Substances
Bortezomib was purchased from Selleckchem and dissolved in DMSO (SIGMA Biochemicals) to a stock solution of 250 mM. Aplidin™ and Aplidin analogs PM01215 and PM02781 were synthesized in Pharmamar and dissolved in DMSO to stock solutions of 250 mM and stored in aliquots at −80 °C. All stocks were further diluted with DMSO to working concentrations of 1 mM and stored at −20 °C. N-acetyl cysteine (NAC, Sigma Biochemicals) was dissolved in distilled sterile water, and 30 % H2O2 was purchased from Merck. Thapsigargin was purchased from Life Technologies and dissolved in DMSO (SIGMA Biochemicals) to a stock solution of 1 mM.
Cell culture
Human endothelial cells from different donors (HUVECs, n = 3) were purchased from PromoCell after immunohistochemical testing (vWF+, CD31+, ASMA-). HMECs were cultivated in endothelial cell growth medium (EGM2) with recommended supplements (PromoCell) on collagen type I (Sigma Biochemicals) -coated ventilated plastic flasks. Cells were passaged using the DetachKit (PromoCell) consisting of 30 mM HEPES, 0.04 %/0.03 % trypsin/EDTA solution and trypsin-neutralizing solution (TNS).
Human mesenchymal cells from bone marrow of various donors (n = 3) were purchased from PromoCell after their analysis by flow cytometry (CD31+, CD44+, CD45-, CD105+). Cells were cultivated in RPMI1640 medium (Sigma Biochemicals) with 10 % bovine calf serum (Hyclone) and 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine (all PAA Laboratories GmbH) on uncoated plastic material.
Human PBMNCs from healthy donors (n = 3,) were prepared as described elsewhere [16]. In brief, blood samples from blood donors were collected in anticoagulant (EDTA) tubes and transferred to Leucosep® tubes (Greiner Bio-One) containing Ficoll (LSM1077 Lymphocyte separation medium, PAA) for density gradient centrifugation. Thereafter, mononuclear cell faction was washed in PBS, characterized by flow cytometry (size, granularity, CD45+ expression) and used for experiments.
All primary cells were characterized by flow cytometry using a panel of cell type-specific markers (Additional file 1: Table S1) and were tested for the absence of HIV1/2, HBV, HCV and mycoplasma. Only cells of low passages were used for experiments. OPM-2 multiple myeloma cells (AC55) were purchased 2012 directly from DSMZ (Germany), authenticated by us (STR-profiling, flow cytometry: CD138+/CD38+) and cultivated in RPMI1640 medium (Sigma Biochemicals) with 10 % bovine calf serum (Hyclone) and 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine (all PAA Laboratories GmbH) on uncoated plastic material. OPM-2 cells were lentivirally transfected to express eGFP and propagated in the presence of blasticidin (2.5 μg/mL, Invitrogen) before usage for in vivo experiments.
Western Blot analysis
Cells were harvested and lysed in a RIPA buffer (Cell Signaling) containing protease inhibitors (Complete Mini EDTA-free; Roche Applied Science). Total protein (20 μg) was denatured, separated with 4 -20 % SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to an Immuno-Blot™ polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking the membrane in 5 % non-fat milk powder dissolved in phosphate-buffered saline (PBS), membranes were incubated overnight in 3 % non-fat milk powder or 5 % BSA at 4 °C with primary antibodies. Afterwards, membranes were incubated with an HRP-conjugated secondary antibody (Dako Cytomation) diluted 1:2,500. After washing, a chemoluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membrane, which was then exposed in the Chemidoc XRS station (Bio-Rad Laboratories). Antibodies used for Western Blot analysis were alpha tubulin (clone B5-1-2; Sigma Biochemicals), p27Kip1 (clone G173-524, BD Pharmingen) and p53 (clone PAb1801, Calbiochem), p21Cip1 (BD Biosciences), p16INK4A (BD Biosciences), VEGF-R2 (Calbiochem), vasohibin (R&D Systems, Clone 411208), GRP78/HSPA5 (R&D Systems, Clone 474421), and XPB1 (SCBT, M-186). Mouse-anti JNK (Santa Cruz Biotechnology, clone D-2), rabbit anti-phospho-p44/42 MAPK (Erk1/2 D13.14.4E), mouse anti-P44/42 MAPK (Erk1/2, clone L34F12), rabbit anti-phospho-Akt (Ser473), rabbit anti-Akt (pan) and rabbit-anti-phospho-JNK (all purchased from Cell Signaling).
