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  • The repair pathway of DNA DSBs through HR and

    2020-01-15

    The repair pathway of DNA-DSBs through HR and the role of BRCA1 in repair kinetics by HR is also an important factor to unravel the radiation sensitization of novel small molecule inhibitors. The involvement of the breast cancer susceptibility gene 1 (BRCA1) in tumorigenesis and its role in facilitating DNA resection and HR during S and G2 phases of the Diclazuril has been well documented (Yu and Chen, 2004, Huen et al., 2010). BRCA1 is involved in the activation of cell cycle check points and plays a major part in maintaining chromosomal stability. Radiation exposure phosphorylates BRCA1 at S1423 thus facilitating DSB repair by the HR pathway. Inhibition of these kinase enzymes through a targeted approach not only sensitizes cancer cells to radiation, but also results in a decrease in cancer cell multiplication rate (Kim et al., 2002, Pajonk et al., 2005). In the absence of DNA repair cells will undergo apoptosis (Martin, 2001).
    Material and methods
    Results
    Discussion In this study, we analysed the radiation sensitization and mechanism of action of two novel substituted benzoxazines, LTU28 and LTU31. Both compounds showed a dose dependent radiation sensitization in both HT29 and A549 cell lines with the maximum effect observed at 0.3 μM for both the compounds. Previous studies have shown the DNA-PK inhibitor NU7441 sensitized breast cancer cell lines to radiation and doxorubicin and retarded the repair of DSBs (Zhao et al., 2006, Shaheen et al., 2011, Ciszewski et al., 2014). To confirm that LTU28 and LTU31 are radiosensitizing the cells by inhibiting the repair of DSBs, we analysed the gamma H2AX levels to quantify DSBs. The histone H2AX plays a key role in DNA-DSB repair by rapidly phosphorylating at serine residues to form gamma H2AX foci formation near the DSBs (Redon, Boon, Johnson, Bonner, & Rogakou, 1999). A previous study has reported that hypoxia can also induce phosphorylation of H2AX to gamma H2AX Diclazuril (Wrann, Kaufmann, Wirthner, Stiehl, & Wenger, 2013). However, measuring phosphorylated H2AX is considered to be a reliable method for analysing the presence of DSBs and various studies have related the effect of gamma H2AX and DSB repair kinetics (Wang et al., 2005, Ismail et al., 2007, Prendergast et al., 2011). In our study, both LTU28 and LTU31 prolonged the presence of gamma H2AX and delayed DSB repair for 24 h. At 0.3 μM, LTU28 showed slightly more potency than LTU31 in inhibiting DSB repair following radiation. The number of DSBs was slightly higher in HT29 following radiation alone and treatment with LTU28 + 6Gy but was less in A549 cells. A few studies have shown differences in expression levels of gamma H2AX after radiation exposure (MacPhail et al., 2003, Francisco et al., 2008). Variations in DSB repair and differences in DNA-PKcs levels have been reported earlier in a radiosensitization study of ovarian cancer cell lines. (Langland et al., 2010). The cell line dependent variations in various enzyme expression levels could be a factor in the differences in the degree of DSB repair inhibition seen in this study. The delay in DSB repair after treatment with LTU28 and LTU31 in combination with radiation was accompanied by an increase in cell death which was measured as hypodiploidy (sub G1). Previous studies have shown the presence of DNA fragmentation or hypodiploidy expressed as sub G1 to be a reliable marker for cell death (Sun et al., 2010, Yu et al., 2011). Our data showed that the percentage of DNA fragmentation was higher after treating both the cell lines with LTU28 + 6Gy but LTU31 + 6Gy did not show the same effect. To analyse the amount of apoptosis represented in this cell death, an apoptosis assay using a monoclonal antibody to single stranded DNA was performed (Kunkl et al., 2000, Bressenot et al., 2013). Our results showed that the cell death induced by LTU31 in combination with radiation was mostly through apoptosis and was higher than in the treatment group LTU28 + 6Gy.