Recombinant human TNF-α was purchased from Peprotech, (Mexico DF, Mexico). DMEM medium, fetal bovine serum, MTT reagent, staurosporine, camptothecin, and propidium iodide were obtained from Sigma (St. Louis, MO, USA). FITC-Annexin-solution was kindly provided by Dr. Gustavo Amarante-Mendes (Department of Immunology, ICB-USP, Brazil). Ac-DEVD-AMC antibody, caspase-3 specific substrate and the pan-caspase inhibitor zVAD-FMK were purchased from Calbiochem (San Diego, CA, USA). Power Sybr®Green master mix was purchased from Applied Biosystems (San Diego, CA, USA). All PCR primers were purchased from BioNEER (Great Seneca Highway Rockville, MD, USA). Recombinant human FAM3B (rhFAM3B) produced in bacterial hosts was obtained from Novus Biologicals (Littleton, CO, USA). Rabbit polyclonal antibodies against Bcl-2, Bax, Bcl-XL, Caspase-3, Caspase-8, and Caspase-9 for western blot analysis were purchased from Cell Signaling (San Diego, CA, USA). Mouse monoclonal anti-β-actin and anti-Bax antibodies and rabbit polyclonal anti-Bcl-2 antibodies for immunohistochemical analyses were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti- PCNA (Proliferating Cell Nuclear Antigen) was obtained from Dako (Glostrup, Denmark). Mouse monoclonal antibody against human FAM3B was obtained from R&D Systems (Minneapolis, MN, USA). Anti-rabbit and anti-mouse IgG HRP-linked antibodies were purchased from Cell Signaling (San Diego, CA, USA). All other chemicals of analytical grade were obtained from E. Merck & Co., Inc. (Darmstadt, Germany). Obatoclax Mesylate (GX15–070), an antagonist of Bcl-2 and ABT-737, a BH3-mimetic pan-inhibitor of Bcl-xL, Bcl-2, and Bcl-w were purchased from Sigma (St. Louis, MO, USA).
Cell culture and treatments
LnCAP, PC-3, and DU145 prostate tumor cells, and the 293 T embryonic kidney immortalized cells were originally obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 10 U mL−1 penicillin, and 10 U mL−1 streptomycin (Life Technologies, Gaithersburg, MD), at 37 °C in a humidified atmosphere containing 5% CO2. For functional assays, DU145 cells were seeded at a density of 5 × 105 cells/mL (except in the MTT assays) and subsequently cultured in presence/absence of several cell death inducers as described below, in order to measure cell viability and cell death.
Cloning of FAM3B cDNA into lentiviral and plasmidial vectors
Human FAM3B was amplified by RT-PCR from human pancreatic islets mRNA. The PCR primers specifically targeting the cDNA encoding the full-length sequence of FAM3B, including BamHI and EcoRI restriction sites (underlined) were as follows: 5’-GGATCCCAAGATCTCCAAGGATTCG-3′ (forward) and 5’-GAATTCTTTTTACAGATGCTTTCAG-3′ (reverse). PCR products were purified from agarose gels after electrophoretic separation and cloned into pGEM-T vector (Promega, Madison, WI). Clones having appropriately sized inserts after digestion with restriction enzymes were sequenced using the T7 promoter primer (ABI PRISM Big Dye Terminator cycle sequencing kit, Applied Biosystems, Branchburg, NJ). Subsequently, this cDNA was cloned into BamHI and EcoRI sites of the pCSC-SP-PW lentiviral mammalian expression vector (Addgene plasmid 12,335, kindly provided by Dr. Ricardo Garcia Correa) to produce pCSC-FAM3B plasmid for lentivirus production. Subsequently FAM3B cDNA was cloned into pcDNA 3.1 mammalian expression vector (Life Technologies, CA, USA) by using the same restriction sites. pcDNA-FAM3B plasmid was used to produce FAM3B secreted protein in conditioned media of 293 T cell cultures.
