Cell cultivation and treatments
Experiments were performed on colorectal carcinoma cell lines DLD1 (CCL-221, ATCC, Sigma-Aldrich, St. Louis, MO, USA) and HCT116 (CCL-247, ATCC, Sigma-Aldrich), line derived from clear cell renal cell carcinoma (ccRCC) (ECACC, 03112702, Sigma-Aldrich) and non-cancerous cell line derived from epithelial cells – EA.hy926 (ATCC, CRL-2922TM). Cell lines were cultured in RPMI medium (Sigma-Aldrich) or Dulbecco Minimal Essential Medium (DMEM; Sigma-Aldrich) with a high glucose (4.5 g/L) and L-glutamine (300 μg/mL), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and penicillin/streptomycin mixture (Calbiochem, San Diego, CA, USA; penicillin 100U/mL; streptomycin 100 μg/mL). Cells were treated with paclitaxel (PTX; Selleckchem, Pittsburgh, PA, USA; 20 nmol/L), vincristine sulfate salt (Vin; Sigma-Aldrich; 100 nmol/L) a slow-releasing sulfide donor GYY4137 (GYY; Cayman Chemical, Ann Arbor, MI, USA; 10 µmol/L), for 24 h.
Generation of CBS-knockout DLD1 cell line
CBS-knockout DLD1 cell line, hereafter referred DLDx, was established by using the CRISPR/Cas9 (CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9)) gene editing method. The CBS CRISPR guide RNA sequences (GATTTCGTTCTTCAGCCGCC and TGTGCCCTCAGGGATCGGGC) were designed by the laboratory of Feng Zhang at the Broad Institute in order to efficiently target the CBS gene with minimal risk of off-target Cas9 binding elsewhere in the genome [25, 26]. Lentiviral transfer plasmids lentiCRISPRv2_CBS-1 and lentiCRISPRv2_CBS-3 (GenScript, Leiden, Netherlands) contained a lentiCRISPRv2 backbone and single above-mentioned oligos cloned into the single guide RNA (sgRNA) scaffold. To produce lentiviral particles, transfer plasmids lentiCRISPRv2_CBS-1 or lentiCRISPRv2_CBS-3 were co-transfected into HEK293T cells with the packaging plasmids pMD2.G (Addgene, Watertown, MA, USA) and psPAX2 (Addgene). Virus-containing medium was collected after 48, 60, and 72 h and passed through a 0.45 μm low protein-binding filter. Lentiviruses were concentrated using PEG 6000 and sedimented by centrifugation (1500 × g, 4°C for 30 min). As a positive control to monitor transduction efficiency, CRISPR-lenti human EMX1 positive control transduction particles (CRISPR11V-1EA, Sigma-Aldrich) were used. Similarly, as a negative control, CRISPR-lenti non-targeting control transduction particles (CRISPR12V-1EA, Sigma-Aldrich) were used. This control includes a guide RNA sequence that does not target known human, mouse and rat genes. DLD1 cells, plated the day before at a density of 0.25×105 cells per 6 cm plate, were infected with each lentivirus, or their combination. DLD1 cells transducted by control lentivirus particles were called DLD-PC (positive control) and DLD-NC (negative control). Twenty-four hours after transduction, cells were selected by puromycin (Puromycin, InvivoGen, USA) and then the CBS protein knockout was confirmed by immunofluorescence (IF) and Western blot analysis (WB).
Cells grown on glass coverslips were fixed in ice-cold methanol. Nonspecific binding was blocked by incubation with PBS containing 3% bovine serum albumin (BSA; Sigma- Aldrich) for 60 min at a room temperature. Cells were then incubated with primary antibodies diluted in PBS with 1% BSA (PBS-BSA) for 1 h at 37°C. The antibody specific to human CBS (1:100 dilution, AP6959c, Abgent, San Diego, CA, USA), the anti-beta tubulin antibody (1:1000 dilution, ab231082, Abcam, Cambridge, UK) and the anti-beta actin antibody (1:250 dilution, ab6276, Abcam) were used. Afterwards, cells were washed four-times with PBS with 0.02% TWEEN (Sigma-Aldrich) for 10 min, incubated with Alexa Fluor-594 goat anti-mouse/anti-rabbit (1:1000 dilution, Thermo Fisher Scientific, Waltham, MA, USA) or IgG Alexa Fluor-488 donkey anti-rabbit IgG (1:1000 dilution, Thermo Fisher Scientific) in PBS-BSA for 1 h at 37°C, and washed as described previously. Finally, cover-slips were mounted onto slides in mounting medium with a blue-fluorescent DNA stain 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Cells were visualized by epifluorescence microscopy using Nikon Eclipse Ti-S/L100 (Nikon, Japan); NIS elements software (Nikon, Tokyo, Japan) was used to process images and to evaluate the resultant pictures.
