The Molecular Effects of a PARP-1/-2 Inhibitor on Proton and Photon Radiotherapy in Head and Neck Cancer Cells.

PURPOSE/OBJECTIVE(S) The responses of human head and neck cancer (HNSCC) cells to photon (XRT) and proton (PRT) radiotherapy can be enhanced by a poly-(ADP-ribose) polymerase (PARP)-1 and PARP-2 inhibitor (PARPi) with undefined underlying mechanisms. We hypothesize that elucidating these mechanisms will facilitate a personalized selection of PARPi in combination with XRT or PRT. Herein, the effects of PARPi on XRT and PRT induced cell death, cell cycle distribution, and related protein expression in HNSCC cells were defined. MATERIALS/METHODS Human papillomavirus (HPV)-negative HN5, SqCC/Y1, and HPV-positive UPCI-SCC-154, UM-SCC-47 cell lines were tested. Cells were exposed to a 4 Gy PRT or XRT with or without PARPi (1 µM, 1 hour [h] before radiation). Cell cycle distribution, cell necrosis and apoptosis (staining with Annexin-VFITC) were determined using terminal deoxy-nucleotidyltransferase dUTP nickend labeling assay and were analyzed by flow cytometry. Cell mitotic catastrophe (co-staining for cytoplasm with γ- tubulin for nucleus with Dapi), and senescence (assessed using SA-β-gal) were examined. Protein expressions were determined by western blot or by flow cytometry. RESULTS PARPi significantly increased XRT and PRT induced cellular senescence at 6 days in the two HPV+ cell lines, with higher percentages of senescence in PRT than XRT cells (P all < 0.05); in the two HPV- cell lines, the PARPi significantly increased the ratio of XRT-induced senescence (P all < 0.01). PARPi caused significantly more cells to undergo mitotic catastrophe after PRT (in 3 cell lines) or XRT (in the two HPV- cell lines), with higher rates of mitotic catastrophe induced by PARPi in PRT vs. XRT group (P all < 0.05). The PARPi led to a trend (non-significant) of increased cell necrosis at 48 h after PRT (in 3 cell lines) or XRT (in 2 cell lines). No effect of PARPi on XRT or PRT induced apoptosis was noted. At 24 h, PARPi significantly prolonged XRT (in two HPV- cell lines) and PRT (in HN5 cells) induced cell cycle arrest at G2/M phase; trends of this prolongation were noted after PRT in SqCC/Y1 and UPCI-SCC-154 cells, and after XRT in UPCI-SCC-154 cells. PARPi caused trends of increasing ratios of specific protein (53BP1 and RAD51 [DNA repair related]; Ki-67 [cell cycle related]) abundant cells at 24 h after PRT (in two HPV- cell lines); and increased cleaved caspase-3 expression after XRT or PRT in four cell lines tested. CONCLUSION In conclusion, PARPi induced higher levels of mitotic catastrophe in combination with PRT versus XRT, promoted XRT and PRT induced senescence, while it led to a trend of increased cell necrosis after PRT and XRT. These upregulating effects of PARPi were cell line dependent. Cell line based delayed DNA damage repair and increase in radiation-induced cell cycle arrest at G2/M phase may have served as parts of the underlying mechanisms. Further studies on personalized therapy of XRT or PRT in combination with PARPi to improve the efficacy of radiotherapy are warranted.