The cell line FADU originating from a hypopharyngeal carcinoma was grown in RPMI-1640 medium (Seromed, Munich, Germany), supplemented with 10% fetal calf serum (FCS). The HLaC78 cell line originated from a larynx carcinoma (18) and was maintained similar to FADU cells in RPMI-1640 medium. Mucosal keratinocytes were prepared from tonsillar tissue according to standard protocols (19). In brief, the mucosa was cut into small pieces and incubated overnight with 0.2% dispase (Sigma-Aldrich, Steinheim, Germany) in Dulbecco’s modified Eagle’s medium (DMEM; Seromed). The epithelium was separated with sterile forceps and digested with 0.1% trypsin (Seromed) for 20 min at 37°C. Residual trypsin was inactivated by addition of FCS. Mucosal keratinocytes were collected by centrifugation and cultured in defined keratinocyte serum-free medium (Keratinocyte-SFM; Invitrogen, Karlsruhe, Germany).

Dried and cut Galium verum L. leaves (Herba galii lutei) were kindly provided by Dr Ivo Pischel, PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Tea was prepared as follows: 100 ml boiling water was poured over 15 g of dried and powdered Galium leaves. After cooling, the supernatant was cleared by centrifugation and sterile filtration. Aliquots were frozen at −80°C. One batch of frozen Galium extract was used for all experiments. Identification of the extract ingredients is presented elsewhere (20).

To measure gene expression rates, real-time TaqMan^® PCR (Applied Biosystems) was performed. RNA was isolated from cell lines and primary cells with the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Darmstadt, Germany) was used for cDNA reverse transcription. Real-time PCR was performed in triplicates on a real-time PCR cycler (Applied Biosystems) using the TaqMan gene expression assays for MDR-1, MMP-9/MMP-2 and TIMP-1/-2. Relative quantification was calculated according to the 2^−ΔΔCT method (21). Expression values were normalised to the expression of GAPDH as an endogenous control which proved to be expressed most stably throughout the cell lines.

Cells were seeded at 5,000 cells/well in 96-well plates. Cells were treated with increasing concentrations of Galium verum aqueous extract (50 and 100 μl/ml) for 48 h. Controls were kept in medium supplemented with 100 μl/ml water. Cell proliferation was measured after 48 h by replacing the culture medium with medium containing 1 mg/ml MTT. After a 4-h incubation, MTT staining solution was replaced by isopropanol, and the cells were incubated at 37°C for 45 min. The colour conversion of MTT to a blue formazan dye was measured with an ELISA reader at a wavelength of 570 nm. The amount of formazan dye is in direct proportion to the number of metabolically active cells in the culture. Relative toxicity was calculated as the percentage of surviving cells by setting control cells treated with vehicle as having 100% surviving cells.

The alkaline single-cell microgel electrophoresis technique (comet assay) was applied to detect DNA strand breaks and alkali labile plus incomplete excision repair sites in single cells. Slide preparation was performed as previously described by Buehrlen et al (22). The evaluation of the slides was carried out on a DMLB fluorescence microscope (Leica Microsystems, Wetzlar, Germany) with a filter system incorporating a green excitation filter (515–560 nm band pass), a dichromatic beam splitter (580 nm long pass), and an emission filter (590 nm long pass) at a magnification of ×4,003. For every sample, two slides with 50 randomly selected cells each were counted (total of 100 cells). For analysis of the DNA fragmentation the Comet 5.5 Image System (Kinetic Imaging, Liverpool, UK) was used. For analysis, the olive tail moment (OTM) as a product of the median migration distance and the percentage of DNA in the tail was used (23).

Tumour spheroids were generated by seeding 5,000 cells/well of HLaC78 and FADU cells on ultra-low attachment (ULA) 96-well round-bottomed plates (Corning, Amsterdam, The Netherlands) (24). The surface of the flat-bottomed 96-well plates was coated with 125 μg/ml Matrigel^® (Becton-Dickinson, Heidelberg, Germany) for 2 h at room temperature. Wells were washed twice with phosphate-buffered saline (PBS) and subsequently blocked with 1% bovine serum albumin in PBS for 1 h. For 3 days on the ULA (see above) plates, pre-cultivated spheroids (see above) of HLaC78 and FADU cell lines were transferred to the coated wells with a multichannel pipette. Spheroids were incubated with or without the Galium decoction (33.3 μl/ml). Migration was recorded by photographing spheroids after 1 and 18 h with a Leica DMI 4000 inverted fluorescence microscope (Leica Microsystems). Quantification of migrated cells was carried out using ImageJ software [National Institutes of Health (NIH) USA].

Cell lines were treated with 33.3 μl/ml Galium verum extract for 48 h. After 48 h, the cells were seeded after incubation with Galium extract at equal cell numbers in multi-wall plates. Complete medium (MEM or RPMI) was replaced after attachment by Opti-MEM (Invitrogen, Karlsruhe, Germany), which is a complete, serum-free medium. Conditioned medium was collected after 18 h and concentrated using Amicon^® Ultra-4 Centrifugal Filters (Merck Millipore, Darmstadt, Germany). Five microliters of the concentrated medium was subjected to electrophoresis on 10% SDS-polyacrylamide gels under non reducing conditions (25), containing 1 mg/ml gelatin (Sigma-Aldrich, Traunstein, Germany). After electrophoresis, gels were renatured two times for 30 min in 2.5% Triton X-100 and developed overnight in developing solution (50 mM Tris-HCl, pH 6.8, 0.2 M NaCl, 10 mM CaCl₂, 0.02% Brij-35) at 37°C. Subsequently they were stained with Coomassie brilliant blue, destained and dried.

