph.D. ©2006, University of Wisconsin-Madison). (B) Chemical structures of cis-bixin and several related carotenoids. (C) Colony-forming assay for cytotoxicity using A549 cells shows that cis-bixin is more cytotoxic than are tested related carotenoids. (D) Colony-forming assays in a variety of cancer cell lines, including A549 (lung), U2OS (osteosarcoma), PC3 (prostate), HCT-118 (colon), MCF-7 (breast), DRO (anaplastic thyroid), and BHP5-16 (papillary thyroid) indicate broad anticancer effects of cis-bixin. All drug exposures were for 24 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Cis-bixin–induced cytotoxicity in myeloma cells is associated with apoptosis. (A) Electron microscopic images (×2,500) of OCI-MY-5 myeloma cells treated with DMSO diluent or 200 μM cis-bixin, demonstrating induction of morphologic apoptosis (arrows). (B) Concentration-dependence of apoptosis induction by cis-bixin in OCI-MY-5 myeloma cells. Apoptosis was assessed with Hoechst 33258 nuclear staining after 24-h exposures to indicated cis-bixin concentrations; error bars indicate ±1 standard deviation. (C) Cis-bixin induces PARP cleavage in OCI-MY-5 cells (24-h drug exposures).

Cis-bixin inhibits thioredoxin and thioredoxin reductase activities, but not glutathione reductase activity. (A, B) Effects of cis-bixin on thioredoxin reductase activity. DTNB was used as the substrate, and the rate of change in absorbance at 405 nm, as shown in the inset in (A), reflects activity of thioredoxin reductase. (B) Effects of cis-bixin compared with those of related carotenoids cis-norbixin, acitretin, and crocin on thioredoxin reductase activity. (C) Effects of cis-bixin on thioredoxin activity as assessed by the insulin precipitation assay, with rate of change in absorbance at 620 nm shown as a percentage of diluent control activity. (D) Effects of cis-bixin on glutathione reductase activity. DTNB was used as the substrate; absorbance was monitored at 405 nm and plotted as percentage of control of diluent activity. Error bars, +1 standard deviation; statistical significance, *p < 0.02 and **p < 0.001; each panel contains representative results of one of three distinct experiments conducted in triplicate, except for reference drug experiments in (B), performed in duplicate.