FH535 increases the radiosensitivity and reverses epithelial-to-mesenchymal transition of radioresistant esophageal cancer cell line KYSE-150R

Human esophageal squamous cancer cell line KYSE-150 were purchased from the American Type Culture Collection (Manassas, VA). Radioresistant cell line KYSE-150R was previously established in our department with gradient dose irradiation [18,19]. Both KYSE-150 and KYSE-150R were cultured in RPMI-1640 (Gibco, Life Technologies Inc., Grand Island, NY) with 100 unit/ml of penicillin, 100 mg/ml of streptomycin, and 10% fetal bovine serum at 37°C in a humidified incubator containing 5% CO₂. The cell lines were sub-cultured every 2 to 3 days following digestion at room temperature with 0.5 ml trypsin/EDTA per well (Sigma-Aldrich Ltd, UK). The viability was reported as the percentage of the viable cells number to the total cells number. There was an average viability of over 95% determined by Trypan Blue staining. FH535 was purchased from Calbiochem (merck), and dissolved in DMSO at a concentration of 10 mM.

Total proteins were extracted from the two cells groups (KYSE-150 and KYSE-150R) in the exponential growth phase using the cell lysis buffer, which containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail (Merck Millipore, Darmstadt, Germany). Nuclear and cytoplasmic proteins were isolated using Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech, Shanghai, China). Protein content was measured using the Bradford protein assay. Samples were denatured at 100°C for 10 minutes. Thirty microgram proteins were separated by 8% or 10% SDS polyacrylamide gels and transferred to nitrocellulose membrane (0.45 μm; Bio-Rad). Incubation with 5% nonfat dry milk in TBS-Tween 20 (TBST) for 2 h at room temperature was conducted to block nonspecific binding sites. Subsequently, membranes were incubated with rabbit antibodies against β-catenin (H-102) (1:500) (ab2365, Cambridge, Mass., USA), Beta-Catenin Phospho (pS33/S37) (1:1,000) (9561 s, Cell Signaling Technology Danvers, USA), GSK3β (1:1,000) (1561–1, Epitomics, Inc., Burlingame, Calif., USA). P-GSK3β (1:1,000) (2435–1, Epitomics, Inc., Burlingame, Calif., USA)., Slug (1:1,000) (T0587, Epitomics, Inc., Burlingame, Calif., USA).E-Cadherin (1:1,000) (ab53033, Cambridge, Mass., USA), Vimentin (R28) (1:1,000) (#3932, Cell Signaling Technology Danvers, USA) and Cyclin D1 (1:1,000) (#2922, Cell Signaling Technology Danvers, USA) overnight at 4°C. Incubation with mouse antibodies against β-actin (1:10,000) (ab8226, Cambridge, Mass., USA) and GAPDH (1:4,000) (sc-32233, Santa Cruz, Calif., USA) were conducted for normalization control. After washing, membranes were again incubated with HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, 1:5,000) for 1 hour at room temperature. Membranes were then treated with enhanced chemiluminescence (ECL, Pierce, Thermo, USA) after washing. Proteins were visualized with ECL and Kodak film exposed for one minute without light. The relative band intensity was determined by Gel-Pro Analyzer 3.1 software (Media Cybernetics).

Total RNA was extracted from the two cell groups (KYSE-150 and KYSE-150R) using Trizol Reagent (Invitrogen) according to the manufacture’s protocol and reversely transcribed into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Thermo Scientific, USA). SYBR Green (ABI) PCR was performed in triplicate using the ABI PRISM 7300 Sequence Detection System. All samples were normalized to the signal generated from β-actin and GAPDH (Sangon Biotech, Shanghai, China). Primer sequences of Wnt pathway and EMT associated genes were presented in Table 1 Data was shown as fold change (2^−ΔΔCt) and analyzed initially using Opticon Monitor Analysis Software V2.02 (MJ Research, Waltham, MA, USA). Triplicates were run for each sample in three independent experiments.

