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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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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
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Fig2: 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

Mentions: To investigate the effect on cell viability of the application of Ad.TRAIL in combination with mitomycin C and hyperthermia, LS174T cells were infected with Ad.TRAIL (MOI 25) and treated with 5 µg/mL mitomycin C for 24 h. After treatment, the cells were heated at 42 °C for 1 h and incubated at 37 °C for 3 h and then cell viability was determined by trypan blue dye exclusion assay. As shown in Fig. 2a, synergistic cytotoxic effect was observed in Ad.TRAIL + mitomycin C or Ad.TRAIL + mitomycin C + hyperthermia compared with any single treatment (p < 0.05). We further investigated whether the combinatorial treatment-induced cytotoxicity was associated with apoptosis. Data from the flow cytometry assay demonstrate that the combinatorial treatment-induced cytotoxicity was associated with apoptosis (Figs. 2b, c); apoptotic death cells were observed in the lower right quadrant (early apoptotic death) and the upper right quadrant (late apoptotic death) of the plots. The results from Figs. 2b, c clearly demonstrate that apoptosis was time-dependent during combined treatment. Figures 2b, c clearly show that treatment with Ad.TRAIL in combination with mitomycin C and hyperthermia enhanced synergistic induction of apoptotic death. These synergistic effects were due to an increased activation (cleavage) of caspase 8/9/3 and thus, the hallmark feature of apoptosis, PARP cleavage (Fig. 3a). Similar results were observed in LS180, CX-1 and HCT116 cell lines (Figs. 3b, c, d). These results indicate that synergistic induction of apoptosis by combinatorial treatment with mitomycin C/Ad.TRAIL/hyperthermia is mediated through an increase in caspase activation.Fig. 2


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)

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
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196052&req=5

Fig2: 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
Mentions: To investigate the effect on cell viability of the application of Ad.TRAIL in combination with mitomycin C and hyperthermia, LS174T cells were infected with Ad.TRAIL (MOI 25) and treated with 5 µg/mL mitomycin C for 24 h. After treatment, the cells were heated at 42 °C for 1 h and incubated at 37 °C for 3 h and then cell viability was determined by trypan blue dye exclusion assay. As shown in Fig. 2a, synergistic cytotoxic effect was observed in Ad.TRAIL + mitomycin C or Ad.TRAIL + mitomycin C + hyperthermia compared with any single treatment (p < 0.05). We further investigated whether the combinatorial treatment-induced cytotoxicity was associated with apoptosis. Data from the flow cytometry assay demonstrate that the combinatorial treatment-induced cytotoxicity was associated with apoptosis (Figs. 2b, c); apoptotic death cells were observed in the lower right quadrant (early apoptotic death) and the upper right quadrant (late apoptotic death) of the plots. The results from Figs. 2b, c clearly demonstrate that apoptosis was time-dependent during combined treatment. Figures 2b, c clearly show that treatment with Ad.TRAIL in combination with mitomycin C and hyperthermia enhanced synergistic induction of apoptotic death. These synergistic effects were due to an increased activation (cleavage) of caspase 8/9/3 and thus, the hallmark feature of apoptosis, PARP cleavage (Fig. 3a). Similar results were observed in LS180, CX-1 and HCT116 cell lines (Figs. 3b, c, d). These results indicate that synergistic induction of apoptosis by combinatorial treatment with mitomycin C/Ad.TRAIL/hyperthermia is mediated through an increase in caspase activation.Fig. 2

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