Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis

Abstract

Background: TRAIL (tumor necrosis factor related apoptosis inducing ligand) is an apoptosis inducing ligand with high specificity for malignant cell systems. Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues.

Methods: Tumour cell systems derived from breast- (MDA MB231), lung--(NCI H460) colorectal--(Colo 205, HCT-15) and head and neck cancer (FaDu, SCC-4) were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry.

Results: The combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL.

Conclusions: Based on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced.

Figure 1

Time course of induction of apoptosis in six solid tumour cell lines. Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 12 to 48 h after treatment with TRAIL 0,1 ng/ml and 10 Gy alone, and after simultaneous and sequential application of combined therapy. Data represent means of three independent experiments; bars ± SD

Figure 2

Isobolographic analysis of apoptosis induction after combined treatment. Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 48 h after combined treatment. Envelopes of additivity were calculated with data of 3 independently performed experiments. Datapoints below the curves resemble a synergistic effect, datapoints between the curves demonstrate an additive effect and above the curves a subadditive response of combined treatment. A : tumour cells were treated simultaneously with 10 Gy and 0,1 ng/ml TRAIL. B: cells were irradiated with 10 Gy 12 h prior to treatment with 0,1 ng/ml TRAIL.

Figure 3

Westernblot analysis of Caspase-8 activation and PARP-cleavage. Lysates were prepared as described in methods. In six tumour cell lines(Colo 205, NCI H460,, HCT-15, MDA MB 231, FaDu and SCC-4) caspase-8 and PARP cleavage was analysed in untreated cells (lane 1), 24 h after treatment with TRAIL 0,1 ng/ml (lane 2) and 10 Gy alone (lane 3), after simultaneous (lane 4) and sequential application (lane 4) of combined therapy. Every cell line shows a different cleavage pattern according to the rate of apoptosis induction. β-Actin staining was used as loading control. The mapped blots represent each one of three independently performed immunoblots.

Figure 4

Surface expression of TRAIL receptors after irradiation with 10 Gy in six tumour cell lines. Quantification of receptor expression was performed 6 to 48 h after irradiation by FACS analyis using the Quantibrite™ evalution system from BD(Heidelberg, Germany) according to manufacturer's instructions. Data shown are from one representative experiment (n ≥ 3). A: Cell surface expression of R1/DR4 B: Cell surface expression R2/DR5

Figure 5

Microscopic evaluation of normal tissue cells. Lack of apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei. Micrographs depict cells of eight different normal tissues, 48 h after irradiation alone and after sequential treatment with 10 Gy 12 h previously to application of 1,0 ng/ml TRAIL.