Data from 60 inpatients experiencing skin toxicity RTOG score grade 2 and higher (grade 2+) during radiotherapy at the Department of Radiation Oncology, University Hospital Freiburg, Germany, were assessed retrospectively. All patients received our standard skin care program, which consisted of once-daily treatment of irradiated skin areas with a moisturizing crème (Eucerin Lotion 3% Urea, Beiersdorf AG, Hamburg, Germany) starting at day one of radiation treatment (linear accelerator Clinac, Varian, 6MeV photons; daily fractions of 1.8–2.0 Gy). Frequency of application was increased to twice or three times daily if a radiation-induced erythema occurred, and stopped with the occurrence of moist desquamation. Irradiated skin areas with grade 2+ lesions were treated three times daily with either green or black tea preparations for 10 minutes from the first day moist desquamation was observed. Skin toxicity was scored by nurses on a daily basis before every application of tea extracts. Treatment with tea extracts was discontinued immediately after the disappearance of moist desquamation. Nine patients (four with head and neck cancer, four with cancer in the pelvic region and one patient with gliosarcoma) were released from the hospital with persisting grade 2+ skin toxicity. These patients were censored and the dataset was analyzed for the duration of skin toxicity grade 2+ using the Kaplan-Meier-Method. A p-value ≤ 0.05 (log-rank test) was considered statistically significant. The study was approved by the local ethics board of the University Hospital Freiburg, Germany.

Cultures of RAW 264.7 murine macrophages (a generous gift of Dr G. Hildebrandt, Department of Radiation Oncology, University of Leipzig, Germany) and MCF-7 breast cancer cells (DMSZ, Braunschweig, Germany) were grown in 75 cm² flasks (Greiner) at 37°C in a humidified atmosphere at 5% CO₂ in DMEM medium (Cell Concepts, Umkirch, Germany), supplemented with 10% heat-inactivated FCS and 1% penicillin/streptomycin (Sigma).

Monocytes from healthy human donors were prepared following a standardised protocol (Ficoll gradient preparation, Amersham-Biosciences) using a completely endotoxin-free cultivation 1011. Using 50 ml tubes, 25 ml Ficoll were loaded with 25 ml blood of buffy coats from healthy blood donors. The gradient was established by centrifugation at 1,800 r.p.m., 20°C for 40 min by using slow acceleration and brakes. Peripheral blood mononuclear cells in the interphase were carefully removed and re-suspended in 50 ml pre-warmed phosphate buffered saline (PBS, Invitrogen) followed by centrifugation for 10 min at 1,600 r.p.m. and 20°C. The supernatant was discarded and the pellet washed in 50 ml PBS and centrifuged as described above. The pellet was then re-suspended in 50 ml RPMI-1640 low endotoxin-medium (from Invitrogen) supplemented with 10% human serum (PAA). After counting the amount of cells in a particle counter (Euro Diagnostics, Krefeld), cells were seeded in 24-well plates for ELISA or six-well plates for RNA analysis (1–2 × 10⁶ cells/well) and incubated at 37°C/5% CO₂. The medium and the non-adherent cells (lymphocytes) were removed and fresh RPMI-1640 medium containing 1% human serum was added.

Two tea bags (1.5 g each) tea were covered with 50 ml Millipore water (black tea: boiling water; green tea: water at 70°C) for five minutes. The resulting extracts were passed through a 0.8 μM and a 0.22 μM filter to free the extracts from insoluble material. Small aliquots were stored until usage at -20°C in the dark. Given a estimated total catechin content of 10–40 mg/g for green tea 12, tea preparations used in our study, were expected to have an estimated total catechin concentration of 25–100 μg/ml. Epigallocatechin-gallate (Sigma-Aldrich) was solubilized in water and small aliquots of the stock solution were stored until usage at -20°C in the dark.

Exponentially-growing RAW 264.7 murine macrophages were irradiated at room temperature using a ¹³⁷Cs-laboratory irradiator (IBL 637, CIS bio international) at a dose rate of 0.78 Gy/min. Control cells were sham-irradiated.

Approximately 10,000 RAW 264.7 murine macrophages per well were plated into 96-well plates in 100 μl RPMI medium per well. After 24 h, tea extracts were added to the media. Three hours later, the cells were irradiated with 0, 2, 4, 6, and 8 Gy. After five days, 20 μl MTT (5 mg/ml) was added to each well and cells were subsequently incubated for four hours at 37°C. Fifty microliters of a 20% SDS solution (0.02 N HCL) was added to each well and absorbance at 560 nm was measured after overnight incubation at 37°C (VersaMax, Molecular Devices).

