Reduction of DNA Damage by Curcumin and Celecoxib in Epithelial Cell Cultures of the Oropharynx after Incubation with Tobacco Smoke Condensate

Materials and Methods

Biopsies. Tissue samples of macroscopically healthy oropharyngeal mucosa were harvested during surgery of oropharyngeal carcinoma. Only mucosa that had to be resected for surgical reasons was used to avoid additional stress for the patients. The study was approved by the Ethical Commission of the Medical Department, Ludwig Maximilians University Munich (no. 221/08).

Mini organ cultures. Specimen were dissected into cubes of 1 mm³, excluding deeper layers, and were washed three times in bronchial epithelial cell basal medium (BEGM; Promocell, Heidelberg, Germany). Cubes were placed in 24-well plates, and coated with 0.75% Noble Agar (Difco, Detroit, MI, USA) and were then dissolved in Dulbecco's modified Eagle's medium (Gibco, Eggenstein, Germany) containing fetal calf serum (10%) (Gibco), non-essential amino acids (Gibco) and amphotericine B (Gibco). After 14-20 days in 250 μl BEGM, and 37°C with 5% CO₂ and 100% relative humidity, MOCs were completely coated with epithelium. BEGM was replaced every second day during cultivation. Multiwell plates were changed every week.

Incubation and cell separation. MOCs were either incubated with 25 μl curcumin (1 μM; Sigma, Steinheim, Germany) or celecoxib (0.1 μM Pfizer, New York, NY, USA) for 1 h at 37°C and washed twice afterwards. Dimethylsulfoxide (DMSO, 166 mM; Merck, Darmstadt, Germany) served as the negative control. A proportion of the MOCs were then incubated with 25 μl smoke condensate (0.7 mg/dl) [as described in (4)] for 18 hours. Again, DMSO served as a negative control. MOCs were washed twice with BEGM before they underwent enzymatic digestion [10 mg hyaluronidase (Boehringer, Mannheim, Germany); 10 mg collagenase (Roche, Mannheim, Germany); 50 mg protease (Sigma)] for 45 min at 37°C. To preserve the physiological character of the samples, no metabolic activation was used before the incubation period. Viability was tested with trypan blue staining.

Comet assay. The procedure of the comet assay for both cell types was mainly based on the protocol of Singh et al. (10). Special slides were designed for the comet assay with a frosting of 5 mm along the long edges (76 mm × 26 mm; Langenbrinck, Emmendingen, Germany), prepared with 85 μl of 0.5% normal melting agarose (Biozym, Hameln, Germany). Following enzymatic digestion the viability of the cells was again examined using trypan blue staining. Having obtained viabilities of between 90 and 100%, the remaining aliquots were suspended with 75 μl of 0.7% low-melting agarose (Biozym) and applied to the prepared slides. Alkaline lysis (10 ml DMSO, 1 ml Triton-X©, 89 ml alkaline lysis buffer) followed for one hour. The slides were then dried and placed into a horizontal gel electrophoresis chamber (Renner, Dannstadt, Germany), and were covered with alkaline buffer solution containing NaOH (10 mM) and Na₂EDTA (200 mM) at pH 13.2. After a 20 min DNA unwinding period, electrophoresis was started at 25 V and 300 mA for 20 min. Following neutralization (0.4 M Tris, pH 7.5; Merck, Darmstadt, Germany), the cells were stained with 85 μl ethidium bromide (20 μg/ml, Sigma). The slides were covered with coverslips and stored for less than three days in humidified boxes at 5°C.

Digital analysis. The cells in the comet assay were investigated using a DMLB© microscope (Leica, Heerbrugg, Switzerland) with an adapted CCD camera (Cohu Inc.; San Diego, Ca, USA). Forty representative cells were investigated per slide, using two slides for each aliquot tested. For data analysis, the median of each slide set was used. The comets were measured using an image analysis system (Comet++™; Kinetic Imaging, Liverpool, UK). Comet analysis was performed blinded to one examiner, in order to reduce observer-based divergence. To quantify DNA damage, the olive tail moment was used (OTM: median DNA migration distance × relative amount of DNA in the tail of the comet) (11). Eighty cells per slide and two slides per patient were evaluated. While it is the subject of controversy in discussions, OTM is still considered the most informative measure in the comet assay (12).

Comet-FISH. For hybridization, the protocol of McKelvey-Martin et al. (13) was used with only minor changes. After neutralization with saline sodium citrate buffer (SSC) (0.3 M NaCl, 30 mM sodium citrate), the slides were sequentially dehydrated with alcohol (70, 85 and 100%) and dried at 37°C. Hybridization mixture was added, containing (all quantities are listed per slide) hybridization buffer (formamide with dextran sulfate, 14 μl), DNA probes (2 μl, LSI EGFR Dual Color Probe-Hyb Set) (Abbott, Il, USA)) and Aqua bidest (4 μl). The DNA probes hybridized to the centromere of chromosome 7 and the EGFR gene on the same chromosome simultaneously. The centromere served as a reference gene, due to its close location on the same chromosome as the EGFR gene.

After coverage and sealing of the prepared slides and incubation at 74°C for 5 min on a precision hot plate, the slides were placed into a wet chamber for 12-16 h at 37°C. Before detection of probes, the slides were washed three times each in 50% formamide in 2 × SSC (Abbott) and incubated for 10 min in 2 × SSC and in 0.1% detergent tergitol NP-40 in 2 × SSC for 5 min.

Staining and analysis. DAPI (10 μl of 42 ng/ml) with Antifade (both from Abbott) was applied after air-drying of the slides followed by storage at −20°C protected from light. DNA fragmentation was visualized using a fluorescence microscope and digital analysis (Comet++; Kinetic Imaging™). Forty cells per slide and two slides per patient were analyzed. Analogous to the OTM, the Munich chromosomal tail moment (MCTM) was used to estimate the degree of EGFR damage. The MCTM is the product of the median DNA migration distance in a gene and the gene fluorescence in the tail of the comet divided by the overall gene fluorescence measured in a cell (14).

Statistical analysis. Statistical analysis was performed using the SPSS 16.0™ software (IBM, Armonk, NY, USA). DNA damage for all patient samples was compared using the Wilcoxon's test. The general level of significance accepted was p≤0.05. Bonferroni correction was used where necessary. Standard box-plots (lower quartile, median, upper quartile) were used to illustrate the results. Dots denote mild statistical outliers [between 1.5 and 3 times interquartile range (IQR)]; asterisks denote extreme statistical outliers (more than 3 times IQR).