Background To evaluate factors associated with osteoradionecrosis of the jaw (ORNJ) in patients with head and neck squamous cell carcinoma (HNSCC), focusing on jaw-related dose–volume histogram (DVH) parameters. Methods We retrospectively reviewed the medical records of 616 patients with HNSCC treated with curative-intent or postoperative radiation therapy (RT) during 2008–2018. Patient-related (age, sex, history of smoking or alcohol use, diabetes mellitus, performance status, pre-RT dental evaluation, pre- or post-RT tooth extraction), tumor-related (primary tumor site, T-stage, nodal status), and treatment-related (pre-RT surgery, pre-RT mandible surgery, induction or concurrent chemotherapy, RT technique) variables and DVH parameters (relative volumes of the jaw exposed to doses of 10 Gy–70 Gy [V10–70]) were investigated and compared between patients with and without ORNJ. The Mann–Whitney U test was used to compare RT dose parameters. Univariate and multivariate Cox regression analyses were used to assess factors associated with ORNJ development. Kaplan–Meier analyses were performed for cumulative ORNJ incidence estimation. Results Forty-six patients (7.5%) developed ORNJ. The median follow-up duration was 40 (range 3–145) months. The median time to ORNJ development was 27 (range 2–127) months. DVH analysis revealed that V30–V70 values were significantly higher in patients with than in those without ORNJ. In univariate analyses, primary tumor site, pre-RT mandible surgery, post-RT tooth extraction, and V60 > 14% were identified as important factors. In multivariate analyses, V60 > 14% (p = 0.0065) and primary tumor site (p = 0.0059) remained significant. The 3-year cumulative ORNJ incidence rates were 2.5% and 8.6% in patients with V60 ≤ 14% and > 14%, respectively (p < 0.0001), and 9.3% and 1.4% in patients with oropharyngeal or oral cancer and other cancers, respectively (p < 0.0001). Conclusions V60 > 14% and oropharyngeal or oral cancer were found to be independent risk factors for ORNJ. These findings might be useful to minimize ORNJ incidence in HNSCC treated with curative RT.

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The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane-enclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa-2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome-specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn- and Mn-superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa-2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non-irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa-2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up- and five downregulated miRNAs were identified in 5 and 8 Gy-irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription-quantitative PCR identified miR-6823-5p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.

Micro-slit-beam radiation therapy (MRT) using synchrotron-generated X-ray beams allows for extremely high-dose irradiation. However, the toxicity of MRT in central nervous system (CNS) use is still unknown. To gather baseline toxicological data, we evaluated mortality in normal mice following CNS-targeted MRT. Male C57BL/6 J mice were head-fixed in a stereotaxic frame. Synchrotron X-ray-beam radiation was provided by the SPring-8 BL28B2 beam-line. For MRT, radiation was delivered to groups of mice in a 10 × 12 mm unidirectional array consisting of 25-μm-wide beams spaced 100, 200 or 300 μm apart; another group of mice received the equivalent broad-beam radiation therapy (BRT) for comparison. Peak and valley dose rates of the MRT were 120 and 0.7 Gy/s, respectively. Delivered doses were 96–960 Gy for MRT, and 24–120 Gy for BRT. Mortality was monitored for 90 days post-irradiation. Brain tissue was stained using hematoxylin and eosin to evaluate neural structure. Demyelination was evaluated by Klüver–Barrera staining. The LD50 and LD100 when using MRT were 600 Gy and 720 Gy, respectively, and when using BRT they were 80 Gy and 96 Gy, respectively. In MRT, mortality decreased as the center-to-center beam spacing increased from 100 μm to 300 μm. Cortical architecture was well preserved in MRT, whereas BRT induced various degrees of cerebral hemorrhage and demyelination. MRT was able to deliver extremely high doses of radiation, while still minimizing neuronal death. The valley doses, influenced by beam spacing and irradiated dose, could represent important survival factors for MRT.

BackgroundIn our previous study, monogalactosyl diacylglycerol (MGDG) purified from spinach was found to have cytotoxic effects in human cancer cell lines. This study further assessed whether MGDG can enhance the cytotoxic effects of radiation in human pancreatic cancer cells in vitro and in vivo.MethodsGlycoglycerolipids from spinach including MGDG were extracted from dried spinach. The cytotoxicity of MGDG were evaluated by the MTT assay using four human pancreatic cancer cell lines (MIAPaCa-2, AsPC-1, BxPC-3 and PANC-1) and normal human dermal fibroblasts (NHDFs). The effects of radiation and MGDG alone or in combination in MIAPaCa-2 cells was analyzed with the colony forming and apoptosis assays, western blotting and cell cycle and DNA damage analyses (γ-H2AX foci staining and comet assay). The inhibitory effects on tumor growth were assessed in a mouse xenograft tumor model.ResultsMGDG showed dose- and time-dependent cytotoxicity, with half-maximal inhibitory concentrations (IC50) in PANC-1, BxPC-3, MIAPaCa-2 and AsPC-1 cells at 72 h of 25.6 ± 2.5, 26.9 ± 1.3, 18.5 ± 1.7, and 22.7 ± 1.9 μM, respectively. The colony forming assay revealed fewer MIAPaCa-2, BxPC-3 and AsPC-1 cell colonies upon treatment with both MGDG and radiation as compared to irradiation alone (P < 0.05). The combination of MGDG and radiation induced a higher proportion of apoptosis in MIAPaCa-2 cells; this effect was associated with increased mitochondrial release of cytochrome c and activation of cleaved poly (ADP-ribose) polymerase and caspase-3. DNA damage was detected and DNA repair mechanisms were more frequently impaired in cells receiving the combination treatment as compared to either one alone. Tumor growth was inhibited to a greater degree in mice treated by intratumoral injection of MGDG combined with irradiation as compared to either one alone (P < 0.05).ConclusionsThis is the first report demonstrating that MGDG enhances the cytotoxicity of radiation to induce apoptosis of cancer cells in vitro and in vivo. Our findings indicate that this therapeutic combination can be an effective strategy for the treatment of pancreatic cancer.