Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells

Abstract

In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.

Fig. 1

Production of secretory TRAIL and its cytotoxicity. a 293 cells were non-transfected (lane 1), or transfected with 4 µg pAdlox empty vector (lane 2) or pAdlox-FETZ vector (lane 3, 4) and then incubated for 48 h. After incubation, 30 µg of cell lysate protein (lane 1, 2, 3, and 4) or 30 µL of cell culture medium (lane 5, 6) were immunoblotted with anti-TRAIL antibody. b 293 cells were infected with Ad.TRAIL (MOI 10) in the presence/absence of caspase inhibitor (20 µM). Cell lysates were prepared in lysis buffer and immunoblotted with anti-TRAIL. c LS174T cells were infected by various MOI of Ad.TRAIL or Ad.GFP for 24 h and harvested. Cell lysates were immunoblotted with anti-PARP antibody. d HCT116 and LS174T cells were infected with Ad.TRAIL (MOI 50) and harvested at various time points. Cell lysates were immunoblotted with anti-PARP antibody. Actin was used as an internal standard

Fig. 2

Synergistic induction of cytotoxicity by treatment with Ad.TRAIL in combination with mitomycin C and hyperthermia in LS174T cells. LS174T cells were treated with Ad.TRAIL (MOI 25) or/and mitomycin C (5 µg/mL) for 24 h and exposed to normothermia (37 °C) or hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. a Cell survival was analyzed by the trypan blue dye exclusion assay. b Cells were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI). c Cells were treated with Ad.TRAIL (MOI 25) and mitomycin C (5 µg/mL) for 4, 8, and 16 h and exposed to hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. Apoptosis was detected by the flow-cytometric assay

Fig. 3

Synergistic induction of apoptosis by treatment with Ad.TRAIL in combination with mitomycin C and hyperthermia in colon cancer cells. LS174T (a), LS180 (b), CX-1 (c) and HCT116 (d) cells were treated with Ad.TRAIL (MOI 25 or 50) or/and mitomycin C (3.5 or 5 µg/mL) for 24 h and exposed to normothermia (37 °C) or hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. After treatment, the cleavage of caspase 8/9/3 or PARP was detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane

Fig. 4

Ad.TRAIL in combination with mitomycin C and hyperthermia-induced activation of the JNK-Bcl-xL pathway, Bak oligomerization, mitochondrial membrane potential change and cytochrome c release. LS174T cells were treated with Ad.TRAIL (MOI 25) or/and mitomycin C (5 µg/mL) for 24 h and exposed to normothermia (37 °C) or hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. a After treatment, cell lysates containing equal amounts of protein were separated by SDS-PAGE and immunoblotted with anti-phospho-JNK, anti-JNK, anti-phospho Bcl-xL or anti-Bcl-xL antibody. b Cell lysates were immunoprecipitated with anti-Bcl-xL antibody or IgG and immunoblotted with anti-Bak antibody. The presence of Bcl-xL and Bak in the lysates was examined. Asterisk (*) is IgG light chain (LC). c Mitochondrial and cytosolic fractions were isolated and were cross-linked with 1 M DSP (dithiobis, succinimidyl propionate) for 30 min and then subjected to immunoblotting with anti-Bak antibody. Bak monomer (×1) and multimer (×2, ×3) are indicated. COX IV and actin were shown as an internal standard of mitochondrial fraction and cytosolic fraction, respectively. d Cells were stained with JC-1 and then analyzed by flow cytometry. e Cytochrome c release into cytosol was determined by immunoblotting for cytochrome c in the cytosolic fraction. Actin was used to confirm the equal amount of proteins loaded

Fig. 5

Role of Bcl-xL in apoptosis. CX-1 cells were stably transfected with HA-Bcl-xL WT, HA-Bcl-xL S62A or HA-Bcl-xL S62D plasmid and then treated with Ad.TRAIL (MOI 25) and/or mitomycin C (5 µg/mL) for 24 h and exposed to normothermia (37 °C) or hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. a After treatment, lysates containing equal amounts of protein were separated by SDS-PAGE and PARP cleavage was detected by immunoblotting. Actin was used as an internal standard. b Cell lysates were immunoprecipitated with anti-HA antibody or mock antibody (IgG) and immunoblotted with anti-Beclin-1 or anti-HA antibody (upper panels). The presence of Beclin-1 and actin in the lysates was examined (lower panels)

Fig. 6

Role of Beclin-1 in Ad.TRAIL in combination with mitomycin C and hyperthermia-induced cell death. LS174T, HCT116 and HCT116 Beclin-1 knock-in (D133A/D146A) cells were treated with Ad.TRAIL (MOI 25) or/and mitomycin C (3.5 or 5 µg/mL) for 24 h and exposed to normothermia (37 °C) or hyperthermia (42 °C) for 1 h, and then incubated for 3 h at 37 °C. a LS174T cell lysates were immunoblotted by Beclin-1 antibody. Arrows indicate cleaved Beclin-1. b Parental HCT116 (HCT116 WT) and HCT116 D133A/D146A cell lysates were immunoblotted with anti-PARP, anti-caspase 8/9/3, or anti-Beclin-1 antibody. Actin was shown as an internal standard