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.

5-Acetyl-6,7,8,4′-tetramethylnortangeretin induces apoptosis in multiple myeloma U266 cells

Multiple myeloma(MM)is one of the most common hematological malignancies and to date,it remains an incurable disease.In this study,we evaluated the inhibitory effect of 5-acetyloxy-6,7,8,4′-tetramethoxyflavone(5-ATMF),a compound from aged citrus peel extracts,on the MM U266 cell line.We found that the compound inhibited cell growth and induced cell apoptosis in multiple apoptotic assays.The apoptotic proteins caspase-3,caspase-9,and PARP(poly ADP-ribose polymerase)were cleaved when cells were treated with 5-ATMF.Along with the apoptosis process,the anti-apoptotic protein,Bcl-2(B-cell lymphoma-2),was significantly downregulated and the pro-apoptotic protein,Bax(Bcl-2 associated X protein),upregulated along with the release of cytochrome C(Cyt c)and the reduction of mitochondrial membrane potential(MMP).Notably,we found that the phosphorylation of Bad(Bcl-2/Bcl-XL-associated death promoter)was decreased but Bad remained unchanged in this process.On adding 5-ATMF to U266 cells,we observed that 5-ATMF repressed the phosphorylation of Akt(PKB Protein Kinase B PKB),thereby increasing the release of Cyt c and inhibiting the phosphorylation of Bad.These effects were enhanced by Ly294002,an inhibitor of Akt.These results suggested that 5-ATMF exerts a pro-apoptotic effect on U266 cells,possibly associated with the mitochondrial apoptotic pathway induced by the PI3K/Akt/Bad pathway.