Peptide synthesis

CIGB-300 and CIGB-300-biotin-conjugated were synthesized as previously described by Perea et al. [12]. Briefly, the peptides were synthesized on solid phase and purified by reverse-phase high-performance liquid chromatography. The catalytic peptide sequence was fused to the TAT fragment of the HIV-1 virus, which confers the compound the ability to pass through the cell membrane [31, 32]. Purity was verified by mass spectrometry.

Reagents and antibodies

Medium for cell culture was obtained from Life Technologies Inc. (Rockville, MD,USA). Fetal bovine serum (FBS) was from Internegocios (Buenos Aires, Argentina). Acrylamide and all other reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad (Richmond, CA, USA). Phorbol 12-myristate 13-acetate (PMA) and 4,5,6,7-Tetrabromo-2-azabenzimidazole (TBB) were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Recombinant human tumor necrosis factor-alpha (TNFα) was purchased from ImmunoTools (Friesoythe, Germany). Bortezomib (VELCADE®) was purchased from Millennium Pharmaceuticals (Cambridge, MA, USA) and cisplatin (MARTIAN®) was purchased from Kampel Laboratory (Buenos Aires, Argentina). Anti-NF-κB p65 (D14E12 XP®, 1:1000) was purchased from Cell Signaling (Danvers, MA, USA), β-CATENIN antibody (#610,153, 1:1.000) was from BD Transduction Laboratories (San Jose, CA, USA), Horseradish peroxidase-conjugated anti-mouse (1:5000), Mouse monoclonal anti c-MYC (sc-40, 1:100) and Mouse monoclonal anti CYCLIN E (sc-247, 1:200) were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-rabbit (1:5000) and β-ACTIN antibody (1:10.000) were from Sigma-Aldrich Co. Monoclonal anti PSMA3 (α7/C8) antibody (ab109532, 1:100), Rabbit Monoclonal anti NF-κB p65 (phospho S529) (ab109458, 1:1000) and Rabbit polyclonal anti BAX (ab69643, 1:1000) were from Abcam (Cambridge, MA, USA), Streptavidin-FITC (SNN1008, 1:300) and CYCLIN D1 antibody (RB-9041, 1:10000) were from Invitrogen-Thermo Fisher (Waltham, MA), Goat Anti-Rabbit IgG H&L Alexa Fluor® 647 (ab150113, 1:250) and Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 (ab150079, 1:1000) from Abcam. Hybond-P membranes for blotting and chemiluminescence reagents (ECL) were from Amersham-GE Healthcare (Buckinghamshire, UK).

Cell lines and culture conditions

We used two human non-small cell lung cancer (NSCLC) derived cell lines: the adenosquamous cell carcinoma NCI-H125 (CRL-5801) and the adenocarcinoma NIH-A549 (CCL-185). The murine L Wnt-3A cell line (CRL-2647) was used in order to produce conditioned media (CM) containing the wnt3a factor, following ATCC’s instructions. All cell lines were obtained from ATCC. H125 was grown in RPMI medium supplemented with 10% FBS and 80 µg/ml gentamycin. A549 and L cells were grown in DMEM media, with the above mentioned supplementation. All cell lines were cultured at 37 °C in a humidified air atmosphere with 5% CO 2 .

Treatments

For Wnt/β-CATENIN pathway studies, H125 sub-confluent monolayers were first incubated during 24 h with 50% of CM containing the wnt3a factor and then treated for another 6 h with a low-lethal dose of CIGB-300 (IC 25 ) or during 2 h with the CK2 competitive inhibitor TBB (25 μM) as a control. For NF-κB studies, the H125, A549 or A549-cispR cells were incubated for 15 or 45 min respectively with a low-lethal dose CIGB-300 (IC 25 ) with or without PMA (16 nM), TNFα (10 ng/ml) or the IC 50 of cisplatin (1.0 µM for H125 and 5.0 µM for A549). For the analysis of NF-κB downstream targets, H125, A549 or A549-Rcisp cells were incubated for 2 h with CIGB-300 (IC 50 ). In the case of Bortezomib, cells were previously pretreated with 8 nM of this drug during 4 h.

