TNFα Cooperates with IFN-γ to Repress Bcl-xL Expression to Sensitize Metastatic Colon Carcinoma Cells to TRAIL-mediated Apoptosis

Abstract

BackgroundTNF-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 FindingsThe 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/SignificanceTNFα 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.

NF-κB Directly Regulates Fas Transcription to Modulate Fas-mediated Apoptosis and Tumor Suppression

“…Western blotting analysis was performed as described previously (35). Anti-cleaved caspase 8 was obtained from R&D Systems.…”

“…Electrophoresis Mobility Shift Assay (EMSA) of NF-B Activation-NF-B activation was analyzed using an NF-B probe (AGT TGA GGG GAC TTT CCC AGG C; Santa Cruz Biotechnology, Inc.) and probes with NF-B consensus sequences of the Fas promoter region (supplemental Table 2) as described previously (35). Briefly, the end-labeled probes were incubated with nuclear extracts for 20 min at room temperature.…”

“…Because certain tumor cells are resistant to apoptosis and IFN-␥ sensitizes tumor cells to apoptosis induction (35), IFN-␥ treatment was included for each cell line. It is clear that FasL inhibited the growth of all nine cell lines in vitro (Fig.…”

Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells

“…Western blotting analysis was carried out as previously described (15). The blots were probed with the following antibodies: anti-IRF8 (C-19, Santa Cruz) at a 1:200 dilution; anti-Cytochrome C (BD Biosciences) at 1:500; anti-Bax (Santa Cruz) at 1:2000, anti-cleaved PARP (Cell Signal) at 1:500, and anti-β-actin (Sigma, At Louis, MO) at 1:8000.…”

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K

“…Immunological approaches to colon cancer therapy have evolved substantially over the past several years. [1][2][3] Numerous tumor-associated antigens have been described in colon cancer, which display spontaneous T-cell responses to tumor antigens in patients without prior immunotherapy, suggesting immunogenicity of colon cancer. [4][5][6] The 90K glycoprotein is composed of 90 kDa subunits, and it has been identified in culture fluid of human breast cancer cells.…”