TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response

Abstract

A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.

Fig 1. Combined effect of TRAIL and doxorubicin in prostate carcinoma PPC-1 cells.

PPC-1 cells grown to subconfluency in 384 well plate were treated with TRAIL (0.1 ng/ml) and increasing concentrations of doxorubicin. The level of cytotoxicity was determined by an ATPLite reagent. T, TRAIL; Dox. doxorubicin. P < 0.01.

Fig 2

(A) upper panel, the structure of ML100 and its structurally related analogs. Lower panel, effect of ML100 and its structurally related analogs on TRAIL-mediated apoptosis in cancer cells and human hepatocytes. Subconfluent prostate carcinoma PPC-1 and PC3, glioma U251 cells, and human primary hepatocytes in a 96-well plate were pre-incubated for 4 h with the indicated concentrations of compounds followed by TRAIL treatment at the constant concentration of 0.1 ng/ml (PPC-1), 1 ng/ml (PC-3 and U-251), and 1000 ng/ml (hepatocytes) for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by an ATPLite reagent. Open and closed circles refer to compound sole and compound/TRAIL combined treatment, respectively. (B) isobologram analysis of the combined treatment of TRAIL and ML100 in cancer cells. Subconfluent melanoma MDA-MB-435, prostate carcinoma DU145 and PC-3, and leukemia THP1 cells in a 96-well plate were treated with TRAIL and ML100 at a ratio of 1 ng/ml of TRAIL: 0.1 μM of ML100 (1: 0.1). Cells were pre-incubated with chemicals for 4 h followed by addition of TRAIL and incubation for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by an ATPLite reagent. T, TRAIL; C, compound.

Fig 3

(A) a pharmacophore analog of ML100, NSC130362, exhibited potent anti-cancer activity and was non-toxic to human hepatocytes. The effect of NSC130362 (inset) on the viability of MDA-MB-435, DU145 cells and hepatocytes was determined in a TRAIL-based combined treatment as described in the legend for Fig 2. *, P < 0.05. (B) both ML100 and NSC130362 synergistically induced caspase 3/7 activity in MDA-MB-435 cells. MDA-MB-435 cells were pre-incubated with ML100 and NSC130362 for 2 h followed by TRAIL treatment for 2 and 6 h, respectively. Caspase 3/7 activity was measured by Caspase 3/7 Lux assay (Promega). *, P < 0.05.

Fig 4. Cell surface expression of DR5 and DcR1/DcR2 in MDA-MB-435 carcinoma cells.

MDA-MB-435 cell were pretreated with either ML100 NSC130362 for 4 h and 24 h. Cells were then labeled with biotin and lysed. Labeled cell surface-associated DR5, DcR1, and DcR2 were captured by streptavidin-agarose beads. The precipitated samples were analyzed by Western blotting with the specific respective antibodies and horseradish peroxidase-conjugated secondary antibodies. The level of the cell surface DR4 was below the detection limits. Molecular weight markers are on the left.

Fig 5. NSC130362 binds GSR in a concentration-dependent manner.

(A) GSR and NSC130362 absorbance profile. (B) Absorbance of GSR + NSC130362 in the flow-through after desalting column. (C) inset. The structure of the GSH-NSC10362 adduct. Absorbance of GSR + NSC130362 +GSH in the flow-through after desalting column. All absorbance values in (B and C) were corrected for absorbance of individual NSC130362, GSH, and the GSH-NSC130362 adduct in the flow through. (D) GSR activity in the absence or presence of either NSC130362 or the GSH-NSC130362 adduct. *, P < 0.05.

Fig 6

(A) NSC130362 inhibited GSR activity (upper panel) and caused depletion of intracellular GSH (lower panel). MDA-MB-435 cells were treated with 30 μM of NSC130362 for 6 h and the levels of GSH and GSR activity were measured using GSH detection and GSR activity kits (Cayman), respectively. (B) upper panel, GSR siRNA potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. MDA-MB-435 cells and human hepatocytes grown to subconfluency were transfected with GSR siRNA (10 nM/well) using SilentFect transfection reagent (BioRad). After 2 days, cells were treated with 10 ng/ml of TRAIL for an additional 24 h. Lower panel, Western blotting of GSR. Subconfluent cells were transfected with GSRsiRNA №1 and №3 as well as with scrambled siRNA (10 nM each) in a 6-well plate. Two days after transfection cells were lysed and the level of GSR was analyzed by Western blotting with the GSR antibodies and horseradish peroxidase-conjugated secondary antibodies. (C) GSH but not general caspase inhibitor Q-VD-OPh completely blocked NSC130362 activity. Subconfluent MDA-MB-435 cells in a 96-well plate were pre-incubated for 4 h with either GSH (10 mM) or general caspase inhibitor Q-VD-OPh (100 μM) (CI) followed by treatment with NSC130362 (10 μM) for 4 h and subsequent incubation with TRAIL (10 ng/ml) for an additional 24 h. (D) Hydrogen peroxide potentiated TRAIL activity in MDA-MB-435 cells. Subconfluent MDA-MB-435 cells in a 96-well plate were pre-incubated for 4 h with hydrogen peroxide (10 μM) followed by treatment with TRAIL (10 ng/ml) for an additional 24 h. At the end of all treatments, the ratio of dead cells was determined by an ATPLite reagent. *, P < 0.05.

Fig 7

(A) MDA-MB-435 cells that survived after NSC130362 treatment had elevated levels of GSH. Subconfluent MDA-MB-435 cells in a 6-well plate were treated for 6 h with NSC130362 (10 and 30 μM), or DMSO followed by staining with mBCl (40 μM) for 10 min and subjected to subsequent flow cytometry analysis. Mean fluorescence intensity was: 2.55 (unstained cells), 6.47 (DMSO-treated cells), 8.60 (10 μM NSC130362-treated cells), 11.60 (30 μM NSC130362-treated cells), 38.30 (10 μM ML100-treated cells), 37.30 (30 μM ML100-treated cells). (B) NSC130362 induced ROS generation and peroxidation of mitochondrial membrane lipid. Subconfluent MDA-MB-435 cells in a 6-well plate were treated for 6 h with either NSC130362 (10 and 30 μM) or DMSO followed by staining with DHE (10 μM) and NAO (5 nM) for 20 min and subjected to subsequent flow cytometry analysis.

Fig 8. Combined treatment of NSC130362 and oxidative stress inducers ATO, Myr, and BSO efficiently induced apoptosis in a variety of cancer cells but not in primary human hepatocytes.

The effect of NSC130362/ATO (A), NSC130362/Myr (B), and NSC130362/BSO (C) combined treatment in breast carcinoma cells and MDA-MB-435 melanoma cells. The effect of NSC130362/ATO and NSC130362/Myr (D) combined treatment in pancreatic, prostate, and lung carcinoma cells as well as in AML cells from cancer patients. Subconfluent cells in a 96-well plate were pre-incubated for 4 h with ATO (3 μM), Myr (100 μM), or BSO (10 μM) followed by treatment with NSC130362 (10 μM) for an additional 24 h. At the end of the treatment, the ratio of dead cells was determined by an ATPLite reagent. *, P < 0.05.

Fig 9

(A) PK studies of NSC130362 in the mouse bloodstream. (B) the synergistic effect of ATO and NSC130362 in MIA PaCa-2 cells. The effect of ATO and NSC130362 on the viability of MIA PaCa-2 cells was determined as described in the legend for Fig 8. (C) the ATO/NSC130362 combined treatment retards growth of MIA PaCa-2 xenografts in immunodeficient mice. (D) H&E-stained sections of mouse liver and heart. Magnification, 40x. *, P = 0.03.