Bortezomib sensitizes human esophageal squamous cell carcinoma cells to TRAIL-mediated apoptosis via activation of both extrinsic and intrinsic apoptosis pathways

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive human cancers, and novel treatment modalities are required. We investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) in combination with the proteasome inhibitor bortezomib (Velcade) on human ESCC cell lines. Bortezomib enhanced the susceptibility to TRAIL in 12 of the 15 ESCC cell lines tested, although most showed low sensitivity to TRAIL as a single agent. The enhancement of TRAIL-induced apoptosis by bortezomib was caspase dependent. Increased processing of caspase-8 often accompanied enhancement of TRAIL-induced apoptosis by bortezomib. However, the increased cell surface expression of death receptors observed on bortezomib treatment did not seem to be crucial for this effect. For some ESCC, bortezomib treatment resulted in a more efficient recruitment of caspase-8 and the Fas-associated death domain to the death-inducing signaling complex. Additional downregulation of the cellular FLICE-inhibitory protein long isoform [c-FLIP(L)] could cooperate in the activation of the extrinsic pathway in some cases. For other ESCC, the crucial effect of bortezomib treatment seemed to be increased signaling via the intrinsic apoptotic pathway on subsequent exposure to TRAIL. Thus, bortezomib could sensitize ESCC to TRAIL apoptosis by multiple molecular mechanisms of action. Therefore, the combination of bortezomib and TRAIL might be a novel therapeutic strategy for ESCC patients who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway.

FIGURE 1

Enhancement of sensitivity to TRAIL in ESCC cell lines. (A) 15 ESCC cell lines were seeded on 96-well plates, and treated with TRAIL at 20, 100, and 500 ng/mL for 18 h. (B) The cells were treated with bortezomib (100 nM) or medium alone for 2 h, and subjected to a further 16–18 h of incubation with or without the addition of TRAIL (100 ng/mL). (C) KE4, TE8, and TE9 cells were incubated in the various concentrations of bortezomib (◆ 0, □ 5, ▲ 20, and ○ 100 nM) and TRAIL as described in (B) in the presence or absence of zVAD-fmk (100 μM). Cell viability was assessed by an MTS assay, and the percentage decrease in cell number (±SD) compared to the medium control is shown in (A–C). (D) TE9 cells were also stained by annexin-V-FITC (green), PI (red), and hoechst33342 (blue) at 6 h and 18 h after the addition of TRAIL. Representative images with bortezomib (100 nM) and TRAIL (500 ng/mL) are shown with the values (±SD) of % apoptotic cells as described in the “Material and methods”. B, botrezomib; T, TRAIL.

FIGURE 2

Caspase activities with bortezomib/TRAIL treatment in KE4, TE8, and TE9 cells. Cells were incubated in the presence or absence of bortezomib (100 nM for KE4 and TE9 cells, and 20 nM for TE8 cells) for 2 h prior to the addition of vehicle or TRAIL (100 ng/mL). (A) After a further 6 h, cell extracts were prepared, and western blotting was performed with anti-caspase-8, anti-caspase-9, anti-cleaved caspase-3, anti-PARP, and anti-β-actin Abs. (B) Cells were incubated with JC-1 dye during the last 15 min of culture. Mitochondrial depolarization was evaluated as indicated by a decrease in the red-to-green fluorescence intensity ratio. Representative results for TE8 cells are shown. (C) Cells were incubated in the presence or absence of bortezomib for 6–8 h. TRAIL was added for a further 2 h (500 ng/mL), and the enzyme activities of caspase-8, caspase-9, and caspase-3 in the cells were measured as described in the “Materials and methods”. B, bortezomib; T, TRAIL; CL, cleaved form.

FIGURE 3

(A) Contribution of bortezomib-induced upregulation of death receptors to TRAIL-induced apoptosis. KE4 and TE9 cells were incubated with TRAIL at 500 ng/mL for 1 h and then washed five times with medium to remove unbound TRAIL. Cells were left untreated or sensitized with bortezomib (100 nM) for 12 h, either with or without the further addition of TRAIL (500 ng/mL). Apoptotic cells were evaluated by an active caspase-3 assay as described in the “Materials and methods”. (B) Expression of c-FLIP and FADD proteins following bortezomib treatment. Cells were incubated in the presence or absence of various concentrations of bortezomib for 18 h. Cell extracts were then prepared, and western blotting was performed with anti-c-FLIP, anti-FADD, and anti-β-actin Abs. B, bortezomib (Bzb); T, TRAIL.

FIGURE 4

Enhanced susceptibility to TRAIL-induced apoptosis in bortezomib-treated cells following specific downregulation of c-FLIP with siRNAs. KE4 and TE9 cells were transfected with siRNAs targeting c-FLIP or control (Ctrl) siRNAs, and cultured for 48 h. (A) Cells were lysed and analyzed for c-FLIP and β-actin expression by western blotting. (B) Transfected cells were incubated in the presence or absence of bortezomib (100 nM) for 2 h, and for a further 10–12 h with the addition of TRAIL at various concentrations. % apoptotic cells (±SD) were assessed after hoechst33342 nuclear staining as described in the “Materials and methods” (Ctrl siRNA: ◇ vehicle, ◆ bortezomib, c-FLIP siRNA: □ vehicle, ■ bortezomib). B, bortezomib.

FIGURE 5

Effects of bortezomib on DISC formation upon TRAIL stimulation. KE4 and TE9 cells were incubated with or without bortezomib (Bzb; 100 nM) for 8 h. (A) Surface expression levels of DR4 and DR5 were analyzed by flow cytometry. Representative histograms from three independent experiments with similar results are shown (solid line, isotype control; shaded area, untreated; dashed line, bortezomib-treated). The percentage increase (average ± SD) of expression with bortezomib is presented as described in “Materials and methods”. (B, C) Cells were further incubated with His-tagged rhTRAIL cross-linked by anti-6 × histidine mAb (1 μg/ml) for 30 min. The cells were lysed and the precipitated DISC was analyzed by western blotting. Representative images from three independent experiments are shown in (B).

FIGURE 6

Effects of bortezomib on the expression of various proteins involved in apoptosis or cell cycle arrest. KE4, TE8, and TE9 cells were treated with various concentrations of bortezomib (Bzb) for 18 h, and the cell extracts were then prepared. The given proteins were detected using western blot analysis.