Abstract 69: Proton versus photon irradiation induced cell death in head and neck cancer cells with different human papillomavirus status

Abstract

Abstract Proton radiotherapy (PRT) has shown to be less toxic in the treatment of head and neck cancers (HNSCC) than photon radiotherapy (XRT). XRT causes multiple types of cancer cell death by inducing DNA double-strand breaks. The DNA damage induced by PRT is more complex and has more severe biological consequences versus XRT. The present study was undertaken to uncover the cell death caused by PRT versus XRT in human papillomavirus positive (HPV+, frequently p53 wild-type, p16 positive; p53, p16 are key factors for cell death) and HPV-negative (HPV-, frequently p53 mutant, p16 negative) HNSCC cells. HNSCC cell lines HN5, SqCC/Y1 (HPV-) and UMSCC-47, UPCI-SCC-154 (HPV+) were used. Single doses of 4 Gy irradiation induced cell apoptosis, necrosis, mitotic catastrophe, and senescence were determined. Clinical 200-MeV proton beams (18 cm × 18 cm field) or 6-MV X-ray beams (25 cm × 25 cm field) were used. Cells were positioned in the centers of the irradiation fields (in the middle of the spread-out Bragg peak for PRT). At 4 hours (h), 24h and 48h after PRT or XRT, cell necrosis and apoptosis were determined by Annexin VFITC-conjugated staining. XRT and PRT caused increased cell necrosis in the HPV– SqCC/Y1 (moderate at 24h, significant at 48h [p all < 0.05]) and HN5 (moderate at 24h, 48h) cells, at slightly higher rates in PRT versus XRT treated cells; and in the HPV+ UPCI-SCC-154 (moderate at 4h) and UMSCC-47 (moderate at 48h) cells, with no difference between PRT versus XRT treatment groups. XRT and PRT induced a small percentage of cell apoptosis (range: 1.1 ± 0.5 to 7.6 ± 2.9). Cell apoptosis was only moderately increased in the HPV+ UMSCC-47 (at 4h, 24h, 48h) and HPV– SqCC/Y1 (at 24h, 48h) cells, with slightly more apoptosis seen in the XRT versus the PRT group. Cell senescence was assessed using a well-established senescence-associated biomarker (SA-β-gal). XRT and PRT induced significantly increased cellular senescence at both 4 and 6 days in all four cell lines (p all < 0.01), with PRT treatment leading to a significantly higher percentage of senescence in cells than XRT (p all < 0.01). Mitotic catastrophe, an important cell death mechanism by which solid tumors respond to clinical radiotherapy, was determined using co-staining for cytoplasm with γ- tubulin (with Texas red) and for nucleus with Dapi (blue). XRT and PRT both led to a significantly increased number of cells undergoing mitotic catastrophe at 4h, 24h, 48h and 72h (p all < 0.05) in all four cell lines, with significantly higher rates seen in PRT treated versus XRT treated (p all < 0.05). Moreover, the peak of cell mitotic catastrophe induced by PRT occurred at 48h in the HPV– cells, versus at 72h in the HPV+ cells; while no obvious peak was observed in the XRT treated group. In conclusion, mitotic catastrophe and senescence are the major types of cell death induced by both XRT and PRT, in which PRT inducing higher levels of both types of cell death as compared to XRT. Citation Format: Li Wang, Shichao Han, Jinming Zhu, Xiaochun Wang, Yuting Li, Zeming Wang, Eric Lin, Xiaofang Wang, David P. Molkentine, Pierre Blanchard, Yining Yang, Ruiping Zhang, Narayan Sahoo, Michael Gillin, Xiaorong Ronald Zhu, Xiaodong Zhang, Jeffrey N. Myers, Steven J. Frank. Proton versus photon irradiation induced cell death in head and neck cancer cells with different human papillomavirus status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 69.