Cell viability and apoptosis determination

To assess the effect of CIGB-300 or cisplatin on cell viability, 1 × 104 cells/well were seeded into 96 well plates in standard culture conditions. Twenty-four hours later cells were treated with different doses of CIGB-300 (25–400 μM) or cisplatin (0.3–30 μM) during 72 h. Cell proliferation was evaluated after 72 h using the MTS assay (Celltiter 96TM Non Radioactive Proliferation Assay, Promega), as described by the manufacturer. Data were analyzed using the R package “ic50” [33] in order to determine the IC 50 values for each drug. Alternatively, proliferative potential was determined by assessing cell number. Briefly, cells were seeded onto 35 mm Petri dishes, treated and after 72 h wells were washed twice with PBS, trypsinized, centrifuged, resuspended in a proper volume and finally counted using an hemocytometer and trypan blue exclusion. For apoptosis determination, A549 and A549-cispR cells growing on glass coverslips were treated with 300 μM of CIGB-300 for 18 h and then stained with Acridine orange (10 μg/ml) and Ethidium bromide (10 μg/ml). Visualization was performed with a Nikon, Eclipse E400 epifluorescence microscope. Uniform green nuclei with organized structure were considered as live cells. Bright green or orange to red nuclei were classified as early or late apoptotic cells respectively. Uniformly orange to red nuclei with organized structure were ascribed as necrotic cells. The percentage of dead cells was determined for each cell line.

3D cultures and peptide internalization assay

Multicellular tumor spheroids were obtained through the hanging-drop method [34]. Growth kinetic was evaluated using spheroids formed from 1000 initial cells. For the 3D growth inhibition assay, each “drop” containing a single spheroid was placed in 500 μl of culture media supplemented with FBS onto 48 well plates on day 3. Two days later spheroids were treated or not with different doses of CIGB-300 (IC 25 and IC 50 obtained for monolayer cultures). Complete culture media, supplemented or not with CIGB-300 was replaced every 48 h. At the same time, images were taken using an optical light microscope. The area, circularity and roundness of spheroids were measured using a custom macro for ImageJ, and finally the volume was calculated. For peptide internalization kinetics, 2-day-old spheroids were treated with the biotinylated variant of CIGB-300 for 5–60 min. Spheroids were fixed in formaldehyde (4% in PBS) for 30 min and embedded in paraffin. Sections of 5 μM were obtained using microtome on polylysine-coated slides. Finally, immunocytochemistry was performed using a Streptavidin–biotin method with the VECTASTAIN Elite ABC KIT from Vector Laboratories (Burlingame, CA, USA) following manufacturer’s instructions. Sections were counter-stained with Mayer’s hematoxylin. Images were taken using a light microscope, at 400× magnification. Immunoreactive staining was quantified transforming each image into polar coordinates and using the color detection tool of the ImageJ plugin Immunohistochemistry Image Analysis Toolbox [35].

Separation of nuclear and cytoplasmic fractions

In order to determine β-CATENIN expression levels, cytoplasmic proteins were extracted using saponin buffer. Briefly, monolayer cultures were incubated for 20 min with ice-cold 0.1% saponin lysis buffer (25 mM Hepes, 75 mM potassium acetate and 0.1% saponin plus phosphatase and protease inhibitors). The extraction procedure was carried out twice and the extracts were pooled prior to their centrifugation. For NF-κB detection, nuclear and cytoplasmic proteins were separated by differential centrifugation following Abcam protocol. Briefly, semiconfluent monolayers were washed twice with ice-cold PBS and then collected in 250 μl of Fractionation Buffer (HEPES 20 mM, KCl 10 mM, MgCl 2 2 mM, EDTA 1 mM and 2-Mercaptoethanol 1 M) with protease and phosphatase inhibitors by scrapping with a Teflon scrapper. Cell suspensions were passed 10 times through a 25 gauge needle, and leaved on ice for 20 min. Then samples were centrifuged at 700g for 5 min. The pellets, which contain nuclei, were washed repeating all the procedure, and finally resuspended in Nuclear Buffer (Fractionation Buffer plus 10% glycerol and 0.1% SDS). Supernatants were centrifuged again at 10,000g for 5 min and the supernatant, containing the cytoplasm and membrane, was collected.

Western blot analysis

Semiconfluent monolayers were washed twice with ice-cold PBS and then lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris–HCl pH 8.0, 1 mM EDTA, 0.5% deoxycholate) with protease and phosphatase inhibitors by scrapping with a Teflon scrapper. Samples were run in 10% SDS-PAGE, and the gels blotted to Hybond-P membranes. Membranes were blocked for 1 h in 5% skim milk in TBS, 0.1% Tween-20. Membranes were then incubated with the first antibody overnight at 4 °C, and with a secondary antibody coupled to horseradish peroxidase [1 h at room temperature (RT)]. Detection was performed by chemiluminescence. Bands were digitalized with a Photo/Analyst Express System (Fotodyne Inc. Hartland, WI, USA) and signal intensity was quantified with ImageJ software.