Recombinant human FAM3B protein assays
In order to verify the effects of exogenous FAM3B on cell viability 1 × 106 DU145 cells were seeded onto 2 cm diameter dishes and treated with recombinant human FAM3B protein (rhFAM3B) obtained in bacterial host. Because TNF-α alone did not induce death in DU145 cells [14, 15], TNF-α-induced activity was enhanced by the addition of cycloheximide (CHX), a protein synthesis inhibitor. Cells were maintained in two experimental conditions: group 1 was cultured under non-stimulated proliferation conditions (DMEM media +0.5% SFB) and group 2 was treated with several TNF-α doses (0–20 ng/mL) + CHX (1 mM). After 12 h cells were treated with rhFAM3B: group 1 cells with several concentrations (0–1000 pM) and group 2 cells only with 1000 pM. Cell viability was monitored by MTT assay after 5 days and 24 h, respectively. All experiments were performed in triplicate and were repeated at least three times independently.
Conditioned media assays
293 T cells were plated at a density of 5 × 106 cells/ in 10-cm-diameter dish and transfected with pcDNA3-FAM3B or pcDNA-empty vectors, in OptiMEM I media using Lipofectamine 2000)® (Life Technologies, CA, USA). Media was changed 4 h after transfection to equal volumes of DMEM media +10% fetal bovine serum (SFB). After 24 h, conditioned media (CM) was harvested, cleared to remove cell debris, and either used immediately or stored at −80 °C. The FAM3B secretion in the CM was verified by western blot. After 12 h in culture, 1 × 106 DU145 cells were treated with CM in several dilutions (1:1, 1:10, 1:100) and cell viability/death was monitored at 24, 48, and 96 h by MTT assay and DNA fragmentation. CM from transfected cells with pcDNA-empty vector was used as control.
Lentivirus-mediated transduction in DU145 prostate tumor cells
Lentiviral particles were produced by using a four-plasmid transfection system (pCSC-FAM3B or PCSC-empty plasmids with three other packaging plasmids) in the 293 T cell line, as described previously [16, 17]. After transient transfection, the lentivirus-containing supernatants were collected, filtered, and concentrated by centrifugation. DU145 cells were infected with lentivirus particles in the presence of 4 mg/mL polybrene (Sigma, St. Louis, MO, USA). After infection, expression of FAM3B was confirmed at mRNA and protein levels by qRT-PCR and western blot, respectively. DU145 cells infected with virus carrying empty vector were used as control in all subsequent experiments.
DU145 cells were seeded on 96-well microtiter plates (1 × 104/well) and treated with following death inducers: a) 2 ng/mL TNF-α + 1 μM cycloheximide (CHX), b) 1 μΜ staurosporine (STS), c) 20 nM camptothecin (CMP), d) 50 μM Etoposide (ETO), and e) serum starvation medium (0.05% SFB). Cell treatment at various times was followed by addition of 0.05 μg/mL of 3 (4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) (MTT) and 2 h incubation at 37 °C. After a brief centrifugation, supernatant was carefully removed and 100 μL DMSO was added to each well. After insoluble crystals were completely dissolved, absorbance at 570 nm was read on a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA).
DU145 cells were seeded on 6-well microtiter plates (3 × 105/well) and treated with 0.2, 2.0, and 20 ng/mL TNF-α + 1 μM CHX. After 48 h of treatment cell culture medium was collected and centrifuged to harvest floating dead cells. The adherent cells were harvested by trypsinization and mixed with the previously collected cells. After centrifugation at 1000 g × 5 min, cells were rinsed twice with phosphate-buffered saline (PBS) and lysed in a hypotonic buffer, pH 7.4, containing 50 μL/mL propidium iodide (PI) (Invitrogen), sodium citrate 0.1% m/v, and Triton X-100 0.1% m/v. Cellular DNA fragmentation was measured on a FACS-Calibur ® flow cytometer using the CellQuest ® program (BD, San Jose, CA, USA).
After harvesting, approximately 1 × 106 DU145 cells were washed once with ice-cold PBS followed by annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). Cells were then resuspended in binding buffer and incubated with the fluorescein isothiocyanate-labeled annexin-V solution (FITC-annexin) (1:500 V/V in binding buffer) and 10 μg/mL PI for 15 min at room temperature in the dark. Cells were washed again with annexin-V binding buffer and immediately analyzed by flow cytometry.
