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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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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)
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Fig5: 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)

Mentions: To examine whether phosphorylation of the S62 residue on Bcl-xL is important for apoptosis, CX-1 cells were stably transfected with plasmid containing HA-Bcl-xL WT, phosphor-defective HA-Bcl-xL S62A or phosphor-mimic HA-Bcl-xL S62D. Figure 5a shows that HA-Bcl-xL S62A, but not HA-Bcl-xL WT and HA-Bcl-xL S62D, inhibited apoptosis. These results suggest that the phosphorylation of Bcl-xL plays an important role in the combinatorial treatment-induced apoptosis. We previously reported that phosphorylation of Bcl-xL affects interaction between Bcl-xL and Beclin-1, causing dissociation of Beclin-1 from Bcl-xL [57]. Data from immunoprecipitation assay show that overexpression of dominant-negative mutant type of Bcl-xL S62A, but not wild type Bcl-xL WT or dominant-positive mutant type of Bcl-xL S62D, suppressed the dissociation of Beclin-1 from Bcl-xL (Fig. 5b). Several researchers reported that Beclin-1 has two cleavage sites at D133 and D146 residues [58] and that Beclin-1 is cleaved by caspase 8, and C-terminal fragment of Beclin-1 localizes at the mitochondria and then induces cytochrome c release [58, 59]. Figure 6a shows that the combinatorial treatment enhanced Beclin-1 cleavage. On Fig. 6b, data from Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells show suppression of cleavage of PARP and caspase 8/9/3 (apoptosis). Beclin-1, a mammalian homolog of yeast autophagy-related protein 6 (Atg6), functions in autophagy by initiating autophagosome formation [60]. However, it has been suggested that crosstalk between apoptosis and autophagy is associated with caspase-mediated cleavage of Beclin-1 which destroys its pro-autophagic activity and can then amplify mitrochondrion-mediated apoptosis through the cleaved C-terminal fragment [58]. Our data suggest that the combinatorial treatment-induced synergistic apoptotic death is mediated through Beclin-1 cleavage.Fig. 5


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)

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)
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Related In: Results  -  Collection

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Fig5: 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)
Mentions: To examine whether phosphorylation of the S62 residue on Bcl-xL is important for apoptosis, CX-1 cells were stably transfected with plasmid containing HA-Bcl-xL WT, phosphor-defective HA-Bcl-xL S62A or phosphor-mimic HA-Bcl-xL S62D. Figure 5a shows that HA-Bcl-xL S62A, but not HA-Bcl-xL WT and HA-Bcl-xL S62D, inhibited apoptosis. These results suggest that the phosphorylation of Bcl-xL plays an important role in the combinatorial treatment-induced apoptosis. We previously reported that phosphorylation of Bcl-xL affects interaction between Bcl-xL and Beclin-1, causing dissociation of Beclin-1 from Bcl-xL [57]. Data from immunoprecipitation assay show that overexpression of dominant-negative mutant type of Bcl-xL S62A, but not wild type Bcl-xL WT or dominant-positive mutant type of Bcl-xL S62D, suppressed the dissociation of Beclin-1 from Bcl-xL (Fig. 5b). Several researchers reported that Beclin-1 has two cleavage sites at D133 and D146 residues [58] and that Beclin-1 is cleaved by caspase 8, and C-terminal fragment of Beclin-1 localizes at the mitochondria and then induces cytochrome c release [58, 59]. Figure 6a shows that the combinatorial treatment enhanced Beclin-1 cleavage. On Fig. 6b, data from Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells show suppression of cleavage of PARP and caspase 8/9/3 (apoptosis). Beclin-1, a mammalian homolog of yeast autophagy-related protein 6 (Atg6), functions in autophagy by initiating autophagosome formation [60]. However, it has been suggested that crosstalk between apoptosis and autophagy is associated with caspase-mediated cleavage of Beclin-1 which destroys its pro-autophagic activity and can then amplify mitrochondrion-mediated apoptosis through the cleaved C-terminal fragment [58]. Our data suggest that the combinatorial treatment-induced synergistic apoptotic death is mediated through Beclin-1 cleavage.Fig. 5

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