KYRE-150R Cells were grown on covered slips and treated with 20 μM FH535 (purchased from Calbiochem and dissolved in DMSO) for 24 h. After washing three times with PBS, cells were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 20 min at room temperature. The fixed cells were permeabilized with PBS containing 0.3% Triton X-100 (Solarbio, Beijing, China) for 20 min. Then, the cells were soaked in 10% normal goat serum (Solarbio, Beijing, China) with PBS for 30 min. The cells were then incubated with various primary antibodies (β-catenin, E-cadherin, Vimentin,1:200 dilutions) overnight at 4°C. After rinsing in PBS, the cells were then incubated with Alexa Flura® 488 conjugated, goat anti-rabbit IgG (H + L) (Thermo Scientific, Waltham, USA) at 1: 1000 dilution for 1 hr at 37°C at a dark condition. The nuclei were visualized by 1 μg/ml DAPI (Beyotime, Shanghai, China) and incubate for 5 min at room temperature. Images were obtained using a DM4000B Automated Upright Microscope System (Leica, Wetzlar, Germany).

KYSE-150R cells, 1x10E3 cells/well, were seeded in a 96-well plate. After an overnight incubation, the cells were subcultured in three groups including: FH535 treated in different concentrations (5 μM, 10 μM, 15 μM, 20 μM), DMSO treated with the same volume as FH535 for 24 hours, and irradiation treated with a dose of 8 Gy high energy X-ray from a linear accelerator (Vairan 2300C/D, Salt Lake, USA). Cell counting kit-8 (CCK-8) assays (Dojindo) were performed following the protocol of the kits. Results were measured according to the absorbance at 450 nm using a microplate reader (Thermo Scientific Varioskan Flash, USA).

KYSE-150R cells, 1 × 10³ cells/well, in the exponential growth period were seeded in a 6-well plate for 24 h. FH535 was added with a concentrations of 20 μM for the following 24 hours. Then, the radioresistant KYSE-150R cells were irradiated by doses of 0, 2, 4, 6, 8, or 10 Gy with X-ray from a linear accelerator (Vairan 2300C/D, Salt Lake, USA) at an average dose rate of 100 cGy/min. Immediately following the irradiation, the cells were incubated for 10 days at 37°C in a 5% CO₂ environment to allow the colony formation and then fixed with pure ethanol. Visible colonies consisting of at least 50 cells were stained with 0.5% crystal violet (Sigma, Germany) and counted. The surviving fraction (SF) were estimated.

Neutral comet assay method was used to detect the DNA fragmentation associated with apoptosis. KYSE-150R cells were seeded at 2 × 10⁵ cells/well into 6-well plates and incubated overnight. From the three cell samples, two samples were then treated with 20 μM FH535 and DMSO for 24 hours, respectively. The other KYSE-150R sample were treated with 8 Gy irradiation. The Cells were collected and suspended in PBS with a concentration of 5 × 10⁵ cells/mL. To prepare the slide, the cell suspension (10 μl) was mixed with 90 μl of 0.75% low temperature melting agarose (LMA) in PBS at 37°C and pipetted onto fully frosted slides, which precoated with a layer of 100 μl 1% normal temperature melting agarose (NMA) in PBS. After gelling for 10 minutes at 4°C, the coverslip was gently removed. All the processes were conducted at a dark condition. The slides were placed in precooled lysis solution (2 M NaCl, 30 mM Na₂EDTA, 10 mM Tris, 1% N-Lauroylsarcosine Sodium Salt, pH 8.2-8.5, supplemented with 10% DMSO and 1% Triton X-100) at 4 °C for 2 hours, and then placed in TBE (0.4 mM Na2EDTA, 18 mM Tris, 18 mM boronic acid, pH 8.2-8.5) at 4°C for 30 minutes twice. Electrophoresis was performed for 20 minutes at 25 V (10 mA) in TBE solution after unwinding. Slides were stained with propidium iodide (PI) (5 ug/ml) for 10 minutes and covered with a coverslip. Microscopic analysis were conducted using a fluorescence microscope (Leica DM2500, Germany) equipped with a CCD camera. Non-overlapping cells were captured at 400 magnification. At least 100 cells per slide were analyzed using the Comet Assay Software Project (CASP). A measurement of olive tail moment (OTM) was used to quantify the extent of DNA damage.