All statistical analyses and graphs were performed with GraphPad Prism 4 (Graphpad Software, La Jolla, CA, USA).

To determine detoxification capacities of HLaC78 and FADU cells, and mucosal keratinocytes, quantitative RT-PCR was performed. Expression of p-gp in the HLaC78 and FADU cells, and mucosal keratinocytes (MKs) was tested by TaqMan qRT-PCR. qRT-PCR revealed distinctly increased MDR-1 expression in FADU cells, when compared to HLaC78 cells (Fig. 1). There was no amplification detectable in primary MKs.

The two cell lines (FADU and HLaC78) and primary MKs were treated with increasing concentrations of Galium aqueous extract (Fig. 2). Cell viability and cytotoxicity of the used drug were assessed with the MTT assay. Mean percent inhibition was calculated from at least three independent experiments. Galium extract significantly suppressed the growth of both cell lines (Kruskal-Wallis test, p<0.05). In HLaC78 and FADU cells growth inhibition corresponded to the expression rate of MDR-1 (Fig. 2).

Primary keratinocytes, however, were less affected by high Galium concentrations than HLaC78 cells, although no MDR-1 transcript was detectable in these cells.

There was no obvious correlation between the sensitivity to high Galium concentrations and the proliferation rates of the cell lines/primary cells.

Investigation of invasion and motility was carried out using spheroid-based experiments. First, these experiments better reflect the solid tumour-microenvironment interaction. Second, the widely used Boyden chamber assay proved to be not reproducible in the actual system.

Spheroids of both cell lines were grown in ultra-low attachment plates (ULA plates) wells and were subsequently transferred manually to wells coated with Matrigel. Images of the cells were captured after attachment to ECM (1 h, t=0) and after 18 h (t=18).

For quantification of the cells migrating out of the spheroids, the areas of the spheroids at t=0 and t=18 were photographed, and the images were examined using Image J area calculation. Areas at t=0 were subtracted from the areas measured after 18 h. For each condition (with or without Galium, HLaC78 or FADU cells) at least 10 spheroids were measured.

For evaluation of cell motility, the area at t=0 was set at 100%. The percent of the migrated area was calculated using the following formula:

whereas Δ Area = Area ^t=18 − Area ^t=0.

Representative examples for FADU and HLaC78 cells migrating on Matrigel with or without treatment of Galium are shown in Fig. 3.

Comparing the percentage of the migrated areas of HLaC78 and FADU cells, HLaC78 cells turned out to be highly invasive, compared to the FADU cell line (unpaired t-test, p<0.0001; Fig. 4).

In both cell lines, Matrigel invasion was inhibited significantly by Galium (<0.0001; Fig. 5) decoction at sublethal doses of 33.3 μl/ml (unpaired t-test, p<0.0001). Comparison of the percent reduction of the migrated areas caused by Galium in the two cell lines revealed a stronger invasion inhibition in the aggressively invading HLaC78 cells (5104±287.3%) when compared to FADU cells (723.3±48.79%).

FADU and HLaC78 cell lines were cultivated with or without Galium extract for 4 or 8 h, respectively. Expression levels of MMP-2 and MMP-9 as well as TIMP-1 and TIMP-2 RNA were measured using qRT-PCR. Results are displayed in Fig. 6.

In both cell lines, MMP-9 and TIMP-1 were significantly upregulated after a 4-h incubation with Galium decoction. After 8 h however only TIMP-1 expression remained increased in FADU cells, when compared to untreated controls. HLaC78 cells displayed no significant changes in gelatinase A and B and TIMP mRNA expression after 8 h.

To test the actual proteolytic activities, conditioned media of HLaC78 and FADU cells incubated with or without Galium aqueous extract for 18 h were applied to gelatin zymographic gels.

FADU cells showed higher overall gelatinolytic activity than HLaC78 cells. Gelatin zymography revealed no significant changes in MMP-2/-9 activity after treatment with Galium decoction in the HLaC78 cell line. Galium-treated FADU cells showed even higher MMP-9 and MMP-2 activity, when compared with the untreated control cells (Fig. 7).

The Olive Tail Moment (OTM) was used to evaluate DNA damage. Benzo[a]pyrene, found in tobacco smoke (including cigarette smoke), has been shown to cause genetic damage in lung cells that was identical to the damage observed in the DNA of most malignant lung tumours (25). After a 1-h treatment of MKs with 200 mM benzo[a]pyrene, a significant increase in the mean olive tail moment was observed (Fig. 8). An overnight preincubation with Galium decoction (50 μl/ml) significantly decreased benzo[a]pyrene-induced DNA damage (Fig. 8).