RNA extraction and cDNA synthesis
Total RNA extracted from clinical samples of prostate adenocarcinoma tissues (N = 19) and their control non-neoplasic tissue (N = 28) were kindly provided by Dra. Dirce Carraro from the A.C. Camargo Hospital Biobank. All samples have signed informed consent for use in research, provided and approved by patients and approved by the institutional research ethics committees. Total RNA from dissected tumors of xenotransplanted mice (1–2 mg) and cell cultures (3 × 105 cells/mL) was extracted with TRIzol™ method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quality assessment of mRNA was made by agarose gel analysis of 18S and 28S rRNA bands integrity. 1–2 μg of RNA per sample were treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) and cDNA was synthesized by using the SuperScript™ III Reverse Transcriptase kit (Invitrogen, CA, USA), with random and OligodT primer mixture, according to the manufacturer’s instructions.
Real-time quantitative RT-PCR (qRT-PCR)
Real-time PCR was performed according to the Power Sybr®Green protocol (Applied Biosystems), using the Sequence Detector ABI PRISM 5700, (Perkin-Elmer/Applied Biosystems, Foster City, CA). The nucleotide sequences specific for qRT-PCR gene amplification were as follows: FAM3B 5’-CCAAAATCCCTGCTCTTCATG-3′ (forward) and 5’GCATTCTTGGCATCGTTATTCA-3′ (reverse); Bcl-2 5’-CTGGGATGCCTTTGTGGAA-3′ (forward) and 5’-CAGCCAGGAGAAATCAAACAGA-3′ (reverse); Bax 5’CAAGAAGCTGAGCGAGTGTC-3′ (forward) and 5’-GAAGTTGCCGTCTGCAAACA-3′ (reverse); Bcl-XL 5′- CAGACCCAGTGAGTGAGCAG-3′ (forward) and 5’CCGGTTGCTCTGAGACATTT-3′ (reverese); HPRT 5’-GAAGGTCTTGCTCGAGATGTG-3′ (forward) and 5’-TCCAGCAGGTCAGCAAAGAAT-3′ (reverse). The primers were designed to span an intron within the cDNA sequence target, making the cDNA amplification product easily distinguishable from the genomic product. We used a 2-step amplification protocol with a denaturation temperature of 95 °C and an annealing-extension temperature of 60 °C. Relative gene expression was calculated from cycle threshold values (Ct) using the formula 2–ΔΔCt . The human HPRT gene was used as internal control for each individual sample gene expression.
Approximately 5 × 105 DU145 cells were resuspended in hypotonic lysis buffer (10 mM HEPES, 50 mM NaCl, 2 mM EDTA, 5 mM DTT, 0.1% CHAPS, 1 mM PMSF, and 10% Sucrose, pH 7.4) and stored at –80 °C. The lysate was then subjected to four freeze-thaw cycles before centrifugation at 10,000 g for 10 min. Protein concentrations were determined in the supernatants by the Bradford colorimetric assay (Biorad, USA). The reaction was started at 37 °C by incubating 100 μg of total protein with caspase-specific substrates (caspase-3, DEVD-AFC; caspase-8, IETD-AFC; caspase-9: LEHD-AFC) in assay buffer (0.05 M HEPES, pH 7.4, 10 mM DTT, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose) and allowed to incubate at 37 °C for 45 min. Protease activity was monitored every 30 s within a total period of 30 min at an excitation wavelength of 400 nm and an emission wavelength of 510 nm using a SpectraMax Gemini XPS spectrofluorometer (Molecular Devices, CA, USA). Caspase activity was calculated per microgram of protein, and expressed as a percentage of control activity. Assays were performed in the presence or the absence of the pan-caspase inhibitor 1 μM ZVAD-FMK as control for broad specificity of caspase activity.
Bcl-2 protein family inhibition assays
We performed inhibition assays for Bcl-2 and Bcl-XL in order to verify the contribution of these proteins to the anti-apoptotic effect of FAM3B in DU145 cells. We used two specific inhibitors to perform the experiments: a) Obatoclax Mesylate (GX15–070), an antagonist of Bcl-2, that also acts upon Mcl-1; b) ABT-737, a BH3-mimetic pan-inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with no inhibition observed against Mcl-1. We used these inhibitors alongside TNF-α (1 and 5 ng /ml) + 1 μM CHX to confirm the role of Bcl-2 and Bcl-X in FAM3B-mediated cell death resistance to TNF-α.
