Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells

Abstract

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation

“…Representative images of foci per nucleus are shown in Figure S8 for each cell line in the basal condition, at 2 h and 24 h. Following hypoxia, the kinetics of RAD51 foci were enhanced after photon exposure in all the subpopulations studied. By contrast, no modification occurred after C-ion exposure, a result in accordance with our previous studies [38]. Interestingly, silencing HIF-1α after hypoxia induced a strong decrease of RAD51 in response to both radiations in non-CSCs and CSCs, as shown in Figure 6b.…”

“…Cells were irradiated at a dose rate of approximately 2 Gy/min with 250 kV photons (X-RAD320, Lyon-Sud Medical School, Lyon, France) and C-ions at the initial energy of 290 MeV/n, center of 6 cm Spread-Out Bragg Peak (SOBP) with the Heavy-Ions Medical Accelerator of Chiba, at National Institute of Radiological Sciences (Chiba, Japan) [16,20,37]. Time points were performed from 30 min after irradiation (time needed to eliminate neutrons in the irradiation room and come back to the bench) to 24 h. The radiobiological parameters (α, β, RBE at 10% survival) calculated according to the linear-quadratic model were previously published for the four subpopulations studied [16,38]. Following a comparative study for DNA Damage Repair kinetics between X-rays and C-ions, and since RBEs were dependent on both the populations tested and the oxygen concentration, all experiments were performed at an equivalent physical dose of 2 Gy photons [38].…”

“…Time points were performed from 30 min after irradiation (time needed to eliminate neutrons in the irradiation room and come back to the bench) to 24 h. The radiobiological parameters (α, β, RBE at 10% survival) calculated according to the linear-quadratic model were previously published for the four subpopulations studied [16,38]. Following a comparative study for DNA Damage Repair kinetics between X-rays and C-ions, and since RBEs were dependent on both the populations tested and the oxygen concentration, all experiments were performed at an equivalent physical dose of 2 Gy photons [38]. Institutional safety was approved and followed for culture of human cell lines.…”

“…After radiation exposure (30 min to 24 h), cells were fixed for 15 min in 4% PFA, permeabilized, and labeled according to recommendations of the manufacturer for each primary antibody, i.e., 1 h at room temperature (1:1000, mouse anti-phospho-histone H2AX-Ser139 (Merck, Kenilworth, NJ, USA); 1:750 rabbit anti-Rad51 (Abcam, Cambridge, GB); 1:250, rabbit anti-53BP1 (NovusBio, Littleton, CO, USA); 1:250, rabbit anti-phospho-ATM-S1981 (Abcam); 1:50 mouse anti-HIF-1α (BD Transduction, San-José, CA, USA), and 1:500, rabbit anti-phospho-DNA-PKcs-S2056 (Abcam)) and secondary antibodies, i.e., for 1 h in the dark at room temperature (1:500, anti-IgG rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA); 1:250, anti-IgG mouse Alexa Fluor 555 (Abcam)). Nuclei were stained for 15 min with 1 µg/mL DAPI (Sigma, Kanagawa, Japan), then slides were mounted using Fluoromount (Merck) and stored at room temperature in the dark until analysis [38] (n = 2).…”

Succinate Pathway in Head and Neck Squamous Cell Carcinoma: Potential as a Diagnostic and Prognostic Marker

“…Radiotherapy is widely applied in HNSCC, either alone or in multimodal therapy strategies comprising surgery, radiotherapy and chemotherapy. Despite the many technical innovations in recent years, there remains a significant rate of radioresistance in patients [ 35 , 36 ], highlighting an urgent medical need for new concepts in radiotherapy practice. In this regard, it has been suggested that the succinate pathway is related to patient outcome or response to cancer therapy [ 37 ].…”