Moderate Bcl-2 overexpression effectively protects NCI-H460 cells from TRAIL-induced apoptosis.

<p>(A) Expression of FLAG-tagged Bcl-2 in NCI-H460 cells. Equal amounts of whole cell lysates from NCI-H460 wild type and NCI-H460/Bcl-2 were analyzed by immunoblotting. Note that the tagged Bcl-2 protein appears at a somewhat lower molecular weight range as compared to wild-type Bcl-2. Equal loading was confirmed by re-probing the membrane with α-tubulin specific antibody. (B) Detection of Bcl-2 by flow cytometry. Fixed cells were permeabilized and stained with anti-Bcl-2 or corresponding isotype control primary antibody and PE-labeled secondary antibody. Cells were analyzed by flow cytometry. (C) Cytotoxicity assays of NCI-H460 and NCI-H460/Bcl-2 cells using Db-scTRAIL. Cells were treated with increasing concentrations of Db-scTRAIL for 24 h, viable cells were stained with crystal violet and quantified at 550 nm. All values were normalized to values from unstimulated cells. Shown are mean values ± SD calculated from triplicates. Data shown is representative of three independent experiments. (D) Db-scTRAIL induces apoptosis in NCI-H460. Cells were co-treated with Db-scTRAIL and z-VAD-fmk (20 μM) or only the carrier DMSO for 24 h. Viable, apoptotic (Annexin V-FITC-positive) and necrotic (PI-positive) cells were determined after flow cytometric analysis. (E) Db-scTRAIL induces cytochrome c and Smac release into the cytosol in wild-type NCI-H460 cells only. Cells were left untreated or treated with Db-scTRAIL (0.5 nM) for the indicated time periods. Cytosolic extracts were prepared and subjected to western blotting with cytochrome c and Smac specific antibodies. Equal loading was confirmed by reprobing the membrane with α-tubulin antibody. (F) ABT-737 resensitizes NCI-H460/Bcl-2 cells. Cells were preincubated with 2.5 μM ABT-737 alone (filled triangle) or in combination with 50 μM z-VAD-fmk (filled circles) or DMSO as control (open squares) and treated with Db-scTRAIL. Viable cells were quantified by crystal violet staining after 24 h. Data represents mean values ± SD calculated from triplicates and shown is a representative of three independent experiments.</p

XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells.

<p>(A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates <i>via</i> immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.</p

Mathematical modeling of the XIAP/Smac interaction in NCI-H460 in response to Db-scTRAIL.

<p>(A) The average numbers of XIAP and Smac in NCI-H460 in an unstimulated cell. XIAP and Smac molecules were quantified in NCI-H460 cells by western blotting. Mean ± SD is shown and were calculated from three independent experiments. (B) Regions of different cellular responses. Dependent on the initial amounts of XIAP and Smac, regions of enhanced or reduced apoptosis are sketched. Red regions represent XIAP* amounts lower or equal to 25% XIAP* compared to the wild type. Green color illustrates XIAP* amounts exceeding 250% of wild type. Different experimental scenarios (I-VI) are indicated by arrows. (C) Initial conditions of XIAP and Smac and the resulting steady state of XIAP are shown for the different scenarios. Green color indicates increased resistance to Db-scTRAIL, red color represents sensitivity, respectively.</p

Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells.

<p>(A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).</p

Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps.

<p>(A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.</p