Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor, NU7441

Abstract

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.

Modulation of ionizing radiation-induced effects by NU7441, KU55933 and VE821 in peripheral blood lymphocytes

“…On the other hand, it significantly increased radiation-induced cytotoxicity and genotoxicity, indicating no direct relation between early H2AX phosphorylation and DNA damage repair capacity in human peripheral lymphocytes. Our results and data reported by others show NU7441 to be a potent radiosensitizing agent (Song et al, 2013;Ciszewski et al, 2014;Tichy et al, 2014). However, lymphocytes and highly differentiated (G 0/ G 1 ) tissues might be significantly affected by its non-selective action.…”

“…NU7441 has not yet been used in oncological therapy. However, in vitro studies have demonstrated its ability to enhance the cytotoxicity of IR in cancer cell lines while expressing low toxicity in vivo, which validates this substance for further testing (Nutley et al, 2005;Zhao et al, 2006;Yu et al, 2012;Tichy et al, 2014). Another PI3-K inhibitor is KU55933.…”

NU7441 Enhances the Radiosensitivity of Liver Cancer Cells

“…NU7441, a derivative from LY294002, is a synthetic and specific DNA-PK inhibitor. In in vitro experiments, NU7441 exerted radiosensitizing activity in breast cancer, leukemia, and prostate cancer [15][16][17]. However, no studies have been performed to examine the effect of NU7441 on liver cancer cells.…”

DNA-PKcs interference sensitizes colorectal cancer cells to a mTOR kinase inhibitor WAY-600

“…1A). Significantly, two DNA-PKcs inhibitors, NU7026 [21] and NU7441 [22], remarkably enhanced WAY-600-mediated activity, resulting in dramatic HT-29 cell death (Fig. 1A).…”