PMC

Model of deoxycholyltaurine-induced post-EGFR signaling in human colon cancer cells. Signaling via the MEK/ERK pathway stimulates cell proliferation. Signaling via the PI3K/Akt pathway stimulates nuclear translocation and transcriptional activity of NF-κB, thereby protecting cells from stress-induced apoptosis. Dashed arrow between deoxycholyltaurine and EGFR indicates a mechanism involving release of the EGFR ligand HB-EGF [,]. Circled P indicates key phosphorylations.

Effects of deoxycholyltaurine (DCT) on tumor necrosis factor-α (TNF-α)-induced NF-κB nuclear translocation in HT-29 and H508 human colon cancer cells. Representative immunoblots (A) and line graphs of immunoblot densitometry (B) show time-course for effects of TNF-α (100 ng/ml) and DCT (100 μM), alone or in combination, on increases in nuclear p65 NF-κB. Cells were incubated with test agents for the indicated times and nuclear extracts were immunoblotted for p65 NF-κB and a loading control, histone H2A. Results are representative of three separate experiments.

Effects of inhibiting post epidermal growth factor receptor (EGFR) signaling on EGF- and deoxycholyltaurine (DCT)-induced NF-κB nuclear translocation in H508 human colon cancer cells. Representative immunoblots show the effects on NF-κB nuclear translocation of incubating H508 cells with 100 μM DCT and 10 ng/ml EGF, alone and in the presence of the following agents: A. Inhibitors of EGFR activation (PD168393, 2 μM and AG1478, 0.1 μM); B. PI3K inhibitors (LY294002, 50 μM and wortmannin,100 nM); C. Src (pp2, 10 μM) and MEK inhibitors (PD98059, 25 μM); and D. and E. NF-κB inhibitors (SN50, 20 μg/ml; Bay11-7082, 10 μM; PDTC, 10 μM; and MG-132, 50 μM). Results shown are representative of three separate experiments.

Effects of bile acid on tumor necrosis factor-α (TNF-α; 100 ng/ml)-induced apoptosis in HT-29 and H508 human colon cancer cells. A. Images of TNF-α-induced apoptosis in HT-29 cells. a, Control cells (left) and cells treated for 24 h with TNF-α alone (middle) or TNF-α plus deoxycholyltaurine (DCT; 100 μM) (right). b. Annexin-V staining. Original magnification, ×200. B. Actions of increasing concentrations of 1 to 300 μM DCT on TNF-α-induced apoptosis. Percentage of apoptotic cells in HT-29 cells was calculated using Annexin-V staining. Values are mean±SE from 3 experiments. **p<0.01 compared with cells treated with TNF-α alone. C. Changes in PARP degradation in HT-29 cells treated with TNF-α (100 ng/ml) alone or with DCT (100 μM) at the times indicated. Levels of p85 PARP in cell extracts were measured by immunoblotting using a monoclonal antibody that does not recognize 116-kDa PARP. Protein loading was verified by immunoblotting with anti-β-actin antibody. Three experiments were performed that showed similar results. D. Percentage of apoptotic H508 cells determined as described for HT-29 cells in A. Cells were treated with DCT (100 αM) and TNF-α (100 ng/ml), alone or in combination. Values are mean±SE from 3 experiments. ***p<0.001 compared with cells treated with TNF-α alone. E. Changes in PARP degradation in H508 cells treated with TNF-α (100 ng/ml) alone or with DCT (100 μM) at the times indicated. Levels of p85 PARP in cell extracts were measured by immunoblotting. Three experiments were performed that showed similar results.

Effects of treatment with DCT on colon cancer cell apoptosis induced by ultraviolet (UV) radiation. A. Increasing UV doses (10–200 J/m²) caused a progressive increase in H508 colon cancer cell apoptosis. B. Microscopic images of UV-induced apoptosis in H508 cells. a, Control cells (left) and cells treated with UV alone (10 J/m²) (middle) or with UV plus 100 μM DCT (right). (b) Annexin-V staining. Original magnification, ×200. C. Percentage of apoptotic cells in H508 cells treated as shown in B. Values are mean±SE from 3 experiments. **p<0.01 compared to cells treated with UV alone. D. Changes in UV (10 J/m²)-induced apoptosis in H508 cells treated with DCT (100 μM), alone or plus an NF-κB inhibitor, Bay11-7082 (10 μM), and an Akt inhibitor, API-2 (5 μM). Values are mean±SE from 3 experiments. *^,***p<0.05 and 0.001, respectively, compared to cells treated with UV plus DCT alone. E. Effect of inhibiting Akt and NF-κB activity on UV-induced PARP degradation. H508 cells were pre-incubated with Bay11-7082 (10 μM) and API-2 (5 μM) for 30 min before exposure to DCT (100 μM) and UV (10 and 50 J/m²). Levels of p85 PARP in cell extracts were measured by immunoblotting using a monoclonal antibody that does not recognize 116-kDa PARP. Protein loading was verified by immunoblotting with anti-β-actin antibody. Three experiments were performed that showed similar results.

