B88 and KOSC-2 cells are human squamous cell carcinoma (SCC) cell lines established from SCCs of the tongue . B88 was a generous gift from the Kanagawa Cancer Center Research Institute (Kanagawa, Japan). KOSC-2 was purchased from the JCRB Cell Bank (Osaka, Japan). In this study, both cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cultures were maintained in a humidified incubator at 37°C in 5% CO2.
Drug screening was performed using the DiscoveryPak HDAC Inhibitor Set (splitomicin, apicidin, valproic acid, trichostatin A, 4-phenyl butyric acid [4-PBA]) obtained from BioVision (Milpitas, CA, USA). Cetuximab was purchased from Merck Serono (Burlington, MA, USA). B88 and KOSC-2 cells were plated at 10,000 cells/well in a 96-well plate. After incubation for 24 h, HDACi, cetuximab (500 mg/ml), or both were added. After 24 h of incubation, the cell toxicity was evaluated using a Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan). Absorbance at 450 nm was measured using a microplate reader (Thermo Scientific Multiskan FC, Thermo Fisher Scientific Life Sciences, Waltham, MA, USA).
RNA extraction and quantitative RT-PCR
The EGFR mRNA levels were determined at 24 h and 48 h post-treatment with 4-PBA (5 mM), cetuximab (500 mg/ml), or both by RT-qPCR. Total RNA was extracted from B88 cells using TRIzol reagent (Invitrogen, La Jolla, CA, USA) according to the manufacturer’s protocol. Two micrograms of cellular RNA were converted to cDNA following the manufacturer’s instructions for the specific reverse transcription kit (Invitrogen).
Quantitative RT-PCR was performed using primers specific for EGFR (Forward: 5'-AAGTGTAAGAAGTGCGAAGG-3'. Reverse: 5'-GGAGGAGTATGTGTGAAGGA-3'.). The degree of expression was quantified using a relative standard curve and normalized to GAPDH that was amplified using specific primers (Forward: 5'-GTCAACGGATTTGGTCGTATT-3'. Reverse: 5'-GATCTCGCTCCTGGAAGATGG-3'.).
Cell viability, cytotoxicity, and caspase 3/7 activities
Cell viability and caspase 3/7 activities were evaluated in B88 cells treated with 4-PBA (5 mM), cetuximab (500 mg/ml), or both using the ApoToxGlo Triplex Assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The ApoToxGlo Triplex Assay combines three assay chemistries to assess viability, cytotoxicity, and caspase activation events in the same cell-based assay. Briefly, 10 µl of viability/cytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110 substrates were added to all wells. After incubation for 1 h at 37°C, the Relative Fluorescence Units (RFU) was determined at 370 Ex/535 Em for cell viability and at 485 Ex/535 Em for cytotoxicity. To determine the effect on caspase-3/7 activity of B88 cells treated with 4-PBA and cetuximab, 10 µl of luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD were added to all wells. The Relative Luminescence Units (RLU) from the caspase-3/7 activated was measured after 30 min of incubation. The fluorescence and luminescence were detected using a FilterMax-F5 (Molecular Devices LLC, Sunnyvale, CA, USA).
TdT-mediated dUTP-biotin nick end labeling assay
For the in situ analysis of DNA fragmentation, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using an ApopTag peroxidase in situ apoptosis detection kit (Merck Millipore, MA, USA) according to the manufacturer's instructions. The apoptotic index (average percentage of apoptotic cells labeled by the TUNEL method) was determined as the number of DAB-stained cells versus the total number of cells using light microscopy. A minimum of 1,000 cells were counted in random fields.
Cells were lysed in a radioimmunoprecipitation assay buffer 24 h after drug treatment. The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples were run on sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and detected by ECL chemiluminescence, and images were captured with ImageQuant LAS-500 analyzer (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Primary antibodies against the following proteins were used: EGFR, p-EGFR, ERK, Akt, p-AKT, cleaved-PARP, and GRP78 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); B-cell lymphoma 2 (bcl-2), CCAAT/enhancer-binding protein homologous protein (CHOP), and p-ERK (Cell Signaling Technology, Beverly, MA, USA); H3K9,14ac, H3K27ac, and H4K16ac (MAB Institute, Nagano, Japan); β-actin (Sigma-Aldrich, St. Louis, MO, USA).
All experiments were performed in triplicate and the values represent the average of at least three independent experiments. We expressed all data as mean ± standard deviation. One-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test was used to analyze the differences between the combination of cetuximab and HDACi vs. individual treatments. p Values < 0.05 were considered significant. We used SPSS software for Windows (V26.0; SPSS Inc., Chicago, IL, USA) for all statistical analyses.