DKK-3 ELISA
Cells were treated for 72 h with 10 nM solution of the respective compounds. For quantitative measurement of DKK-3 in supernatants a commercially available ELISA (human DKK-3 DuoSet; DY1118, R & D Systems) was used according to the manufacturer’s guidelines.
Immunofluorescence and confocal microscopy
Cells were seeded on collagen-coated eight-well culture slides (Falcon BD Labware) and incubated with 10 nM of Aplidin, PM01215 and PM02781 for 5 h. Living cells were stained with CellRox®Green reagent to monitor intracellular oxidative stress, and nuclei were stained with NucBlue (Molecular Probes, Life Technologies) according to the manufacturer’s protocol. Confocal microscopy was performed with a spinning disc confocal microscopic system (Ultra VIEW VoX; Perkin Elmer, Waltham, MA, USA) that was connected to a Zeiss AxioObserver Z1 inverted microscope (Zeiss). Images were acquired with Velocity software (Perkin Elmer) using a 63x oil immersion objective with a numerical aperture of 1.42.
Flow cytometry
Cell death was evaluated by human FITC-labeled Annexin V (Enzo®) and 7-amino-actinomycin D (7-AAD, Beckman Coulter) staining. Therefore, cells were resuspended in 200 μL Annexin V Binding Buffer (Abcam) with 2 μL Annexin V and 2 μL PI (20 μg/mL), incubated for 15 min, washed and resuspended in PBS/ 5 % FCS prior to analysis. Cells were examined in the FACSCalibur (Becton-Dickinson, Heidelberg, Germany). Cell cycle analysis was performed with the Coulter DNA PREP Reagent kit (Beckman Coulter).
Primary cells were all characterized by staining with a panel of antibodies and flow cytometric analysis (Additional file 1: Table S1). Therefore, we used anti-human EpCAM/TROP1 Phycoerythrin MAb (Clone 158206), anti-human CD31/PECAM-1 APC MAb (Clone 9G11), anti-human VEGF R2/KDR Phycoerythrin MAb (Clone 89106), anti-human CD45 PerCP MAb (Clone 2D1) and anti-human CD14 Fluorescein MAb (Clone 134620, all from R&D systems).
Quantitative RT-PCR analysis
Total RNA was isolated from HUVECs using TRI Reagent (Sigma -Aldrich), according to the manufacturer’s instructions. RNA was purified by cell lyses and nucleic acid extraction using the RNeasy Kit (Qiagen). Thereafter, genomic DNA in the RNA samples was digested with the RQ1 DNAse (Promega). The cDNA was amplified from 1 μg total RNA using the SuperScript II Reverse Transcriptase Kit (Invitrogen Life Technologies). For validation, real time RT-PCR was performed using a SensiMix SYBR No-ROX Kit (Bioline) and a Rotor-Gene 6000 detection system (Corbett Research). Primers were designed to amplify specific GAPDH (for: 5-ctgacctgccgtctagaaaa; rev: 5-gagcttgacaaagtggtcgt), TATA Box Binding Protein (for: 5-ggagccaagagtgaagaaca; rev: 5-agcacaaggccttctaacct), DKK3 (for: 5- tcatcacctgggagctagag, rev: 5-caacttcatactcatcgggg); VASH1 (for: 5-agatccccataccgagtgtg, rev: 5-gggcctctttggtcatttcc), p16INK4A (for 5-caacgcaccgaatagttacg, rev: 5-agcaccaccagcgtgtc), p27KIP1 (for 5-gccctccccagtctctctta, rev: 5-tcaaaactcccaagcacctc), TP53 (for 5-gttccgagagctgaatgagg, rev: 5-ttatggcgggaggtagactg), X-Box Binding Protein 1 XBP1u (for: 5- agtccgcagcactcagac; rev: 5-gaactgggtccttctgggtag) XBP1s (for: 5- agtccgcagcaggtgcaggc; rev: 5-gaactgggtccttctgggtag), HSP5A (for: 5-ctcgactcgaattccaaaga; rev: 5-aaggggacatacatcaagca), and DDIT3 (CHOP) gene (for: 5-cctcctggaaatgaagaggaaga; rev: 5-tcctggttctcccttggtct).
Real time cell proliferation assays
Real time cell proliferation experiments were performed using the RTCA DP instrument (Roche Diagnostics GmbH), which was placed in a humidified incubator maintained at 5 % CO2 and 37 °C. For proliferation assays, cells were seeded in complete medium in 16-well plates (E-plate 16, Roche Diagnostics GmbH) at a density of 2000 cells/well after coating with 10 μg/mm2 fibronectin (Sigma Biochemicals). The plate containing gold microelectrodes on its bottom was monitored every 10 min for 4 h (adhesion process), then once every 30 min, until the end of experiment, for a total of 72 h. Data analysis was performed using RTCA software 1.2 supplied with the instrument.
