The Smac mimetic SM83 sensitizes oncogenic K-Ras expressing CRC cells to Db_αEGFR-scTRAIL.

<p>(a–d) Caco-2tet Ras^G12V cells were seeded into 3D cultures in the presence of doxycycline. (a) Three days later, cultures were left untreated or treated with 5 µM SM83 or 20 µM Z-VAD for 1 h prior to addition of 1 nM Db_αEGFR-scTRAIL. Viability was measured 24 h later by MTT assay and normalized to the untreated control (n = 3). (b) 24 h after treatment, cells were fixed and stained for DNA strand breaks. Tunel-positive cells were counted (n = 2). (c) Three days post seeding, cultures were left untreated (ut) or treated with 5 µM SM83. Cells were recovered from the cultures 24 h later and lysates were analyzed by immunoblotting. Shown is one representative blot of two independent experiments. Tubulin was detected as a loading control. (d) Quantification of Western blots from (c). Protein levels were normalized to the corresponding tubulin control; levels in the untreated cultures were set as 1 (n = 2). (e, f) HCT-116 and LoVo cells were grown in 3D cultures for six days, and then fixed and stained for F-actin and DNA (DAPI) (scale bar: 20 µm) (top panels). Three days post seeding, cultures were left untreated or pretreated with 5 µM SM83 prior to addition of 0.05 nM Db_αEGFR-scTRAIL. Viability was measured 24 h later by MTT assay and normalized to the untreated control (bottom panels) (n = 3).</p

Inducible expression of oncogenic K-Ras^G12V in Caco-2tet cells.

<p>(a) Caco-2tet vector control and K-Ras^G12V cells were treated with 2 µg/ml doxycycline for 72 h (+dox). Cells were harvested and GFP fluorescence was analyzed by flow cytometry. Non-induced cells were used as a control (−dox). (b) Caco-2tet vector control and K-Ras^G12V cells were grown in 2D and treated with doxycycline for the indicated times prior to lysis. GFP, Ras, pERK (T202/Y204) and ERK levels were determined by Western blotting. Tubulin was detected as a loading control. All panels shown are from the same blot. (c) Caco-2tet vector control and K-Ras^G12V cells were seeded into 3D cultures in the absence or presence of doxycycline. Three days post seeding lumen expansion was induced by addition of 100 ng/ml CTX. Cultures were fixed three days later and stained with E-cadherin-specific antibody (green), phalloidin (red) and DAPI (nuclei; blue). Shown are confocal sections of representative cysts (scale bar: 10 µm).</p

Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

<div><p>Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.</p> </div

EGFR-directed targeting determines the bioactivity of Db_αEGFR-scTRAIL.

<p>(a) Caco-2 cells grown in 2D were left untreated or treated with 4 nM Db_αEGFR-scTRAIL or 4 nM Cetuximab for 15 min prior to stimulation with EGF or TGF-α (10 ng/ml) for 10 min. Phosphorylated and total proteins were detected by immunoblotting. Shown is one representative blot of three independent experiments. Tubulin was detected as a loading control. (b) Quantification of Western blots from (a). Phospho-EGFR levels were normalized to the corresponding total protein levels; levels in the untreated control were set as 1 (n = 3). (c, d). Three days post seeding, Caco-2 3D cultures grown in the absence or presence of growth factors were treated with 1 nM Db_αEGFR-scTRAIL. (c) Viability was determined by MTT assay after 72 h and normalized to the respective untreated control (ut). (n = 3) (d) Caspase 3/7 activity was measured after 24 h. Values were normalized to the corresponding untreated control (n = 3). (e) Three days post seeding, Caco-2 3D cultures were either left untreated or treated with 5 nM Db_αEGFR-scTRAIL for 72 h. Surviving cysts in the phase contrast images are indicated by arrows (scale bar: 50 µm). (f) Lysates derived from the 3D cultures shown in (e) were analyzed by immunoblotting. Shown is one representative blot of three independent experiments. Tubulin was detected as a loading control. Specific bands are marked by arrowheads. (g) Quantification of Western blots from (f). Protein levels were normalized to the corresponding tubulin control; levels in the untreated cultures were set as 1 (n = 3).</p

SAXS study of XIAP-BIR2BIR3.

