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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.

Kim SY, Lee DH, Song X, Bartlett DL, Kwon YT, Lee YJ - Apoptosis (2014)

Bottom Line: The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, School of Medicine, University of Pittsburgh, Hillman Cancer Center, 5117 Centre Ave. Room 1.46C, Pittsburgh, PA, 15213, USA.

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.

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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
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Fig4: 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

Mentions: We previously reported that the JNK-Bcl-xL pathway plays an important role in the synergistic effect on apoptosis of treatment with multiple cytotoxic agents [43, 57]. This possibility was examined during treatment with mitomycin C/Ad.TRAIL/hyperthermia. Phosphorylation of JNK and Bcl-xL was observed during treatment with mitomycin C/Ad.TRAIL/hyperthermia in LS174T cells (Fig. 4a). Even with treatment with only mitomycin C, phosphorylation of JNK and Bcl-xL was detected. Moreover, an increase in phosphorylation was observed during combinatorial therapy. Data from immunoprecipitation assay show that the combinatorial treatment induced the dissociation of Bak from Bcl-xL (Fig. 4b). We previously reported that phosphorylation of Bcl-xL alters the interactions between Bcl-xL and Bax and then leads to Bax oligomerization [43]. Since the presence of Bax was not detected in LS174T cells (data not shown), we examined Bak oligomerization. Bak oligomerization occurred during treatment with Ad.TRAIL in combination with mitomycin C with/without hyperthermia (Fig. 4c). Oligomerized Bak may bind to the mitochondria, altering mitochondrial membrane potential (Fig. 4d) and causing a cytochrome c release (Fig. 4e).Fig. 4


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.

Kim SY, Lee DH, Song X, Bartlett DL, Kwon YT, Lee YJ - Apoptosis (2014)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: 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
Mentions: We previously reported that the JNK-Bcl-xL pathway plays an important role in the synergistic effect on apoptosis of treatment with multiple cytotoxic agents [43, 57]. This possibility was examined during treatment with mitomycin C/Ad.TRAIL/hyperthermia. Phosphorylation of JNK and Bcl-xL was observed during treatment with mitomycin C/Ad.TRAIL/hyperthermia in LS174T cells (Fig. 4a). Even with treatment with only mitomycin C, phosphorylation of JNK and Bcl-xL was detected. Moreover, an increase in phosphorylation was observed during combinatorial therapy. Data from immunoprecipitation assay show that the combinatorial treatment induced the dissociation of Bak from Bcl-xL (Fig. 4b). We previously reported that phosphorylation of Bcl-xL alters the interactions between Bcl-xL and Bax and then leads to Bax oligomerization [43]. Since the presence of Bax was not detected in LS174T cells (data not shown), we examined Bak oligomerization. Bak oligomerization occurred during treatment with Ad.TRAIL in combination with mitomycin C with/without hyperthermia (Fig. 4c). Oligomerized Bak may bind to the mitochondria, altering mitochondrial membrane potential (Fig. 4d) and causing a cytochrome c release (Fig. 4e).Fig. 4

Bottom Line: The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, School of Medicine, University of Pittsburgh, Hillman Cancer Center, 5117 Centre Ave. Room 1.46C, Pittsburgh, PA, 15213, USA.

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.

Show MeSH
Related in: MedlinePlus