Proteasome function was measured as described previously 13 with some minor modifications. To obtain crude cellular extracts, cells were washed with PBS, then with buffer I (50 mM Tris, pH 7.4, 2 mM DTT, 5 mM MgCl₂, 2 mM ATP), and pelleted by centrifugation (1,000 × g, 5 min, 4°C). Glass beads and homogenization buffer (50 mM Tris, pH 7.4, 1 mM DTT, 5 mM MgCl₂, 2 mM ATP, 250 mM sucrose) were added and cells were vortexed for 1 min. Beads and cell debris were removed by centrifugation at 1,000 × g for 5 min and 10,000 × g for 20 min at 4°C. Protein concentration was determined by the Micro BCA protocol (Pierce) with BSA (Sigma) as standard. To measure 26 S proteasome activity, 10 μg protein of crude cellular extracts or 1 μg protein of partially purified proteasomes of each sample was diluted with buffer I to a final volume of 200 μl. The fluorogenic proteasome substrate SucLLVY-MCA (chymotrypsin-like, Sigma) was dissolved in DMSO and added in a final concentration of 80 μM in 1% DMSO. Proteolytic activity was continuously monitored by measuring the release of the fluorescent group 7-amido-4-methylcoumarin (AMC) in a fluorescence plate reader (SpectraMax Gemini, Molecular Devices, 37°C) at 380/460 nm.

For preparation of total cytosolic extracts, cells were dislodged mechanically, washed with ice-cold PBS and lysed in TOTEX-buffer (20 mM HEPES, pH 7.9, 0.35 mM NaCl, 20% glycerol, 1% Nonidet P-40, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, PMSF and aprotinin) for 30 minutes on ice. Lysate was centrifuged at 12,000 × g for 5 min at 4°C. Protein concentration was determined using the BCA protocol (Pierce) and bovine serum albumin (BSA, Sigma) as standard. Fifteen micrograms of protein from the resulting supernatant was incubated for 25 minutes at room temperature with 2 μl BSA (10 μg/μl), 2 μl dIdC (1 μg/μl), 4 μl Ficoll-buffer (20% Ficoll 400, 100 mM HEPES, 300 mM KCl, 10 mM DTT, 0.1 mM PMSF), 2 μl buffer D+ (20 mM HEPES, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% NP-40, 2 mM DTT, 0.1 mM PMSF) and 1 μl of the [γ³²P]-ATP labeled oligonucleotide (Promega, NF-κB: AGT-TGA GGG GAC TTT CCC AGG). For a negative control, unlabeled oligonucleotide was added in 50-fold excess. Gel analysis was carried out in native 4% acrylamide/0.5% TBE gels. Dried gels were placed on a phosphor screen for 24 hours and analyzed on a phosphor imager (Typhoon 9410, Amersham).

For the analysis of IL-6, TNF-α, IL-1-β, IL-8 and PGE₂ release, cells were preincubated with the green and black tea solutions in the indicated concentrations for 30 min before stimulation with LPS (10 ng/ml) for additional 24 h. After culture supernatants were harvested and centrifuged for 10 min at 10,000 × g, levels of the cytokines or PGE₂ in the supernatant were measured by ELISA (IL-6, IL-8, TNF-α: Pelikine, distributed by HISS, Freiburg, Germany) or EIA (PGE2: AssayDesign, distributed by Biotrend, Köln, Germany) according to the manufacturer's instructions. All experiments were carried out in triplicates using two-sided student's t-test for statistical analysis (a p-value ≤ 0.05 was considered statistically significant).

During curative-intended radiation treatment of solid cancer, many patients experience grade 2+ skin toxicity, which can cause treatment gaps and thus limit the curability of the cancer. Tea extracts have been applied in our department empirically to terminate acute skin reactions during radiation therapy, but their use has not been investigated systematically to date. In the present study, 60 patients treated with radiation therapy received our standard skin care program. Demographics of all patients are shown in Table 1. The standard skin care program at the Department of Radiation Therapy, University Hospital Freiburg, consisted of thrice-daily topical application of green or black tea extracts to skin regions with grade 2+ toxicity and continued as long as grade 2+ toxicity persisted. Applying these treatments, we found no difference in duration of grade 2+ skin reactions in patients treated for cancer of the head and neck region between green or black tea (Figure 1A, mean duration for green tea 16.5 ± 1.7 days, for black tea 17 ± 1.8 days, p = 0.64; log-rank test). Patients treated for cancer of the pelvic region had a shorter duration of grade 2+ skin toxicity if treated with green tea extracts (Figure 1B, mean duration for green tea 16.3 ± 1.6 days, for black tea 22.2 ± 2.1 days, p = 0.014; log-rank test). The most obvious difference between patients treated for head and neck cancer and patients treated for tumors of the pelvic region was that most of the latter received radiochemotherapy (23/25). Subgroup analysis of patients with cancers of the head and neck region showed no advantage for patients treated with green tea extracts (8/15) during radiochemotherapy. However, the numbers of patients in these subgroups was small and thus the reliability of the statistical analysis was limited.