Proximity Ligation Assay
The proximity ligation assay (PLA) was used for in situ detection of the co-localization between CBS and b-tubulin or b-actin. The assay was performed in a humid chamber at 37°C according to the manufacturer’s instructions (Olink Bioscience, Uppsala, Sweden). Cells were seeded on glass coverslips and further cultured for 24 h. Afterwards, cells were fixed with methanol, blocked with 3% PBS-BSA for 30 min, incubated with a mixture of antibodies against CBS and b-tubulin or b-actin for 1 h, washed three times, and incubated with plus and minus PLA probes for 1 h. Then, the cells were washed (3x5 min), incubated for 40 min with ligation mixture containing connector oligonucleotides, washed again, and incubated with amplification mixture containing fluorescently labeled DNA probe for 100 min. After a final wash, the samples were mounted and the signal was analyzed using a Zeiss LSM 510 Meta confocal microscope with a Plan Neofluar 40_/1.3 oil objective. The following antibodies were used: human CBS (1:100 dilution, AP6959c, Abgent), the anti-beta tubulin antibody (1:1000 dilution, ab231082, Abcam) and the anti-beta actin antibody (1:250 dilution, ab6276, Abcam).
Western blot analysis
Cells were scraped into 10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L phenylmethyl- sulfonyl fluoride (PMSF, Serva, Heidelberg, Germany), protease inhibitor cocktail tablets (Complete EDTA-free, Roche Diagnostics, Indianapolis, IN, USA) and centrifuged for 5 min at 3000 x g at 4°C. Pellet was re-suspended in Tris-buffer containing the 50 µmol/L CHAPS (3-[(3-cholamidopropyl) dimethylammonio] 1-propanesulfonate, Sigma-Aldrich), and then incubated for 30 min at 4°C. Lysate was centrifuged for 15 min at 10 000 x g at 4°C. Protein concentration was determined by Modified Lowry Protein Assay Kit (Thermo Scientific). Fifteen to forty micrograms of protein extract from each sample was separated by electrophoresis on 4-20% gradient SDS polyacrylamide gels. Afterwards, proteins were transferred to Hybond PVDF blotting membrane (GE Healthcare, Life Sciences, Chicago, IL, USA) using semidry blotting (Owl, Irvine, CA, USA). Membranes were blocked in 5% non-fat dry milk in TBS-T (Tris-buffered Saline with Tween-20) for overnight at 4°C and then incubated for 1 h with primary antibody β-actin (1:5000 dilution, ab6276, Abcam), antibody β-tubulin (1:1000, ab108348, Abcam), or membranes were blocked in 5% non-fat dry milk in TBS-T or 5% BSA in TBS-T for 1 h at room temperature and then incubated overnight at 4°C with appropriate primary antibodies: CBS (1:1000 dilution, ab144600, ab135626, Abcam), p53 (1:1000 dilution, ab154036, Abcam) and p53 phospho S20 (1:1000 dilution, ab157454, Abcam). After washing, membranes were incubated with secondary antibodies to mouse (secondary goat anti-mouse antibody; 1:10 000 dilution, ab6789, Abcam) or rabbit (secondary goat anti-rabbit antibody; 1:10 000 dilution, ab97200, Abcam) IgG conjugated to horseradish peroxidase for 1 h at room temperature. For visualization, chemiluminescence detection system (Luminata™ Crescendo Western HRP Substrate, Millipore, Burlington, Mass., USA) was used. Each membrane was digitally captured using an imaging system (C-DiGit, LI-COR).
Appropriate antibody (anti-human CBS, ab135626, Abcam) was incubated with 60 µL washed magnetic beads (Dynabeads M-280), coated with M-280 sheep anti-rabbit IgG (Invitrogen Dynal AS, Oslo, Norway) for overnight at 4°C on a rotator (VWR International, Radnor, PA, USA). As negative controls, the coated beads were incubated with either mouse IgG1_ (MOPC21, Sigma, USA), or with rabbit - globulin (Jackson ImmunoResearch, West Grove, PA, USA). The beads with attached antibody were washed (twice, 200 µL) with phosphate-buffered saline (PBS). Proteins were immunoprecipitated from 0.5 mg of detergent-extracted total protein by incubation for 4 h at 4°C with antibody-bound beads. Bead complexes were washed four times with PTA solution (145 mmol/L NaCl, 10 mmol/L NaH2PO4, 10 mmol/L sodium azide, and 0.5% Tween 20, pH 7.0). Immunoprecipitated proteins were then extracted with 60 µL of 2x Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and boiled for 5 min.
