BLM helicase inhibition synergizes with PARP inhibition to improve the radiosensitivity of olaparib resistant non-small cell lung cancer cells by inhibiting homologous recombination repair

Objective:We aimed to investigate the radiosensitizing efficacy of the poly-ADP-ribose polymerase(PARP)inhibitor,olaparib,and the Bloom syndrome protein(BLM)helicase inhibitor,ML216,in non-small cell lung cancer(NSCLC)cells.Methods:Radiosensitization of NSCLC cells was assessed by colony formation and tumor growth assays.Mechanistically,the effects of ML216,olaparib,and radiation on cell and tumor proliferation,DNA damage,cell cycle,apoptosis,homologous recombination(HR)repair,and non-homologous end joining(NHEJ)repair activity were determined.Results:Both olaparib and ML216 enhanced the radiosensitivities of olaparib-sensitive H460 and H1299 cells,which was seen as decreased surviving fractions and Rad51 foci,increased total DNA damage,andγH2AX and 53BP1 foci(P<0.05).The expressions of HR repair proteins were remarkably decreased in olaparib-treated H460 and H1299 cells after irradiation(P<0.05),while olaparib combined with ML216 exerted a synergistic radiosensitization effect on olaparib-resistant A549 cells.In addition to increases of double strand break(DSB)damage and decreases of Rad51 foci,olaparib combined with ML216 also increased pDNA-PKcs(S2056)foci,abrogated G2 cell cycle arrest,and induced apoptosis in A549 lung cancer after irradiation in vitro and in vivo(P<0.05).Moreover,Western blot showed that olaparib combined with ML216 and irradiation inhibited HR repair,promoted NHEJ repair,and inactivated cell cycle checkpoint signals both in vitro and in vivo(P<0.05).Conclusions:Taken together,these results showed the efficacy of PARP and BLM helicase inhibitors for radiosensitizing NSCLC cells,and supported the model that BLM inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization,as well as providing the basis for the potential clinical development of this combination for tumors intrinsically resistant to PARP inhibitors and radiotherapy.

Self-synthesized second mitochondria-derived activator of caspase(SMAC)mimetic TP-WY-1345 enhances the radiosensitivity of NSCLC cells H1299 by targeting anti-apoptotic protein cIAP1

Objective:To explore the toxicity and cellular uptake of the self-synthesized second mitochondria-derived activator of caspase(SMAC)mimetic TP-WY-1345 polypeptide and its radiosensitizing effect on non-small cell lung cancer(NSCLC).Methods:A fluorescence microscope was used to observe the uptake efficiency of TP-WY-1345 by human NSCLC cells H1299.Toxicity of the TP-WY-1345 peptide in normal cells was examined in human embryonic lung fibroblasts MRC5.CCK-8 and clone formation experiments were performed to detect the radiosensitizing effect of TP-WY-1345 in the H1299 cells.The AutoDock Vina simulation software was employed to predict the binding efficiency of TP-WY-1345 to the inhibitor of apoptosis proteins(IAPs).Moreover,Western blot and qPCR experiments were carried out to determine the protein and gene expressions,and the flow cytometry(FCM)technique was used to detect the apoptosis level under different conditions.Results:TP-WY-1345 can be self-synthesized,and significant green fluorescence was observed in H12992 h and 6 h after incubation with TP-WY-1345.The cell counting and CCK-8 results showed that 10μmol/L and 20μmol/L TP-WY-1345 did not produce a significant toxic effect on the MRC5 cells(P>0.05).Compared with the single ionizing radiation group,the cell viability and clone formation of H1299 cells were significantly inhibited after treatment with TP-WY-1345 at a concentration of 50μmol/L(P<0.05).The sensitivity enhancement ratio(SER)was calculated to be 1.14.Moreover,the binding efficiency of TP-WY-1345 to cIAP1 protein was predicted to be7.3,and this strong binding force was demonstrated by Western blot.TP-WY-1345 inhibited the protein and mRNA expressions of cIAP1 and further increased the apoptosis level of the H1299 cells at 24 h(P<0.01)and 48 h after radiation(P<0.05).Conclusion:SMAC mimetic TP-WY-1345 can enter the H1299 cells and produce a radiosensitizing effect by increasing the level of radiation-induced apoptosis of the cells.There,TP-WY-1345 is expected to become a new generation of radiosensitizing drugs.