Cytotoxicity of Thymus vulgaris Essential Oil Towards Human Oral Cavity Squamous Cell Carcinoma

Materials and Methods

Plant material. The T. vulgaris was cultivated in Ross-on-Wye, Herefordshire, UK. The thyme oil was obtained from a mixture of blossoms, leaves and stipes by steam distillation (T=100°C). The procedure was performed by Norfolk Essential Oils (Norfolk, UK). The T. vulgaris essential oil used in this study was provided by Rehau AG and Co (Rehau, Germany).

Cell culture. The UMSCC1 cell line was originally derived from a male patient with a T2 N0 M0 squamous cell carcinoma of the oral cavity (12). The UMSCC1 cells were cultured in McCoy's medium containing 10% fetal bovine serum (FBS) supplemented with 1% antibiotic-antimycotic (100×), liquid containing 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B/mL utilizing penicillin G (sodium salt), streptomycin sulfate and amphotericin B as Fungizone® antimycotic (in 0.85% saline) purchased from Invitrogen GmbH (Karlsruhe, Germany). The cells were maintained as monolayers in a plastic culture flask at 37°C in a humidified atmosphere containing 5% CO₂.

Gas chromatography. The oil extract was analyzed by gas chromatography using an Agilent Technologies 6890N GC instrument and a HP-5 capillary column (0.25 mm × 60 m; 0.25 μm film thickness; Santa Clara, CA, USA). The procedure was carried out by Phytolab GmbH (Vestenbergsgreuth, Germany). The oil was finally diluted with hexane (1:10).

XTT cytotoxicity assay. The cell proliferation was assessed using a standard 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay kit (Roche, Indianapolis, IN, USA), which measures the metabolic activity of viable cells (13, 14). The cytotoxicity of T. vulgaris essential oil compounds was determined by means of a Cell Proliferation Kit II (Roche Diagnostics, Mannheim, Germany). This test is based on the cleavage of the yellow XTT salt by ubiquitous dehydrogenases leading to the formation of an orange formazan dye. The intensity of the dye is commensurate to the number of metabolically active cells. Fresh stock solutions of thyme essential oil was prepared in DMSO in a dilution series ranging from 0.54 μg/ml to 18 mg/ml in McCoy's medium to perform the XTT test. The cells were suspended to a final density of 1×10⁵ cells/ml. One hundred microliters of the cell suspension were placed into the wells of a 96-well culture plate (Costar, Corning, NY, USA). The marginal wells were filled with 100 μl of McCoy's medium in order to minimize evaporation effects, besides, wells filled with medium were required to determine the background absorbance caused by non-metabolized XTT. A row of wells containing cells served as solvent controls. Each concentration was tested in at least two independent plates containing different batches of cells.

After incubation of 72 hours, the XTT reagent was freshly prepared and added to each well as specified by the manufacturer: XTT-labeling reagent and electron-coupling reagent was mixed in a ratio of 50:1 and 50 μl of this mixture were added to each well of the 96-well plate. The plates were incubated for 3 hours at 37°C, 5% CO₂ in a humidified atmosphere and read out after incubation. Quantification of cell cytotoxicity was performed in an ELISA plate reader (Bio-Rad, München, Germany) at 490 nm with a reference wavelength of 655 nm. The cytotoxic effect of the treatment was expressed as the percentage of viability compared to untreated cells (13). The toxicity of the compounds was determined by means of the formula: Cell viability(%)=Absorbance of sample cellsAbsorbance of untreated cells×100 The simple ligand-binding module of SigmaPlot® software (version 10.0, Systat Software Inc., San José, California, CA, USA) was used for analysis.

RNA isolation. The total RNA of the UMSCC1 cells was extracted from the test samples using an RNeasy® Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions to obtain highly pure RNA. Isolated total RNA was resuspended in the sample buffer provided by the manufacturer. The concentration and quality of the total RNA was verified by electrophoresis using the total RNA Nanochip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only the samples with an RNA index values greater than 8.5 were selected for expression profiling. The RNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All RNA samples were stored at −80°C until microarray analysis.

Probe labeling and Illumina Sentrix BeadChip array hybridization. Biotin-labeled cRNA samples for hybridization with Illumina Mouse Sentrix-8 BeadChip arrays (Illumina Inc., San Diego, CA, USA) were prepared according to Illumina's sample labeling procedure based on a previously published protocol (13). In brief, 250 ng total RNA were used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to gain biotin-labeled cRNA using the MessageAmpII aRNA Amplification kit (Ambion, Inc., Austin, TX, USA). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was purified in a column using the TotalPrep RNA Amplification Kit, and eluted in 60 μl of water. The quality of the cRNA was verified with the RNA NanoChip Assay and quantified on an Agilent 2100 Bioanalyzer and a spectrophotometer (NanoDrop).

Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 50 ng cRNA/μl, in an unsealed wet chamber for 20 h. Spike-in controls for low, medium and highly abundant RNAs were added along with mismatch control and biotinylation control oligonucleotides. The microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 min. After blocking for 5 min in 4 ml of 1% (w/v) blocker casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology Inc., Rockford, IL, USA), array signals were developed by 10 min incubation in 2 mL of 1 μg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned.

Scanning and data analysis. Microarray scanning was conducted using a bead station array scanner, adjusted to a scaling factor of 1 and photomultiplier set to 430. Data extraction was carried out for all the beads individually. Outliers were defined as >2.5 median absolute deviation. All the remaining data points were used for the calculation of the mean average signal for a given probe, and the standard deviation for each probe was calculated. The RNA from the UMSCC1 cell line was subjected to microarray analysis at least twice. The normalized data obtained from the duplicated hybridizations were averaged to obtain a final data set. Reproducibility of the data was assessed by calculating a percent error (standard deviation/mean ×100) for each gene element.

The data were cropped to a final set of 804 elements by eliminating genes with difference in expression exceeding standard deviation. Next, statistical significance was verified by means of empirical Bayes t-test and the false discovery rate was corrected with the Benjamini–Hochberg method. Ultimately, genes with p>0.05 were discarded after the allocation of p-values.

Data analysis was conducted with Chipster analysis software (http://chipster.csc.fi) for DNA microarray expression data by normalization of the signals using the cubic spline algorithm after background subtraction, and differentially regulated genes were defined by calculating the standard deviation differences of a given probe in a one-by-one comparison of samples or groups. For all the genes scored the fold change and p-values were determined.

Signaling pathway analysis. The data obtained from the Chipster were analyzed using pathway analysis using the Ingenuity Pathways Analysis software (version 6.5) by Ingenuity Systems (Redwood City, CA, USA). This software utilizes the raw image obtained from Chipster in order to align the image and determine expression values for all of the elements.

Statistical analysis. Statistical calculations were carried out with the SPSS 10.0 for Windows software package (SPSS Inc., Chicago, IL, USA). The results are expressed as the mean±S.E.M. of five independent experiments. Student's t-test was used for the statistical analyses; p-values <0.05 were considered statistically significant.