Cell culture and drugs
Human primary cultured normal tongue epithelial cells (NTEC, a kind gift from Dr. Sun’s laboratory and Dr. Zeng’s laboratory) (Wen et al. 2014; Song et al. 2006) were maintained in keratinocyte/serum-free medium (Invitrogen Life Technologies, US). Oral tongue squamous cell carcinoma (OTSCC) cell lines SCC-9 and SCC-25 were purchased from the American Type Culture Collection. Tca8113 and CAL27 were purchased from the Committee of the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (HyClone, UK). ABT-199 (Catalogue No. CT-A199) and cisplatin (Catalogue No. 479306) were purchased from ChemieTek and Sigma, respectively.
Plasmid and siRNA transfection
For Bcl-2 overexpression, OTSCC cell lines were transfected with 1.5 µg pEMD or pEMD-Bcl2 plasmids (a kind gift from Clark Distelhorst) as previously described (Wang et al. 2001). BCL-2 knockdown were carried out in TSCC lines by transfecting with 100 nM scramble siRNA or human BCL-2-specific siRNAs using Dharmafect Transfection Reagent (Dharmacon RNAi Technologies) according to manufacture’s instructions. The target sequences of human BCL-2-specific are siRNA BCL-2: AAC ATC GCC CTG TGG ATG ACT.
Cellular protein were extracted by RIPA lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % Nonidet P-40, 1 mM Na3VO4, 1 mM NaF, and 1× protease inhibitor, Life Technologies Inc, US). Total protein content was measured using the bicinchoninic acid protein assay kit (Thermo Scientific, US). Equal amount of proteins were resolved using denaturing sodium dodecyl sulphate–polyacrylamide gel electrophoresis and analysed by western blot using antibodies against BCL-2 and β-catenin (Cell Signalling Technologies, US).
Real-time PCR analysis
The total RNA of NTEC, SCC-25, SCC-9, CAL27 and Tca8113 cells were isolated by using TRIzol Reagent (Life technologies, CA, US). The cDNA was amplified using a SsoFast EvaGreen Supermix kit (Bio-rad. CA) and real time PCR analysis was then performed using CFX96 RT PCR system (Bio-rad, CA). The primers are for human BCL-2 (5′-CTG CAC CTG ACG CCC TTC ACC-3′ and 5′-CAC ATG ACC CCA CCG AAC TCA AAG A-3′) and β-actin (5′-AAG GAT TCC TAT GTG GGC GAC G-3′ and 5′-GCC TGG ATA GCA ACG TAC ATG G-3′).
Measurement of proliferation and apoptosis
Cells were treated with ABT-199, cisplatin or combination for 3 days. Proliferation activity was measured by the CellTiter 96R AQueous One Solution Cell Proliferation assay kit (Promega, US). To monitor cell density, 1 × 105 cells were seeded on Day 1. The cell density were counted daily for 4 days under microscope. We determined the cell density by using Trypan Blue staining and then hemocytometer. Briefly, we take the average cell count from each of the sets of 16 corner squares and multiply by 10,000; then multiply by 2 to correct for the 1:2 dilution from the Trypan Blue addition.
Apoptotic cells were stained with Annexin V-FITC (Beckman Coulter, France) and analysed on a Beckman Coulter. The percentage of Annexin V-positive cells was determined by using CXP software.
Determination of mitochondrial membrane potential, oxygen consumption rate (OCR), reactive oxygen species (ROS) and cellular ATP level
Cells were treated with ABT-199 for 24 h. For mitochondrial membrane potential measurement, the cells were incubated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1, Invitrogen) for 30 min prior to PBS washing. Labelled cells were resuspended in 500 µL PBS and flow cytometry was conducted on a Beckman Coulter FC500. The mitochondrial membrane potential level was analysed using FlowJo version 7.7.1 (TreeStar, Ashland, OR). OCR was measured using a Seahorse XF24 extracellular flux analyser (Seahorse Bioscience, US) as per manufacturer’s instructions (Varum et al. 2011). Cells in 24-well-plate were equilibrated to the un-buffered medium in a CO2-free incubator and then transferred to the Seahorse XF24 extracellular flux analyser for OCR measurement. To measure ROS levels, cells were stained with redox-sensitive probe Carboxy-H2DCFDA in PBS buffer. Labelled cells were re-suspended in PI buffer and the level of Carboxy-H2DCFDA was measured using Beckman Coulter FC500. ATP levels were measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, US) according to the manufacturer’s instructions.
Tongue cancer xenograft in SCID mouse
SCID mice were purchased from Animal Resources Centre Australia. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of Yangtze University. The SCID mice were inoculated with 1 million SCC-9 cells subcutaneously in the flank. The inoculation volume (0.2 ml) comprised a 50:50 mixture of cells in growth media and Matrigel (BD Biosciences). Tumour length and width were measured every 3 days and tumor volume was estimated by applying the following equation: volume = length × width2/2. When tumors reached approximately 200 mm3, the mice were treated with vehicle control, oral cisplatin at 20 mg/kg, oral ABT-199 20 mg/kg or combination of both for 30 days. Tumor frozen section slides were fixed with 4 % paraformaldehyde (Sigma, US). The nuclei and cytoplasm were counterstained with hematoxylin and eosin (H&E). The tumor sections were observed under a confocal microscopy (Zeiss LSM 510, Germany).
All data are expressed as mean ± SD. A non-parametric Student’s t test was performed using the two-tailed distribution. A p value <0.05 was considered statistically significant.