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TNFα cooperates with IFN-γ to repress Bcl-xL expression to sensitize metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Liu F, Hu X, Zimmerman M, Waller JL, Wu P, Hayes-Jordan A, Lev D, Liu K - PLoS ONE (2011)

Bottom Line: TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent.Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis.TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang University, Hangzhou, People's Republic of China.

ABSTRACT

Background: TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Methodology/principal findings: The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo.

Conclusions/significance: TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer.

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TNFα-mediated NF-κB activation on TRAIL-induced apoptosis.A. Analysis of IKKβ-KA-mediated inhibition of NF-κB activation. Left panel: TNFα-induced NF-κB activation kinetics. SW480 cells were treated with TNFα for the indicated time. Nuclear extracts were prepared and used in the EMSA using a double-stranded oligo nucleotide probe containing NF-κB consensus sequence. Middle panel: specificity of NF-κB EMSA. SW480 cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and analyzed for NF-κB activation by EMSA. IgG (lane 4), anti-p50 subunit of NF-κB antibody (lane 5), and excess molar ratio of cold probe (lane 6) were used for the specificity assay. Right panel: inhibition of NF-κB activation by IKKβ-KA mutant. SW480.Vector and SW480.IKKβ-KA cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and used in the EMSA assay as shown above. B. Sensitivity of SW480.Vector and SW480.IKKβ-KA cells to TRAIL-induced apoptosis. Tumor cells were treated with IFN-γ, TNFα, or both IFN-γ and TNFα overnight, followed by incubation with recombinant TRAIL for approximately 24 h. Cells were then stained with PI and analyzed for cell death. C. Quantification of TRAIL-induced cell death. Cell death as shown in B was quantified.
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pone-0016241-g004: TNFα-mediated NF-κB activation on TRAIL-induced apoptosis.A. Analysis of IKKβ-KA-mediated inhibition of NF-κB activation. Left panel: TNFα-induced NF-κB activation kinetics. SW480 cells were treated with TNFα for the indicated time. Nuclear extracts were prepared and used in the EMSA using a double-stranded oligo nucleotide probe containing NF-κB consensus sequence. Middle panel: specificity of NF-κB EMSA. SW480 cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and analyzed for NF-κB activation by EMSA. IgG (lane 4), anti-p50 subunit of NF-κB antibody (lane 5), and excess molar ratio of cold probe (lane 6) were used for the specificity assay. Right panel: inhibition of NF-κB activation by IKKβ-KA mutant. SW480.Vector and SW480.IKKβ-KA cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and used in the EMSA assay as shown above. B. Sensitivity of SW480.Vector and SW480.IKKβ-KA cells to TRAIL-induced apoptosis. Tumor cells were treated with IFN-γ, TNFα, or both IFN-γ and TNFα overnight, followed by incubation with recombinant TRAIL for approximately 24 h. Cells were then stained with PI and analyzed for cell death. C. Quantification of TRAIL-induced cell death. Cell death as shown in B was quantified.

Mentions: Our above data demonstrated that TNFα, when used in combination with IFN-γ, can sensitize metastatic human colon carcinoma cells to TRAIL-induced apoptosis. However, TNFα is also a potent activator of NF-κB [54] and NF-κB has been shown to play a important role in TRAIL resistance [55], [56]. Thus, TNFα may simultaneously activate apoptosis and cell survival pathways, two conflicting biological processes, in human colon carcinoma cells. To determine whether these two conflicting pathways co-exist and interferes with each other, we examined TNFα-induced NF-κB activation and the effects of blocking NF-κB activation on TRAIL-induced apoptosis in human colon carcinoma cell line SW480. SW480 cell line was chosen since we have a well-established NF-κB activation model in this cell line. SW480 cells exhibited spontaneously activated NF-κB activity, albeit at low level. Treatment of the tumor cells with recombinant TNFα rapidly and transiently activated NF-κB (Fig. 4A). Although IFN-γ cooperates with TNFα to enhance TRAIL-induced apoptosis, IFN-γ did not alter TNFα-mediated NF-κB activation (Fig. 4A). It has been shown that it is IKKβ that activate the canonical NF-κB to promote tumor [57]. Next, we stably transfected SW480 cells with empty vector (SW480.Vector) and a vector expressing IKKβ mutant IKKβ-K44A (SW480.IKKβ-KA) [58], and examined the effects of inhibition of NF-κB activation on colon carcinoma cell sensitivity to TRAIL. EMSA analysis indicated that ectopic expression of the IKKβ mutant blocked both constitutively and TNFα-induced NF-κB activation (Fig. 4A).