Capillary tube formation and angiogenic sprouting assays
Cells were incubated for 12 h with 10 nM of the respective compounds. To analyze tube formation, 24-well plates were coated with 200 μL growth factor-reduced matrigel (BD Biosciences). HUVECs were resuspended in 200 μL EGM-2 medium (1 × 105 cells) containing 10 nM of the respective compound and placed on top of the polymerized matrix; tube formation was observed after 6 hours. Tubes were viewed under an inverted transmission microscope (Zeiss Axiovert 200 M) and documented with a digital imaging system (Axiovision Software, Zeiss).
For sprouting assays HUVEC spheroids were generated overnight in hanging-drop culture consisting of 400 cells in EBM-2 medium, 2 % FCS and 20 % methylcellulose (Sigma Biochemicals). Spheroids were embedded in collagen type I from rat tail (Becton Dickinson) and stimulated with 50 ng/ml VEGF (Sigma Biochemicals) in the presence or absence of compounds or control substances (DMSO, bortezomib). Sprouts were also analyzed by inverted transmission-microscopy (Zeiss Axiovert 200 M) and documented by a digital imaging (Axiovision Software, Zeiss). The cumulative sprout length (CSL) was analyzed after printing of high quality pictures and counting by two independent blinded observers.
Chicken chorioallantoic membrane (CAM)
Fertilized chicken eggs (Gallus domesticus, Charles River) were placed in a 75–80 % humidified 37 °C incubator (Grumbach) to allow normal embryo development. On day three eggs were opened, egg shells removed and embryos were placed in a sterile Petri dish in an egg incubator to induce CAM development. On day 8, when CAM and its vasculature were well developed, all experiments were performed. Subsequently, two rings per chicken were grafted on the CAM. Drugs (10 nmol/ring) with VEGF (1 μg/ring) or drugs alone were applied every second day at the center of Permanox™ rings.
On day 6 post-grafting chicken embryos were sacrificed by hypothermia, blood vessels in the ring area were photographed by stereo microscope (Olympus SZW 10) and vessel density was determined by counting with Photoshop CS4 (Adobe).
Human tumor xenograft model in the CAM
OPM-2eGFP multiple myeloma cells (2.5 × 105) were mixed with rat-tail collagen and human mesenchymal stromal cells (0.5 × 105) and the 1 nmol of the respective compounds. Collagen drops (30 μl) were placed on parafilm for 30 min to allow polymerization of the extracellular matrix at 37 °C. Then onplants were transferred to the CAM of 7-day-old chick embryos. After 5 days of in vivo growth, onplants were documented by the Olympus SZX10 stereomicroscope (Olympus) equipped with an Olympus DFPL 2-4x objective lens connected with a digital camera (Olympus E410) and flexible cold light (KL200; Olympus). Excised xenografts were transferred into 0.5 ml RIPA Buffer (Sigma Aldrich, Linz, Austria) and homogenized with an Ultra Turrax homogenizer three times for 5 s on ice. Thereafter, homogenate underwent three freezing/thawing-cycles in liquid nitrogen and 37 °C water bath. After centrifugation, supernatants were diluted in assay buffer. GFP levels were measured by Cell Biolabs’ GFP ELISA Kit (San Diego, CA, USA), using biotinylated anti-GFP antibodies, according to the manufacturer’s protocol.
Senescence-associated beta galactosidase (SA-β-gal) activity assay
Cells were fixed (2 % formaldehyde, 0.2 % glutaraldehyde in PBS) for 5 min at room temperature and rinsed several times in PBS. To measure SA-β-gal activity, cells were incubated in a staining solution (4.2 mM citric acid, 12.5 mM sodium phosphate, 158 mM sodium chloride, 0.21 mM magnesium chloride, 2.21 mg/ml potassium ferrocyanide, 1.68 mg/ml potassium ferricyanide, 1 mg/ml X-Gal, pH 6.0) at 37 °C for 24 h. Cells were washed and embedded in PBS, viewed in an inverted transmission microscope and photographed (Zeiss Axiovert 200, Axiovision software).
Statistical analysis
Statistical analyses were performed with the GraphPad Prism™ software for Windows. Unpaired t-test was used to study differences between the means of one treatment group and control. The average scores across treated groups were not compared. Statistical analyses of quantitative PCR data were performed according to the delta Ct method described by Pfaffl et al. [17].