NF-κB-dependent reporter gene expression

H125 cells were transiently co-transfected with NF-κB-RE-luc Luciferase Reporter Vector pGL4.32 and the Renilla Luciferase Control Reporter Vector pRL-TK (Promega, Madison, WI) in a 10:1 ratio, using Fugene (Promega) following manufacturer’s instructions. Briefly, 1 × 105 cells/well were seeded onto 24-well plates and transfected the next day. Six hours after adding transfection reagents, cells were washed and complete media was added. After an overnight recovery, cells were treated for 6 h with PMA (16 nM), in presence or not of CIGB-300 (IC 25 ) during the first hour. Finally cells were lysed and the luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) and normalized to the constitutive Renilla luciferase activity.

Immunofluorescence microscopy

H125 cells growing on glass coverslips were treated with CIGB-300 for 15 min in presence or not of PMA, washed twice with PBS, fixed in ice cold paraformaldehyde, permeabilized and blocked with PBS containing 5% bovine serum albumin for 1 h at RT. p65 protein was detected by incubation with a specific primary antibody diluted in PBS containing 5% BSA, followed by an incubation with a secondary antibody coupled to Alexa 488 for 1 h at RT. Nuclei were counterstained with DAPI. Images were obtained using an epifluorescence microscope (Nikon, Eclipse TE2000), processed and analyzed using the ImageJ software. Nuclear intensity was measured taking into account the overlapping with DAPI staining. The number of nuclei with highly positive signal was counted and expressed as a percentage of the total intensity for each condition.

Generation of a cisplatin resistant cell line

Cisplatin resistant A549 lung cancer cell line (A549-cispR) was obtained by the chronic administration of cisplatin IC 50 (5.0 μM) during 6 months as previously described [36]. Parental A549 cells were maintained in culture during the same period.

Drugs interaction analysis

To determine the combination index (CI) of CIGB-300 and cisplatin, A549 and A549-cispR cells were treated with both drugs following a constant-ratio analysis. Briefly, drug mixture was serially diluted and added for 72 h to cells seeded onto a 96 well plate. Drugs and media were refreshed every 24 h. The combination ratio used was approximately equal to the IC 50 ratio for each drug. Three points below and above the IC 50 value were used for each mixture. Effects of all the mixture points were displayed using the form CI vs. Fa, where Fa is fraction affected and represents the respective proliferation inhibition parameters (e.g., a Fa of 0.5 is a proliferation inhibition of 50%). CI plot values were obtained from three different experiments using the Compusyn software [37].

Proteasome activity determination

Proteasome activity was assessed in H125 cells using the Proteasome-Glo Chymotrypsin-Like Cell-Based Assay (Promega) following manufacturer’s instructions. Briefly, H125 cells were plated onto 96-well plates and 24 h later treated with CIGB-300 (30 min) or with Bortezomib (4 h). Briefly: 100 µl of Proteasome-Glo™ Cell-Based Reagent were added to each 100 µl of sample and incubated at RT for 10 min. Luminescence emission was measured with a Multimode Microplate Reader, Synergy luminometer (Biotek).

Confocal immunofluorescence

H125 cells growing on glass coverslips were treated with biotin-conjugated CIGB-300 for 30 min, washed twice with PBS, fixed in ice cold paraformaldehyde, permeabilized and blocked with PBS containing 5% bovine serum albumin for 1 h at RT. α7/C8 protein was detected by incubation with a specific primary antibody diluted in PBS containing 5% BSA, followed by an incubation with a secondary antibody coupled to Alexa 647 and with Streptavidin-FITC conjugated for 1 h at RT. Nuclei were stained with DAPI. Images were obtained in a LSM 510 Meta confocal Zeiss microscope. Confocal images were processed for presentation with ImageJ. Background of each channel was subtracted. Pearson’s and Mander’s coefficients were calculated using the JACoP plugin [38]. Single cell analysis for 15–20 representative cells was performed on three independent experiments.

Statistical analysis

Data obtained were evaluated for their statistical significance with the two-tail paired Student’s t test or analysis of variance (ANOVA) with post hoc corrections. Values were considered statistically significant at p below 0.05.