TNFα cooperates with IFN-γ to repress Bcl-xL expression to sensitize metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Liu F, Hu X, Zimmerman M, Waller JL, Wu P, Hayes-Jordan A, Lev D, Liu K - PLoS ONE (2011)

TNFα-mediated NF-κB activation on TRAIL-induced apoptosis.A. Analysis of IKKβ-KA-mediated inhibition of NF-κB activation. Left panel: TNFα-induced NF-κB activation kinetics. SW480 cells were treated with TNFα for the indicated time. Nuclear extracts were prepared and used in the EMSA using a double-stranded oligo nucleotide probe containing NF-κB consensus sequence. Middle panel: specificity of NF-κB EMSA. SW480 cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and analyzed for NF-κB activation by EMSA. IgG (lane 4), anti-p50 subunit of NF-κB antibody (lane 5), and excess molar ratio of cold probe (lane 6) were used for the specificity assay. Right panel: inhibition of NF-κB activation by IKKβ-KA mutant. SW480.Vector and SW480.IKKβ-KA cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and used in the EMSA assay as shown above. B. Sensitivity of SW480.Vector and SW480.IKKβ-KA cells to TRAIL-induced apoptosis. Tumor cells were treated with IFN-γ, TNFα, or both IFN-γ and TNFα overnight, followed by incubation with recombinant TRAIL for approximately 24 h. Cells were then stained with PI and analyzed for cell death. C. Quantification of TRAIL-induced cell death. Cell death as shown in B was quantified.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3022032&req=5

pone-0016241-g004: TNFα-mediated NF-κB activation on TRAIL-induced apoptosis.A. Analysis of IKKβ-KA-mediated inhibition of NF-κB activation. Left panel: TNFα-induced NF-κB activation kinetics. SW480 cells were treated with TNFα for the indicated time. Nuclear extracts were prepared and used in the EMSA using a double-stranded oligo nucleotide probe containing NF-κB consensus sequence. Middle panel: specificity of NF-κB EMSA. SW480 cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and analyzed for NF-κB activation by EMSA. IgG (lane 4), anti-p50 subunit of NF-κB antibody (lane 5), and excess molar ratio of cold probe (lane 6) were used for the specificity assay. Right panel: inhibition of NF-κB activation by IKKβ-KA mutant. SW480.Vector and SW480.IKKβ-KA cells were treated with IFN-γ, TNFα or both IFN-γ and TNFα for 60 min and used in the EMSA assay as shown above. B. Sensitivity of SW480.Vector and SW480.IKKβ-KA cells to TRAIL-induced apoptosis. Tumor cells were treated with IFN-γ, TNFα, or both IFN-γ and TNFα overnight, followed by incubation with recombinant TRAIL for approximately 24 h. Cells were then stained with PI and analyzed for cell death. C. Quantification of TRAIL-induced cell death. Cell death as shown in B was quantified.
Mentions: Our above data demonstrated that TNFα, when used in combination with IFN-γ, can sensitize metastatic human colon carcinoma cells to TRAIL-induced apoptosis. However, TNFα is also a potent activator of NF-κB [54] and NF-κB has been shown to play a important role in TRAIL resistance [55], [56]. Thus, TNFα may simultaneously activate apoptosis and cell survival pathways, two conflicting biological processes, in human colon carcinoma cells. To determine whether these two conflicting pathways co-exist and interferes with each other, we examined TNFα-induced NF-κB activation and the effects of blocking NF-κB activation on TRAIL-induced apoptosis in human colon carcinoma cell line SW480. SW480 cell line was chosen since we have a well-established NF-κB activation model in this cell line. SW480 cells exhibited spontaneously activated NF-κB activity, albeit at low level. Treatment of the tumor cells with recombinant TNFα rapidly and transiently activated NF-κB (Fig. 4A). Although IFN-γ cooperates with TNFα to enhance TRAIL-induced apoptosis, IFN-γ did not alter TNFα-mediated NF-κB activation (Fig. 4A). It has been shown that it is IKKβ that activate the canonical NF-κB to promote tumor [57]. Next, we stably transfected SW480 cells with empty vector (SW480.Vector) and a vector expressing IKKβ mutant IKKβ-K44A (SW480.IKKβ-KA) [58], and examined the effects of inhibition of NF-κB activation on colon carcinoma cell sensitivity to TRAIL. EMSA analysis indicated that ectopic expression of the IKKβ mutant blocked both constitutively and TNFα-induced NF-κB activation (Fig. 4A).

Bottom Line: TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent.Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis.TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Zhejiang University, Hangzhou, People's Republic of China.

ABSTRACT

Background: TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis.

Methodology/principal findings: The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo.

Conclusions/significance: TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer.

Show MeSH
Related in: MedlinePlus