Effects of inhibiting epidermal growth factor receptor (EGFR) activation on NF-κB nuclear translocation and TNF-α-induced apoptosis in HT-29 human colon cancer cells. A. Representative immunoblots showing NF-κB nuclear translocation. Effect of anti-EGFR ligand-binding domain antibody (LA1, 0.1 μg/ml) and EGFR activation inhibitor (AG1478, 0.1 μM) on DCT-induced NF-κB nuclear translocation. HT-29 cells were pre-incubated for 30 min, alone or with EGFR activation inhibitors, prior to treatment with 100 μM DCT. Nuclear extracts were immunoblotted for the p65 NF-κB subunit. Protein loading was verified by immunoblotting using anti-histone H2A antibody. Three experiments were performed that showed similar results. B. Changes in TNF-α-induced apoptosis in HT-29 cells treated with DCT (100 μM), alone or plus EGFR activation inhibitor, LA1 (0.1 μg/ml). Apoptosis was visualized 24 h after exposure to TNF-α (upper panel); images of Annexin-V staining (lower panel). Original magnification, ×200. C. Percentage of apoptotic cells in HT-29 cells pre-treated for 30 min with LA1 (0.1 μg/ml) and AG1478 (0.1 μM). Percentage of apoptotic cells in control and EGFR activation inhibitor-treated cells, and in TNF-α-treated cells alone, with DCT (100 μM) and DCT plus indicated concentrations of EGFR activation inhibitors. Values are means±SE from 3 experiments. ***p<0.05 and 0.01, respectively, compared with cells incubated without EGFR activation inhibitors. D. Effect of inhibiting EGFR activation on TNF-α-stimulated PARP degradation. HT-29 cells were pre-incubated with LA1 (0.1 μg/ml) and AG1478 (0.1 μM) 30 min before exposure to DCT (100 μM) and TNF-α (100 ng/ml). Levels of p85 PARP in cell extracts were measured by immunoblotting using a monoclonal antibody that does not recognize 116-kDa PARP. Protein loading was verified by immunoblotting with anti-β-actin antibody. Three experiments were performed that showed similar results.

Effects of deoxycholyltaurine (DCT) on nuclear translocation and activation of NF-κB in human colon cancer cells. A. DCT stimulates nuclear translocation of p65 NF-κB. a: Representative immunoblots for dose–response for actions of DCT on p65 NF-κB protein in nuclear extracts. HT-29 and H508 cells were incubated for 30 min with indicated concentrations of DCT and nuclear extracts were immunoblotted for p65 NF-κB subunit and the loading control histone H2A. b: Representative immunoblots for time-courses for actions of DCT on p65 NF-κB protein in nuclear extracts. HT-29 and H508 cells were incubated with 100 μM DCT for indicated times and nuclear extracts were immunoblotted for p65 NF-κB subunit and the loading control histone H2A. B. Cellular distribution of NF-κB in H508 (top row) and HT-29 (bottom row) cells. a, Control; b, Cells treated with EGF (10 ng/ml) for 30 min; c, cells treated with DCT (100 μM) for 30 min. Cells were permeabilized and incubated with anti-p65 NF-κB antibody and then with anti-IgG conjugated with TRITC. Nuclei were stained with Hoechst. NF-κB is shown as red, whereas nuclei are shown as blue. Original magnification, ×200. Three experiments were performed that showed similar results. C. Changes in NF-κB-motif binding activity in H508 and HT-29 cells treated with DCT (100 μM, 30 min). Nuclear extracts were isolated from cells with different treatments and the level of p65 NF-κB transcriptional activation was measured using a TransAM NF-κB kit. Values are means±SE from three experiments. *p<0.05 compared with cells incubated without DCT. D. Changes in NF-κB promoter activity as measured by luciferase reporter gene assays. a, Structure of luciferase (Luc) reporter: control p-TAL-Luc and NF-κB-Luc construct with four NF-κB binding sites. b, Level of NF-κB-dependent promoter activity. Cells were transfected with either pNF-κB-Luc or pTAL-Luc using Lipofectamine. Data for NF-κB-dependent promoter activity were normalized using Renilla luciferase activity. Values are means±SE from 3 experiments. ***p<0.05 and 0.01, respectively, compared with cells incubated without DCT.

