Antitumor indolequinones induced apoptosis in human pancreatic cancer cells via inhibition of thioredoxin reductase and activation of redox signaling

Abstract

Indolequinones (IQs) were developed as potential antitumor agents against human pancreatic cancer. IQs exhibited potent antitumor activity against the human pancreatic cancer cell line MIA PaCa-2 with growth inhibitory IC(50) values in the low nanomolar range. IQs were found to induce time- and concentration-dependent apoptosis and to be potent inhibitors of thioredoxin reductase 1 (TR1) in MIA PaCa-2 cells at concentrations equivalent to those inducing growth-inhibitory effects. The mechanism of inhibition of TR1 by the IQs was studied in detail in cell-free systems using purified enzyme. The C-terminal selenocysteine of TR1 was characterized as the primary adduction site of the IQ-derived reactive iminium using liquid chromatography-tandem mass spectrometry analysis. Inhibition of TR1 by IQs in MIA PaCa-2 cells resulted in a shift of thioredoxin-1 redox state to the oxidized form and activation of the p38/c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathway. Oxidized thioredoxin is known to activate apoptosis signal-regulating kinase 1, an upstream activator of p38/JNK in the MAPK signaling cascade and this was confirmed in our study providing a potential mechanism for IQ-induced apoptosis. These data describe the redox and signaling events involved in the mechanism of growth inhibition induced by novel inhibitors of TR1 in human pancreatic cancer cells.

Fig. 1.

Structure of IQs and proposed mechanism of action. A, chemical structure of IQs 1 and 2. B, IQs via reduction and rearrangement can generate a reactive iminium electrophile, which has the potential to alkylate cellular nucleophiles.

Fig. 2.

Induction of apoptosis by IQs in MIA PaCa-2 human pancreatic cancer cells. A and B, IQ treatment induced dose-dependent apoptosis in MIA PaCa-2 cells. Cells were treated with IQ 1 (A) or 2 (B) at various concentrations. Apoptosis was measured 18 h after drug treatment using Annexin-PI staining in combination with flow cytometry. C and D, time course of mitochondrial cytochrome c release induced by IQ treatment in MIA PaCa-2 cells. Cells were treated with 500 nM IQ 1 (C) or 150 nM IQ 2 (D) and collected at indicated time points, and cytochrome c (Cyt. c) levels in the cytosolic fraction were analyzed using immunoblotting analysis. Cytochrome c band intensity was also quantified relative to β-actin and indicated as fold of control underneath the immunoblot (C and D). Immunoblot shown was representative of three independent experiments. E and F, time course of caspase-3 activation and apoptosis induction by IQ treatment in MIA PaCa-2 cells. Cells were treated with 500 nM IQ 1 (E) or 150 nM IQ 2 (F), and at indicated time points, cells were collected; caspase-3 activity (▴) was determined using fluorescent substrates, and apoptosis (□) was measured using annexin-PI staining and flow cytometry. Data represent mean ± S.D. of three independent determinations.

Fig. 3.

Characterization of TR1 inhibition by IQs in cell-free systems. A, dose-dependent inhibition of TR1 activity in cell-free system by IQs. Recombinant rat TR1 (0.5 μM) was preincubated for 5 min with 250 μM NADPH in the presence of NQO2/NRH, then IQ 1 or 2 was added and incubated for 5 min (maximum inhibition was achieved at 5 min). An aliquot of 20 μl sample (from 150 μl total reaction) was taken out for measurement of TR1 activity using DTNB as substrate. B and D, mass spectrometric analysis of TR1-IQ 1 adducts. Theoretical (B) and experimentally determined (C) spectra from LC/MS analysis of TR1 C-terminal tryptic peptide containing one IQ 1-derived iminium and one carbamidomethyl adduct. TR1 was first reduced with NADPH and then treated with IQ 1 in the presence of NQO2/NRH. The proteins were denatured, and unreacted thiols and selenols were derivatized with iodoacetamide. Then, they were digested with trypsin and subjected to LC-MS/MS analysis. Note the distinctive isotope pattern characteristic of selenium-containing ions. D, MS/MS analysis of the C-terminal tryptic fragment of IQ 1-modified TR1. The sequence of the C-terminal peptide is shown (inset) along with b- and y-series ions (arrows). The IQ adduction was determined to be on the Sec residue (IQ), whereas the Cys residue was found to bear a carbamidomethyl modification.

Fig. 4.

Inhibition of TR1 activity by IQs in MIA PaCa-2 cells. A and B, effect of IQ treatment on the activity of TR1, GR, and Gpx in MIA PaCa-2 cells. Cells were treated with IQ 1 (A) or 2 (B) for 1 h. TR1 activity in cells was then measured using the endpoint insulin reduction assay and expressed as a percentage of DMSO-treated control. GR and Gpx activity in cells were also measured and expressed as percentage of DMSO-treated control. C and D, time-dependent inhibition of TR1 by IQs in MIA PaCa-2 cells. Cells were treated with IQ 1 (1000 nM) or IQ 2 (D; 300 nM) for indicated times. TR1 activity in cells was then measured using the endpoint insulin reduction assay. E, a comparison of TR1 inhibitory ability in MIA PaCa-2 cells between IQs and auranofin. Data represent mean ± S.D. of three independent determinations.

Fig. 5.

Effect of IQ treatment on thioredoxin redox state in pancreatic cancer cells. Thioredoxin redox state was determined in MIA PaCa-2 cells 1 h after IQ 1 (A) or 2 (B) treatment as described under Materials and Methods. H₂O₂ treatment (1 mM for 10 min) was included as a positive control. Immunoblot shown represents three independent experiments.

Fig. 6.

Effects of IQ treatment on the activation of the MAPK apoptotic signaling pathway. A and B, IQ treatment induced dose-dependent activation of p38 and JNK MAPKs in MIA PaCa-2 cells. Cells were treated with various concentrations of IQ 1 (A) or 2 (B) for 1 h; p38 and JNK phosphorylation were then detected using immunoblot analysis. C and D, IQ-induced apoptosis was inhibited by pretreatment with p38 and JNK inhibitors. MIA PaCa-2 cells were pretreated with the p38 inhibitor SB203580 (5 μM), the JNK inhibitory peptide l-JNKi (5 μM), or both for 1 h before treatment with IQ 1 (C) or 2 (D) for 18 h. Apoptosis was measured using Annexin-PI staining and flow cytometry. E and F, IQ treatment induced ASK1 activation in MIA PaCa-2 cells. Cells were transiently transfected with the plasmid pCMV-SPORT6-ASK1 or the vector control by electroporation and incubated in complete medium for 16 h. Cells were then treated with IQ 1 (E) or 2 (F) for 30 min. Levels of total ASK1 (top) and phosphorylated ASK1 (bottom) were analyzed using immunoblotting. H₂O₂ treatment (1 mM for 10 min) was included as a positive control. Immunoblots shown represent three independent experiments.

Fig. 7.

A proposed apoptotic signaling cascade induced by IQ treatment in human pancreatic cancer cells. TR1 inhibition by the IQs results in a shift of thioredoxin redox state from the reduced (Trx1_red) to the oxidized (Trx1_ox) form. Oxidized thioredoxin dissociates with ASK1 and results in its activation via phosphorylation. Activated ASK1 as an mitogen-activated protein kinase kinase kinase (MAP3K) would activate mitogen-activated protein kinase kinases (MAP2Ks), including MKK3/6 and MKK4/7, which will in turn activate p38 and JNK MAPKs and induce downstream mitochondrial apoptosis.