Journal: Cell death and differentiation

Article Title: Targeting Bcl-xL in esophageal squamous cancer to sensitize to chemotherapy plus TRAIL-induced apoptosis while normal epithelial cells are protected by blockade of caspase 9.

doi: 10.1038/sj.cdd.4401388

Figure Lengend Snippet: Figure 1 Analysis of determinants of TRAIL mediated cytotoxicity suggests sensitivity of primary esophageal epithelial cells as well as esophageal tumors, while there is some correlation between TRAIL resistance and overexpression of Bcl-xL in TRAIL-resistant esophageal squamous cell carcinomas. (a) TRAIL cytotoxicity toward squamous esophageal cancer cells in the presence or absence of CHX. Each cancer cell line (5 105 cells/well) was seeded onto a six-well plate and the cells were treated with TRAIL crosslinked with anti-6X His Ab. After 16 h of TRAIL treatment, cells were harvested and an active-caspase 3 assay was performed. In case of CHX treatment, the cells were pretreated with CHX (10 mg/ml final concentration) for 6 h before the addition of TRAIL. We defined the cells as TRAIL sensitive when more than 30% of the cell population activated caspase 3. Experiments were performed in duplicate (mean value7S.D.). Human esophageal cancer cell lines were previously described. All the esophageal cancer cell lines were grown with DMEM containing 10% FCS. Primary normal esophageal epithelial cells (EPC2) were established from normal human esophagus (unpublished data, Yasir Suliman, Oliver G Opitz and Anil K Rustgi). EPC2 cells have a diploid karyotype, and express E-cadherin as well as cytokeratins K4, K5, K13, and K14. EPC2 cells were grown in Keratinocyte-SFM medium (Gibco BRL, Rockville, MD, USA) containing bovine pituitary extract (40 mg/ml) and epidermal growth factor (1 ng/ml). For detecting apoptosis mediated by TRAIL, an active-caspase 3 assay was performed using the Cytofix/Cytoperm kit (Pharmingen, San Diego, CA, USA) as previously described. Briefly, 5 105 cells were seeded onto a six-well plate, after which, the cells were treated with TRAIL crosslinked with anti-6X His Ab. After 4 or 16 h of treatment, the cells were harvested, fixed, and then incubated with 0.125 mg/ml rabbit anti-active-caspase 3 Ab (Clone C92-605; Pharmingen) for 20 min in a dark room. After washing, the cells were probed with 0.125 mg/ml of the phycoerythrin (PE)-conjugated goat anti-rabbit secondary Ab (CALTAG Laboratories, Burlingame, CA, USA) for 20 min in a dark room. The intensity of PE was analyzed by flow cytometry using a Beckman–Coulter Epics Elite analyzer. The N-terminal histidine (His)-tagged recombinant human TRAIL (rhTRAIL, Thr-95 to Gly-281) was obtained from R&D Systems (Minneapolis, MN, USA). A mouse monoclonal anti-6X His antibody (R&D Systems) was used for crosslinking of the rhTRAIL. Cells were treated with 50 ng/ml final concentration of TRAIL and 1 mg/ ml of anti-6X His Ab for 4 or 16 h. When the cells were treated with both cycloheximide (CHX) and TRAIL, CHX (10 mg/ml final concentration) was added 6 h before the addition of TRAIL. In the case of adriamycin, cells were pretreated with adriamycin (0.5 mM final concentration) for 16 h before the addition of TRAIL. (b) Relative expression level of signaling molecules involved in TRAIL-mediated apoptosis. Cell lysates were subjected to 12 or 15% SDS-PAGE, and then immunostained with Abs are indicated in the figure. TRAIL sensitivity was defined by the results of active-caspase 3 assay (see Figure 1); S ¼ sensitive, R ¼ resistant. Western blot analysis was carried out as previously described. Blotted membranes were immunostained with anti-DR4 (1 : 500; Pharmingen), anti-DR5 (1 : 500; IMGENEX, San Diego, CA, USA), anti-DcR1 (1 : 500; Pharmingen), anti-DcR2 (1 : 500; Pharmingen), anti-FADD (Clone IF7, 1 : 2000; Upstate Biotechnology, Lake Placid, NY, USA), anti-caspase 8 (Clone 5F7, 1 : 1000; Upstate Biotechnology), anti-FLIP (Clone Dave-2, 1 : 1000; Alexis Biochemicals, San Diego, CA, USA), anti-Bid (1 : 500; Pharmingen), anti-Bax (1 : 500, Pharmingen), anti Bcl-2 (Bcl-2/100, 1 : 500; Pharmingen), anti-Bcl-xL (1 : 500; Pharmingen), anti-caspase 3 (E-8, 1 : 200; Santa Cruz, Santa Cruz, CA, USA), anti-PARP (1 : 2000; Roche Diagnostics GmbH, Mannheim, Germany), anti-TRAIL (1 : 500; PeproTech Inc., Rocky Hill, NJ, USA), or anti-actin (I-19, 1 : 200; Santa Cruz). (c) Bcl-xL is overexpressed in human esophageal squamous cell carcinoma specimens. Bcl-xL staining in normal human esophagus and three archival esophageal squamous cell carcinoma specimens. For the cancer (CA) case labeled #17, the normal esophageal tissue was obtained from the same patient. We used as primary antibody the mouse anti-human Bcl-xL monoclonal antibody (Clone 2H12) obtained from Zymed (San Francisco, CA, USA) at a dilution of 1 : 50 in an overnight incubation at 41C. (d) Active-caspase 3 assay for apoptosis after treatment with either TRAIL alone or TRAIL plus adriamycin (0.5 mM) in the presence or absence of z- LEHD-FMK on EPC2 normal esophageal epithelial cells. EPC2 (5 105) cells were treated with TRAIL (50 ng/ml) for 4 h. (e) Preferential cytoprotective effect of z- LEHD-FMK from TRAIL-mediated apoptosis in esophageal cells. Both EPC2-GFP (2 105 cells) and TRAIL-sensitive TE2 (5 105 cells) were grown in a six-well plate, and the cells were treated with TRAIL for 4 h in the presence or absence of z-LEHD-FMK. The active-caspase 3 assay was performed and the two populations of cells (EPC2-GFP and TE2) were discriminated by the presence or absence of green fluorescence. A bar graph is shown on the right to display the same result quantitatively. Experiments were performed in duplicate (mean value7S.D.). The irreversible caspase inhibitors z-LEHD-FMK (caspase 9 inhibitor), z-IETD-FMK (caspase 8 inhibitor), and z-VAD-FMK (Pan caspase inhibitor) were obtained from R&D Systems. The caspase inhibitor was used at a final concentration of 20 mM and was added 2 h prior to the addition of TRAIL

