Inhibiting nonhomologous end-joining repair would promote the antitumor activity of gemcitabine in nonsmall cell lung cancer cell lines

Abstract

Nonsmall cell lung cancer (NSCLC) is a major type of lung cancer. In current study, we aim to evaluate whether the combination of Ku70/80 heterodimer protein inhibitor STL127705 and gemcitabine would be more favorable approach for the treatment of NSCLC compared with monotreatment with gemcitabine. Clongenic survival assay was used to determine the survival and sensitivity to irradiation. H1299 was stained with fluorescein isothiocyanate-Annexin V, and cell apoptosis was measured by flow cytometry. H1299 cells were transfected with nonhomologous end-joining (NHEJ) repair reporter, and stable cell line was selected by puromycin. NHEJ activity was determined based on the intensity of green fluorescent protein. DNA double-strand breaks (DSBs) were determined by the fluorescence intensity of γH2AX using flow cytometry. The mRNA expressions of Ku70 and Ku80 were determined using quantitative real-time PCR. Combination of STL127705 enhanced sensitivity of NSCLC cell lines to irradiation when compared with treatment with gemcitabine alone. However, small cell lung cancer cell line was not affected. H1299 cells treated with STL127705 in combination with gemcitabine showed a significantly increased apoptosis compared with H1299 cells treated with gemcitabine alone. Moreover, STL127705 treatment dramatically reduced NHEJ activity in H1299 cells when compared with gemcitabine single treatment. Increased DSBs were consistently observed in H1299 when treated with the combination of STL127705 and gemcitabine. However, the mRNA levels of Ku70 and Ku80 were upregulated by the combination treatment. It demonstrated that STL127705 enhanced antitumor activity of gemcitabine. Mechanistically, treatment with STL127705 enhanced DNA damage via inhibiting NHEJ pathway, blocking DNA-PK, and forming Ku70/80 heterodimer, eventually leading to tumor cells apoptosis.

Improving homology‐directed repair by small molecule agents for genetic engineering in unconventional yeast?—Learning from the engineering of mammalian systems

“…Mammalian and yeast Ku70/80 are conserved and its deletion has shown significant enhancement of HR‐mediated genome editing in many species, including yeast (Arras & Fraser, 2016 ; Kooistra et al., 2004 ; Nayak et al., 2006 ; Ninomiya et al., 2004 ). A computational small‐molecule screen against a potential binding pocket within the human Ku70/80 heterodimer yielded the small molecule STL127705, which showed strong inhibition of Ku70/80 binding to DNA and inhibition of the Ku‐dependent activation of the DNA‐PKcs kinase in vitro and in human cell lines, as well as higher cellular susceptible to radiation, indicating a clear potential to diminish DSB repair by NHEJ (Guo et al., 2022 ; Weterings et al., 2016 ). STL127705 has not been tested in genome editing studies.…”