Potent activity of indolequinones against human pancreatic cancer: identification of thioredoxin reductase as a potential target

Abstract

The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cell-free systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.

Fig. 1.

Effect of indolequinone treatment on colony formation in human pancreatic cancer cells. Colony-forming ability was measured in MIA PaCa-2 and BxPC-3 cells after 4- or 72-h indolequinone treatment. Data represent mean ± S.D. of three independent determinations.

Fig. 2.

Induction of caspase-dependent apoptosis by indolequinone 3 in pancreatic cancer cells. A, effect of indolequinone 3 treatment on apoptotic cell death. MIA PaCa-2 and BxPC-3 cells were treated with indolequinone 3 at various concentrations. Apoptosis was measured 24 h after 4-h drug treatment using annexin-PI staining in combination with flow cytometry analysis. B, effect of pretreatment with the pan-caspase inhibitor z-VAD-fmk on indolequinone 3-induced apoptosis. BxPC-3 cells were pretreated with 100 μM z-VAD-fmk for 30 min before indolequinone 3 treatment. Data represent mean ± S.D. of three independent determinations. C, induction of caspase activation by indolequinone 3 in MIA PaCa-2 and BxPC-3 pancreatic cancer cells. The cleaved forms of caspase 3, 7, 8, and 9 were detected in both cell lines using immunoblot analysis 24 h after 4-h drug treatment. Staurosporine (STAU) treatment (500 nM) was included as positive control for caspase activation. Immunoblot shown was representative of at least three independent experiments.

Fig. 3.

Effect of indolequinone 3 treatment on DNA single-strand breaks and DNA cross-links in MIA PaCa-2 and BxPC-3 cells. DNA damage was determined using the comet assay after treating cells with varying concentrations of indolequinone 3 (IQ3) for 1 h. A, DNA single-strand breaks were expressed as percentage of DNA in the comet tail. H₂O₂ treatment (200 μM for 20 min) was included as a positive control. B, comparison of drug-induced DNA single strand breaks in MIA PaCa-2 cells between indolequinone 3 and known redox cycling quinones β-lapachone (β-lap) and streptonigrin (SN). Cells were treated for 1 h. The dose range used for β-lapachone was 1 to 10 μM. C, no DNA cross-links were observed after 1-h indolequinone 3 treatment because there was no decrease in percentage tail DNA in drug-treated cells compared with untreated H₂O₂ control. Results are expressed as the mean ± S.D. of three separate determinations. ^**, significantly different from nontreatment control cells, p < 0.01.

Fig. 4.

Antitumor activity of indolequinones in the NCI-60 cell line panel. Growth inhibition screening in the NCI-60 cell line panel was performed by the NCI/National Institutes of Health developmental therapeutics program. The LC₅₀ mean graph was compared side-by-side for compound 3 (left), 6 (middle), and AW464 (right), a recently established thioredoxin reductase inhibitor. The original graph for compound 3 and 6 were obtained from the National Cancer Institute. [The AW464 part of this figure was adapted from Bradshaw TD, Matthews CS, Cookson J, Chew EH, Shah M, Bailey K, Monks A, Harris E, Westwell AD, Wells G, Laughton CA, and Stevens MF (2005) Elucidation of thioredoxin as a molecular target for antitumor quinols. Cancer Res 65:3911-3919. Copyright © 2005 American Association for Cancer Research. Used with permission.]

Fig. 5.

Inhibition of thioredoxin reductase activity by indolequinone 3. A, inhibition of thioredoxin reductase activity in MIA PaCa-2 cells by indolequinone 3 (▴), indolequinone 9 (▿), and a nontoxic indolequinone analog, ACH983 (▾). Thioredoxin reductase activity in MIA PaCa-2 cells was measured using the endpoint insulin reduction assay after 4 h of drug treatment. Data were expressed as percent of DMSO-treated control. Data represent mean ± S.D. of three independent determinations. B, inhibition of thioredoxin reductase activity in cell-free system by indolequinone 3. Recombinant rat TrxR (0.5 μM) was preincubated for 5 min with 250 μM NADPH in the presence of NQO2/NRH, then indolequinone 3 was added and incubated for 5 min (maximum inhibition was achieved at 5 min). A 20-μl aliquot was taken out for measurement of TrxR activity using DTNB as substrate. □, reaction system contained every component; ▵, nonreduced TrxR (-NADPH) was not inhibited; ▾, nonreduced indolequinone 3 (-NQO2/NRH) resulted in no inhibition. C, alkylation of the C-terminal selenocysteine of recombinant rat TrxR by indolquinone 3 after reduction by NQO2/NRH inhibited subsequent biotinylation of the C-terminal selenocysteine by biotinylated iodoacetamide. Top, biotinylation of TrxR was detected using streptavidin-conjugated horseradish peroxidase in combination with ECL; bottom, the membrane was stripped and reprobed for total TrxR.

Fig. 6.

Inhibition of pancreatic xenograft tumor growth in nude mice after treatment with indolequinones. MIA PaCa-2 xenograft tumors were grown on the flanks of nude mice and then treated with selected indolequinones (1.0 and 2.5 mg/kg i.p.) every other day for 20 days. Mice were weighed twice weekly, and neither the control mice nor the treatment groups suffered significant weight loss or any apparent toxicity. Tumor volume was calculated by the formula (L × W²)/2, where L is the longer measurement of the tumor and W is the smaller tumor measurement. Data represents the mean ± S.E.M. of six mice. ^*, tumor volume in the treatment group was statistically different from controls as determined by the Dunnett's test.

Fig. 7.

A proposed mechanism for the inhibition of TrxR by indolequinones. A, indolequinone activation by two-electron reduction to generate a reactive iminium electrophile. B, reduction of oxidized TrxR by NADPH to generate the reduced C-terminal selenocysteine. C, inhibition of TrxR via alkylation of the reduced C-terminal selenocysteine by the reactive indolequinone iminium electrophile.