CK conducted the experiments
CK conducted the experiments. for both radiation qualities. For 100?nM DNAPKi the survival ratio at 4?Gy more GnRH Associated Peptide (GAP) (1-13), human than doubled from 1.59 under normoxia to 3.3 under hypoxia revealing a strong radiosensitizing effect under hypoxic conditions. In contrast, this ratio only moderately increased after photon irradiation and ATMi under hypoxia. The most effective treatment was combined carbon ion irradiation and DNA damage repair inhibition. Conclusions Carbon ions efficiently eradicate hypoxic tumor cells. Both, ATMi and DNAPKi elicit radiosensitizing effects. DNAPKi preferentially sensitizes hypoxic cells to radiotherapy. Electronic supplementary material The online version of this article (10.1186/s13014-017-0939-0) contains supplementary material, which is available to authorized users. simulation of the Heidelberg Ion Beam Therapy (HIT) beam-line . Dose maps were generated, with dose uniformity found to be within 2% range in the SOBP region. Carbon dose levels for planned 1, 2, 4 and 6?Gy were corrected accordingly to actual prescribed 0.95, 1.9, 3.8, and 5.64?Gy. Software and calculations The survival fractions derived from the clonogenic survival data were fitted according to the linear-quadratic model for photons. A linear model was applied to carbon ion data. The fits as well as OER, RBE, and SER values (Additional?file?1: Table S5 and Table S6) were calculated using an in-house tool based on Minuit package available in ROOT . PE values were plotted with GraphPad Prism 5. To display the oxygen effect, the relative effect of carbon ions, and the sensitization effect of inhibitors, measured data points were used to determine ratios of clonogenic survival at a corresponding dose: Ratios were calculated as survival fractions of hypoxic cells and normoxic cells; survival fractions of cells irradiated with photons and cells irradiated with carbon ions; survival fractions of mock-treated cells and cells treated with inhibitors at the same dose, respectively. Effects were compared at a preferential dose of 4?Gy being a reasonable dose for patients in fractionated therapy. Statistics Data are presented as means and standard deviations (SD). Statistical significance was determined using unpaired (two-tailed). The asterisks represent significantly different values. Data represent average values of at least three independent experiments, each performed with technical quadruplicates (n:4). Results Oxygen effect and relative GnRH Associated Peptide (GAP) (1-13), human effect for photon vs. carbon irradiation under hypoxia Hypoxia increased the survival fraction of A549 cells significantly (between 1.36 to 2.34-fold) at photon doses 4?Gy under hypoxia vs. normoxia (SF survival fraction at indicated dose Table 2 Relative effect of photons vs. carbon ions for A549 cells at the indicated dose SF4Gy survival fraction at 4?Gy photons and 3.8?Gy carbon ions Preferential Radiosensitization of hypoxic cells to GnRH Associated Peptide (GAP) (1-13), human DNAPKi Next, we investigated the inherent and radiosensitizing effect of two novel DNAPK and ATM serine-threonine kinase inhibitors. The PE was not significantly reduced after ATMi treatment. The PE was only significantly reduced by 15% after 1000?nM of DNAPKi (Fig.?2). This is in line with the reported high selectivity and on target potency of these compounds: DNAPKi (M3814) is a highly potent and selective inhibitor of DNA-PK GnRH Associated Peptide (GAP) (1-13), human with subnanomolar potency on its target [20, 21]. The split to closely related PIKK proteins has been measured in biochemical assays and is about 150-fold to PI3K delta and greater than 400-fold to the other family members (ATM, PI3Kalpha C delta, mTOR). The preclinical ATM inhibitor tested is a subnanomolar potent inhibitor with 50-fold selectivity over DNA-PK and greater than 1000-fold selectivity against the other PIKK family members (ATR, PI3Kalpha C GnRH Associated Peptide (GAP) (1-13), human delta, mTOR). Open in a separate window Fig. 2 Lack of cytotoxicity of utilized ATMi and DNAPKi alone at pharmacologically relevant doses. PE of A549 cells Rabbit polyclonal to KIAA0317 after treatment with dose series of ATMi (light grey).