Approximately 1 × 106 cells or 50 mg of tumor tissue were harvested and sonicated in 100 μL of ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 10 mg/mL aprotinin, and 1 mM PMSF). An aliquot was taken for total protein determination according to the Bradford method. Proteins were precipitated by the addition of 3 volumes of cold acetone and pelleted by centrifugation at 12000 g for 10 min. Pellets were solubilized in SDS-β-mercaptoethanol sample buffer by boiling for 4 min. Equal amounts of protein (50–100 μg) were run on 7.5–12% SDS-polyacrylamide gels and electrically transferred to nitrocellulose membranes. After wash, membranes were then incubated overnight with the primary antibodies in PBS with 15% fat-free milk-powder, at working dilutions for the following rabbit polyclonal antibodies against: caspase-3 (1:1000), caspase-9 (1:2000), caspase-8 (1:1000), Bcl-2 (1:200), Bax (1:500) and Bcl-XL (1:500) and mouse monoclonal antibodies against FAM3B (1:500), and β-actin (1:1000). After wash with washing buffer (1X TBS, 0.1% Tween-20) membranes were incubated with secondary antibodies horseradish peroxidase-linked goat anti-rabbit IgG (1:5000) or horseradish peroxidase-linked horse anti-mouse IgG (1:2500) for 2 h at room temperature under agitation. The Enhanced Chemiluminescence System (ECL; Amersham-Pharmacia, Buckinghamshire, UK) was used for detection according to the manufacturer’s instructions. Immunoblot results were quantified by using ImageJ image processing software (freely available at https://imagej.nih.gov/ij/index.html) and normalized by β-actin expression.
Soft agar assays
Cells were plated in 6-well plates using a two-layer soft agar system with 1 × 103 cells per well in a volume of 1 mL per well as described earlier . In brief, a 2.5% agarose stock was prepared in PBS. The bottom 0.6% agar support was prepared in DMEM containing 10% FBS. Cells were harvested, washed, and mixed with the top-agarose suspension at a final concentration of 0.3%, which was then layered onto the bottom agar. The agar plates were incubated at 37 °C changing the medium every 3 days. After 21 days, cells were fixed with 3.7% formaldehyde and then the size and number of colonies was determined using an inverted microscope. All experiments were repeated independently at least three times using triplicate plates.
Xenografts in nude mice
Animal care and experiments were approved by the Animal Care and Use Committee of the Biomedical Institute of São Paulo University, according to the Brazilian Society of Experimental Biology guidelines. Male 6- to 8-week-old nude (nu/nu) mice were obtained from Biomedical Sciences Institute of University of Sao Paulo (Brazil). Exponentially growing 1 × 106 DU145/FAM3B and DU145/control cells were mixed with 200 μL of Matrigel® and injected subcutaneously into the dorsal region of nude mice. After the first week, primary tumor growth was monitored twice a week by measuring the tumor diameter with calipers and calculating tumor volume (mm3) using the standard formula V = (L + W2)/2, where L is the length and W is the width of the tumor mass. Tumors were harvested at varying times thereafter for molecular and immunohistochemical assays.
Histology and immunohistochemistry analyses
Nude mice were sacrificed after 6–8 weeks. Dissected tumors were fixed in formalin (10%) and embedded in paraffin for subsequent histology analysis with haematoxilin-eosin (HE). Immunohistochemistry was performed on 4-μm-thick sections mounted on glass slides precoated with 2% silane. Sections were deparaffinized and rehydrated by conventional techniques, then heated in citrate buffer for antigen retrieval, pretreated with 30% hydrogen peroxide in methanol for endogenous peroxide blockade and preincubated with normal horse serum diluted at 1:70 in nonfat milk at 2% in Tris-buffered saline (TBS) to prevent nonspecific binding. Sections were then incubated overnight at 4 °C with anti-Bax, anti-Bcl-2 antibodies, and anti-PCNA, diluted at 1:100, in a solution containing 1% BSA diluted in TBS. The EnVision Labelled Polymer for peroxydase (Dako Glostrup, Denmark) was used before development with 3-Amino-9-Ethyl-Carbazol (AEC) or DAB substrate. All sections were counterstained with Harri’s hematoxylin and covered with DAKO Glycergel® (Dako Glostrup, Denmark). For negative control experiments, incubation with the primary antibody was not performed.
Data are presented as the mean ± standard deviation (SD). Each experiment was repeated at least three times with triplicate values within each group. Differences between means were analyzed by one-way ANOVA and Student’s t-test. A p value <0.05 was considered statistically significant. Analyses were performed using the GraphPad Prism software version 5.1 (San Diego, CA, USA).