Article Snippet: The irreversible caspase inhibitors z-LEHD-FMK (caspase 9 inhibitor), z-IETD-FMK (caspase 8 inhibitor), and z-VAD-FMK (Pan caspase inhibitor) were obtained from R&D Systems.

Techniques: Over Expression, Caspase-3 Assay, Concentration Assay, Incubation, Cytometry, Recombinant, Expressing, SDS Page, Western Blot, Staining, Labeling

Journal: Cell death and differentiation

Article Title: Targeting Bcl-xL in esophageal squamous cancer to sensitize to chemotherapy plus TRAIL-induced apoptosis while normal epithelial cells are protected by blockade of caspase 9.

doi: 10.1038/sj.cdd.4401388

Figure Lengend Snippet: Figure 2 Targeting Bcl-xL may result in sensitization of TRAIL-resistant esophageal squamous cell carcinomas to TRAIL, while use of caspase 9 blockade still permits death of the cancer cells by TRAIL plus adriamycin. (a) Active-caspase 3 assay for apoptosis after the combination of adriamycin and TRAIL treatment of TRAIL-resistant HCE4, TE3, and TE5 cells. The cells were pretreated with 0.5 mM adriamycin for 16 h followed by incubation for 4 h in the presence of TRAIL. z-LEHD-FMK (20 mM final concentration) was added (as indicated) at 2 h before the addition of TRAIL. (b) Mitochondrial membrane potential change after adriamycin treatment. To detect a change in the mitochondrial membrane potential (Dcm) after treating cells with adriamycin, 3, 30-dihexyloxacarbocyanine iodide (DiOC6 (3)) was used. Briefly, 5 105

Article Snippet: The irreversible caspase inhibitors z-LEHD-FMK (caspase 9 inhibitor), z-IETD-FMK (caspase 8 inhibitor), and z-VAD-FMK (Pan caspase inhibitor) were obtained from R&D Systems.

Techniques: Caspase-3 Assay, Incubation, Concentration Assay, Membrane