Effects of inactivation of NF-κB by ectopic expression of IκBα super-repressor (AdIκBαSR) on colon cancer cells. HT-29 and H508 cells transfected with either recombinant adenoviral vector encoding human IκBαSR cDNA (AdIκBSR) or adenoviral vector lacking IκBαSR cDNA (Adnull) at a multiplicity of infection of 25 plaque-forming units per cell for 48 h followed by incubation with DCT. A. Changes in NF-κB transcriptional activity in HT-29 (a) and H508 (b) cells. Cells were transfected with either pNF-κB-Luc or pTAL-Luc and incubated with DCT (100 μM, 2 h). Transcriptional activity was examined by NF-κB-dependent promoter luciferase activity. Values are means±SE from 3 experiments. **^,***p<0.01 and 0.001, respectively, compared with DCT-treated cells infected with Adnull. B. Changes in TNF-α-induced apoptosis in HT-29 cells infected with AdIκBSR or Adnull. Cells were pretreated with Adnull and AdIκBSR, followed by DCT treatment (100 μM, 2 h), and apoptosis was visualized 24 h after exposure to TNF-α (upper panel); images of Annexin-V staining (lower panel). C. Percentage of apoptotic cells in HT-29 (a) and H508 (b) cells. Percentage of apoptotic cells in control and TNF-α treated cells with various treatments described in B (microscopic images of apoptosis in H508 cells are not shown). Values are means±SE from 3 experiments. *p<0.05 compared with cells exposed to TNF-α and infected with Adnull. D. Effect of exposure to DCT (100 μM, 2 h) on TNF-α (100 ng/ml)-stimulated PARP degradation in HT-29 and H508 cells transfected with AdIκBSR. Levels of p85 PARP in cell extracts were measured by immunoblotting using a monoclonal antibody that does not recognize 116-kDa PARP. Protein loading was verified by immunoblotting with anti-β-actin antibody. Three experiments were performed that showed similar results.

Effects of inhibiting Akt activation in HT-29 cells on NF-κB activation and TNF-α-induced apoptosis. A. Representative immunoblots show NF-κB nuclear translocation. a. Effect of transfection of colon cancer cells with kinase-dead Akt on DCT-induced NF-κB nuclear translocation. Control HT-29 cells and HT-29 cells transfected with plasmids containing wild type (WT) and mutant akt (DN) were incubated with 100 μM DCT for 30 min. b. HT-29 cells were pre-incubated with Akt inhibitor, API-2 (5 μM), for 30 min and then incubated with or without DCT (100 μM). Nuclear extracts were immunoblotted for the p65 NF-κB subunit. Protein loading was verified by immunoblotting using anti-histone H2A antibody. Three experiments were performed that showed similar results. B. Effect of inhibiting Akt activity on NF-κB transcriptional activity as measured by NF-κB promoter activity in HT-29 cells. Control HT-29 cells and cells transfected with plasmids containing wild type (WT) and mutant akt (DNM) were incubated with 100 μM DCT. Transcriptional activity was examined by NF-κB-dependent promoter luciferase activity. Values are means±SE from 3 experiments. ***p<0.05 and 0.01, respectively, compared with cells incubated without DCT. C. Changes in TNF-α-induced apoptosis in HT-29 cells treated with DCT (100 μM), alone or plus an Akt inhibitor, API-2 (5 μM). Apoptosis was visualized 24 h after exposure to TNF-α (upper panel); images of Annexin-V staining (lower panel). Original magnification, ×200. D. Percentage of apoptotic HT-29 cells following treatments described in C. Percentage of apoptotic cells in control and API-2-treated cells, and in TNF-α-treated cells alone, with DCT (100 μM) and DCT plus indicated concentrations of API-2. Values are means±SE from 3 experiments. *^,**^,****p<0.05, 0.01, and 0.001, respectively, compared with control cells incubated without API-2. E. Effect of inhibiting Akt activity on TNF-α-stimulated PARP degradation. HT-29 cells were pre-incubated with API-2 (5 μM, 30 min) before exposure to DCT (100 μM) and TNF-α (100 ng/ml). Levels of p85 PARP in cell extracts were measured by immunoblotting using a monoclonal antibody that does not recognize 116-kDa PARP. Protein loading was verified by immunoblotting with anti-β-actin antibody. Three experiments were performed that showed similar results.