Our clinical observations led us to investigate possible underlying molecular mechanisms by which tea extracts could support skin restitution during and after radiotherapy. A hallmark of radiation-induced inflammation is the release of pro-inflammatory cytokines by immune cells. In order to test if clinically-used tea extracts exhibit anti-inflammatory effects in human immune cells we used human monocytes isolated from buffy coats. Both black tea and green tea extracts caused a significant concentration-dependent decrease of LPS-induced IL-1β, IL-6, IL-8, TNFα and PGE₂ secretion in to the media. While both extracts showed clear anti-inflammatory activity, this inhibitory effect was more pronounced in green tea extracts reflecting the higher efficiency of green tea in the clinical study (Figure 2A–J).

To further study the effects of tea extracts on signal transduction in immune cells, we used RAW 264.7 murine macrophages instead of buffy-coat-derived monocytes as a model because it was necessary to scale up the cell number to yield enough protein for proteasome function assays.

Tea extracts contain polyphenols, which have been shown to inhibit proteasome function. Therefore, we decided to test if tea preparations used in skin care during radiation therapy also have inhibitory effects on the proteasome. Crude extracts of exponentially-growing RAW 264.7 macrophages were incubated with serial dilutions of green and black tea extracts. Chymotryptic, tryptic and pepdidyl-glutamyl 26S proteasome activity were continuously monitored using a fluorogenic peptide assay. Green tea extracts caused a concentration-dependent inhibition of the chymotryptic 26S proteasome activity (46% ± 5.3 at 0.5% (p ≤ 0.001; two-sided Student's t-test), 9.9% ± 1.3 at 1% (p ≤ 0.001; two-sided Student's t-test). Green tea extracts at concentrations of 2% and higher completely inhibited chymotryptic 26S proteasome activity.

Extracts from black tea were even more effective. Concentrations of 0.5 % strongly inhibited chymotryptic 26S proteasome activity to 8% ± 1.1 (p ≤ 0.001; two-sided Student's t-test) of untreated controls. No remaining activity was detected at 1% or 0.5%. Comparable results were found for the peptidyl-glutamyl 26S proteasome activity (Table 2), However, inhibition of the tryptic activity by black tea extracts was not dose-dependent at the concentrations used, and was almost already complete at 0.5% concentration.

Observing an inhibitory effect of proteasome function, we next tested if – as a consequence – NF-κB DNA-binding activity was affected by tea extracts. Surprisingly, for both extracts we observed an increase in NF-κB activity, which was accompanied by a size -shift of the specific bands at 4% and 0.4% concentrations in the gel-shift assays. Further dilutions caused a decrease in NF-κB DNA-binding activity in experiments using black tea extracts, while the same dilutions of green tea extract had no effect (Figure 3, 4). Supershift assays using an anti-p50 or anti-p65 antibody confirmed specificity of the shifted bands (Figure 5). The observed size shift could be completely reversed by pre-incubation of the cells with the pan-caspase inhibitor z-VAD-fmk (50 μM) indicating involvement of caspases (Figure 6). Further exploring the underlying mechanism, we repeated the gel-shift experiments using EGCG in concentrations comparable to concentrations expected in tea extracts 12. NF-κB DNA-binding was increased at very high concentrations of EGCG (400 μM), but showed almost no change at all other concentrations used. A size shift of the DNA-bound complex could not be detected (Figure 7).

The pan-caspase inhibitor z-VAD-fmk non-specifically inhibits a broad panel of caspases. In order to further explore the involvement of caspases we used MCF-7 breast cancer cells, which are known to be functionally deleted in caspase-3 15. Gel-shift experiments using oligonucleotides containing the consensus binding sequences for NF-κB failed to show a size shift of the specific bands for NF-κB (Figure 8).

We next investigated if tea extracts also altered the viability and radiation response of RAW 264.7 murine macrophages. Cells were pre-incubated with different concentrations of green and black tea extracts for four hours and irradiated with 0, 2, 4, 6 and 8 Gy. EGCG at 400 μM and tea extracts at 4% concentrations caused a significant decrease of viability, consistent with activation of the cellular death program. EGCG at 40 μM or green tea extracts (0.4%) did not have a significant cytotoxic effect while black tea extracts (0.4%) significantly decreased viability to 73.6% ± 8 (p ≤ 0.05; two-sided Student's t-test) of untreated control cells. Concentrations of 0.04% of either green tea or black tea had no toxic effect. Additionally, EGCG (40 μM), green tea extracts (0.4%) and black tea extracts (0.4%) caused a slight radioprotection (Figure 9).