Relative viability of the cells was determined by the CellTiter-Glo™Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) on 96-well plate using 3000 cells per well and evaluated by the LumiStar GALAXY reader (BMG Labtechnologies, Germany) after 4 days from plating and treatment with GYY4137. Experiments were performed in octaplicates, repeated at least three times from different cultivations. Values were expressed as means ± S.E.M.
Cell migration assay
Fifty thousand DLD1, DLDx, DLDNC and DLDPC cells per well were plated on ImageLock 96-well plates (Essen BioScience, Ann Arbor, MI, USA), and let to adhere for 24 h. Confluent monolayers were then wounded with wound needle (IncuCyteWoundMaker; Essen BioScience), washed twice and supplemented with fresh culture medium or fresh culture medium with GYY4137. Images were taken every 2 h for the next 24 h in the IncuCyte ZOOM™ kinetic imaging system (Essen BioScience). Cell migration was evaluated by IncuCyte ZOOM™ 2016A software (Essen BioScience) based on the relative wound density measurements and expressed as means of octaplicates ± S.E.M.
Paraffin-embedded tumor tissue was sliced in 5 μm sections. Tissue was pre-treated in automated water bath at 96°C for 20 min using Dako PT link (Dako, Glostrup, Denmark ). After washing in 1x Envision FLEX wash buffer (Dako), slides were incubated with FLEX peroxidase-blocking reagent (Dako) for 10 min. Sections were incubated with primary anti-human Ki67 (MIB-1 FLEX, Dako) antibody at RT for 20 min. Subsequently, sections were incubated with LSAB2 System-HRP, Biotinylated Link for 20 min and then with Streptavidin-HRP for 20 min at RT. Visualization was performed by incubation with 3,3′-Diaminobenzidine (DAB substrate-chromogen solution, Dako) for 2 min. Nuclei were counterstained with hematoxylin (FLEX, Dako, Denmark) for 5 min at RT. Among incubations, slides were washed in 1x FLEX wash buffer (Dako). The slides were mounted with Dako Faramount Aqueous Mounting Medium (Dako) and the stained sections were then analyzed by Axiovert 40C Zeiss microscope and Zen 2.6 software (Zeiss, Jena, Germany).
In vivo experiments
Animal experiments were approved by the Institutional Ethic Committee and by the national competence authority – State Veterinary and Food Administration of the Slovak Republic (project registration No. Ro-2032-3/2020-220) in compliance with the Directive 2010/63/EU and the Regulation 377/2012 on the protection of animals used for scientific purposes. Project was conducted in the approved animal facility (license No. SK UCH 02017). SCID/bg mice (males, 17 weeks old at the beginning of experiment) were bilaterally subcutaneously injected by 1x106 DLD1, DLDx, DLD-PC or DLD-NC cells resuspended in 100 µL of serum-free cultivation medium. Visible xenografts developed after 4 days. Mice were then randomly divided into either a treatment group (intraperitoneal injection of 20 mg/kg GYY4137 diluted in saline, given daily), or a control group (saline). Xenografts were measured every 3 days with a caliper, and the tumor volume (V) was calculated according to the formula: V=length×width2/2, the width being the greatest transverse diameter and length the greatest longitudinal diameter. Mice were kept on standard pelleted food and water ad libitum, monitored daily for the weight loss or other signs of possible toxic effects of the drug. At the experimental endpoints (after 14 days of therapy) mice were sacrificed. Xenografts were resected, weighed and cryopreserved at -80°C for further experiments or stored in buffered formaline.
The results are presented as mean ± S.E.M. Each value represents an average of at least 3 wells from at least three independent cultivations of each type of cells. Statistical differences among groups were determined by one way ANOVA. For multiple comparisons, an adjusted t-test with p values corrected by the Bonferroni method was used (InStat, GraphPad Software). Statistical significance * or + - p < 0.05 was considered to be significant, ** or ++p < 0.01, *** or +++p < 0.001.