<p>A) experimental scattering patterns with associated error bars; blue line: free XIAP-BIR2BIR3; red line: XIAP-BIR2BIR3 complexed with 9a. B) distance distribution functions p(r); color code as in panel A. C) distribution of Rg values of free XIAP-BIR2BIR3; green: random pool; orange: selected ensembles fitting the data; D) distribution of D_max values; color code as in panel C.</p

Caco-2 3D cultures are sensitive to Db_αEGFR-scTRAIL.

<p>Cells were grown in 3D or 2D in medium containing 10% FCS. (a) Three days post seeding, cultures were treated with Db_αEGFR-scTRAIL. Viability was measured 72 h later by MTT assay and normalized to the untreated control (n = 3). (b) Phase contrast images of the 3D and 2D cultures described in (a) treated with the indicated concentrations of Db_αEGFR-scTRAIL for 72 h (scale bar: 50 µm). (c) Three days post seeding, 3D cultures were pretreated with 20 µM Z-VAD as indicated before addition of 1 nM Db_αEGFR-scTRAIL. Viability was measured 72 h later by MTT assay and normalized to the untreated control (ut) (n = 3). (d) 24 h after treatment, cells were fixed and stained for DNA strand breaks. Tunel-positive cells were counted (n = 2). (e) Representative pictures of the Tunel stainings described in (d), Tunel-positive cells (red), DAPI (nuclei; blue). Shown are confocal sections (scale bar: 100 µm). (f) Three days post seeding, cultures were treated with 0.1 nM or 1 nM Db_αEGFR-scTRAIL for 24 h. Caspase 3/7 activity was measured and normalized to the respective untreated control (ut) (n = 3). (g) Four days post seeding, lysates were generated and analyzed by immunoblotting. Shown is one representative blot of three independent experiments. Tubulin was detected as a loading control. Specific bands are marked by arrowheads. (h) Quantification of Western blots from (g). Protein levels were normalized to the corresponding tubulin control; levels in the 2D cultures were set as 1 (n = 3).</p

Caco-2 3D cultures are sensitive to pharmacological EGFR inhibition.

<p>Caco-2 cells were grown in 3D cultures containing 2% FCS in the presence of growth factors (10 ng/ml) or in 2% FCS only (con). (a) Cultures were analyzed by MTT assay at day 6 and normalized to the control (n = 5). (b) Three days post seeding 100 ng/ml CTX was added to induce lumen expansion. Spheroids were fixed on day 6 and stained with phalloidin (F-actin) and DAPI (nuclei). Shown are confocal sections of a representative cyst (scale bar: 10 µm). (c) Three days after seeding, 3D cultures were left untreated or treated with 0.5 µM Cetuximab (Cet) for 72 h. Viability was determined by MTT assay and normalized to the untreated control (ut). (n = 4) (d) Caco-2 3D cultures were left untreated or treated with 0.5 µM Cetuximab for 72 h and analyzed by phase microscopy (scale bar: 50 µm). (e) Three days after seeding, 3D cultures were left untreated (ut) or treated with 0.5 µM Cetuximab (Cet) for 72 h. Cytotoxicity was determined using the CytoTox-Glo Cytotoxicity Assay (n = 3).</p

X-ray data-collection and refinement statistics for the cIAP-BIR3/9a and XIAP-BIR3/9a complexes.

<p>Values in parentheses are for the highest resolution shell.</p>†<p><b>R_merge</b> = Σ |<i>I</i> - (<i>I</i>)|/Σ<i>I</i> × 100, where <i>I</i> is intensity of a reflection and (<i>I</i>) is its average intensity.</p>‡<p><b>R _factor</b> = Σ |F_o - F_c|/Σ |F_o| × 100.</p>§<p><b>R_free</b>is calculated on 5% randomly selected reflections, for cross-validation.</p

Analytical Gel Filtration Chromatograms.

<p>A) XIAP-BIR3 (33 µM) in absence/presence of an excess (1 mM) of 9a. B) XIAP-BIR2BIR3 in absence/presence of an excess (1 mM) of 9a. C) cIAP-BIR3 in absence/presence of an equal amount (33 µM) of 9a.F</p

Dimeric assemblies of cIAP1- and XIAP1-BIR3 bound to 9a.

<p>A) X-Ray structure of cIAP1-BIR3 dimer (cartoon in blue and pale blue) in complex with 9a (green sticks). B) X-Ray structure of XIAP-BIR3 dimer in complex with 9a: the A and F molecules are in orange and pale yellow, respectively, 9a is represented as green sticks (drawn with Pymol).</p