Chemoprevention by Quercetin of Oral Squamous Cell Carcinoma by Suppression of the NF-κB Signaling Pathway in DMBA-treated Hamsters

Materials and Methods

Hamsters. Male Syrian hamsters aged 8-10 weeks weighing between 100 and 110 g were obtained from Vital River Laboratories Company, Beijing, China. All animals were fed with an autoclaved laboratory rodent diet. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with an alternating 12-h light/dark cycle in accordance with the regulations of the ethics committee and the Guide for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006). All animal experiments were approved by the Animal Committee of Nanjing Origin Biosciences, China (OB1511).

Induction of HBP carcinogenesis and treatment. Hamsters were randomized and divided into six groups of 8 animals each. Group 1 served as the normal control without any treatment. Group 2 received quercetin treatment-only, at a dose of 50 mg/kg daily. Groups 3-6 were given DMBA for induction of HBP carcinogenesis. DMBA (0.5%) (Sigma Chemical Company, St. Louis, MO, USA) in liquid paraffin was painted on the right buccal pouches of hamsters with a number-4 brush, three times per week for 14 weeks (17). Group 3 served as the model control and received DMBA only. Group 4, 5 and 6 received simultaneous treatment of quercetin (Sigma Chemical Company, St. Louis, MO, USA) at doses of 12.5 mg/kg (L), 25 mg/kg (M), or 50 mg/kg (H), respectively. Quercetin was administrated via oral gavage daily for 14 weeks. Animal body weights were recorded weekly during the experimental period. The experiment was terminated at 14 weeks and all animals were sacrificed. The right pouch of the animal was grossly inspected to evaluate pre-malignant lesions or tumor development and photographed. The buccal pouch tissues of all animals were collected for further analysis.

Histo-pathological analysis. The pouch tissue was fixed in 10% formalin, dehydrated, paraffin embedded, processed and sliced into 5 μm-thick sections. Each sample was cut into 30 sections. The first, 15th, and final sections were stained with haematoxylin and eosin (H&E) for histopathological analysis. Basal-cell hyperplasia, dysplasia and OSCC were diagnosed by a pathologist who was blinded to the experimental groups, according to the established criteria (18).

Apoptosis analysis. Apoptosis in the pouch tissue was determined by TUNEL staining, using a commercially-available kit (In Situ Cell Death Detection Kit, POD; Roche, Penzburg, Germany). The pouch tissue sections were deparaffinized and dehydrated according to standard protocols. Tissue sections were incubated with Proteinase K working solution for 30 min at 21-37°C. The slides were then washed with PBS (pH 7.2-7.6) twice. The positive control was incubated with DNase I for 10 min at 15-25°C. The negative control was incubated with labeling solution (without terminal transferase) instead of the TUNEL reaction mixture. The slides were then washed with PBS (pH 7.2-7.6) three times. Converter-POD was added on the slides and incubated in a humidified chamber for 30 min at 37°C. The slides were then washed with PBS (pH 7.2-7.6) three times. The DAB substrate was added to the slides, which were incubated for 10 min at 15-25°C. The slides were then washed with PBS (pH 7.2-7.6) three times. The slides were mounted and analyzed by microscopy (Olympus, Melville, NY, USA). The slides were viewed at 400× magnification and apoptotic cells were recognized by the appearance of brown-stained cells. Expression levels were quantified by the average optical density (AOD) of the positive cells in 5 fields/sample with Image-Pro Plus 6.0 software.

Real-time polymerase chain reaction (RT-PCR). Total RNA was isolated from pouch tissue samples in 1 ml of TRIzol reagent (Invitrogen Life Tech, Carlsbad, CA, USA). Then total RNA was treated with DNaseI and subjected to quantitative PCR, which was performed with an ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green I dye. The primers used for PCR reactions were GAPDH (sense 5’-GGGTGTGAACCATGACAAGT-3’; antisense 5’-AGTGGATGCAG GGATGATGT-3’), Bax (sense 5’-TCATGAAGACAGGGGCCTTT-3’; antisense 5’-CTGTCCAGCTCATCTCCGAT-3’), Bcl-2 (sense 5’-CCTGGCATCTTCTCCTTCCA-3’; antisense 5’-CTGACTGGACAT CTCTGCGA-3’), NFκB-p50 (sense 5’-AAAATATCCACCTGCA CGCC-3’; antisense 5’-CCAGGATTGTAGCCCCGTAT-3’) and NFκB-p65 (sense 5’-ACAGATACCACCAAGACGCA-3’; antisense 5’-AGGTCTCGCTTCTTCACACA-3’). The GAPDH gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. Gene expression was calculated using the 2^−ΔΔCt method.

Western blotting. Protein from pouch tissue was extracted with RIPA buffer (Beyotime BioTech, Shanghai, China). Protein concentration was determined with the Bradford protein assay kit (KeyGen BioTech). Total proteins were separated by sodium salt-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membranes were incubated with 5% non-fat milk solution for blocking non-specific binding and then with primary antibodies to NF-κB p50 (Ab7971; 1:400); NF-κB p65 (sc-372; 1:500); Bcl-2 (sc-492; 1:1,000); Bax (sc-493; 1:1,000). All antibodies were from Santa Cruz Biotechnology Inc., except anti-NF-κB p50, which was from ABcam, Shanghai, China. After washing twice with tris-buffered saline, membranes were incubated with an appropriate secondary antibody (1:5,000) conjugated with HRP for 2 h at room temperature. Analysis by electro-chemiluminescence was performed according to the manufacturer's instructions using a Bio-Rad imaging system. Quantity One version software (Bio-Rad, Hercules, CA, USA) was used to quantify the density of bands.

Statistical analysis. The data are expressed as mean ± SD. Statistical analysis for body weight was with the Student's t-test, while tumor incidence was compared by χ²-test. The data for densitometric analysis were analyzed using ANOVA followed by Bonferroni post hoc tests. A value of p<0.05 was considered to indicate a statistically significant difference.