| [Background]Widespread chromosomal and genomic instability exists in MM.MM is also characterized by deranged DNA repair mechanisms.DNA damaging agents cause DNA strand breaks which are divided into single-strand breaks and double-strand breaks.Single-strand breaks are mainly repaired through the base excision repair pathway,and double-strand breaks are repaired mainly through the HR and NHEJ repair pathways.PARP1 plays an important role in the BER pathway.PARP1 is also involved in HR and NHEJ repair pathways.PARP1 regulates autophagy through the PAR modification and nucleoplasmic translocation of HMGB1.And HMGB1 is also involved in DNA damage repair.PARP1/HMGB1 links DNA damage repair and autophagy.Inhibition of PARP1 can inhibit the BER and repress DNA single-strand breaks repair.These single-strand breaks cannot be repaired in time,leading to the accumulation of fatal double-strand breaks,causing cancer cell proliferation arrest and apoptosis.At the same time,PARP1 inhibitors can down-regulate autophagy through the PARP1/HMGB1 pathway and delay the protective autophagy caused by DNA toxic chemotherapy.Thus the PARP1 inhibitors can enhance the effect of DNA toxic chemotherapeutics and reduce the drug resistance,yielding a synergistic anti-tumor effect.[Objective]Inhibition of PARP1 results in collapsed DNA replication fork and double-strand breaks(DSBs).Accumulation of DSBs goes beyond the capacity of DNA repair response,ultimately resulting in cell death.This work is aimed at assessing the synergistic effects of the DNA-damaging agent temozolomide(TMZ)and the PARP inhibitor niraparib(Nira)in human multiple myeloma(MM)cells and exploring its mechanisms.TMZ induces cytoprotective autophagy by activating PARP1.Inhibition of PARP1 can blunt the autophagy,hinder DDR and favor apoptosis,which can enhance the effect of DNA toxic chemotherapeutics.In this study,we used TMZ and Nira,alone or in combination,to detect the viability,proliferation and apoptosis of multiple myeloma cells after treatment by these agents,in order to provide a novel treatment strategy for MM.[Methods]Exposure of myeloma RPMI8226 and NCI-H929 cells with TMZ and/or Nira for 48 hours in vitro,CCK-8 was utilized for cell viability assessment.S-phase ratio and apoptosis were detected by flow cytometry.Soft-agar clone formation assay was applied to detect its clone formation ability.We then calculated the combination index by CompuSyn software.The expression of DNA damage marker yH2A.X was performed through cell immunofluorescence.We also detected the key elements of DNA damage repair pathway including p-ATM,p-CHK2,RAD51 and yH2A.X by western blot.Autophagy-related indicators were assessed by qRT-PCR,western blot and transmission electron microscopy.Immunofluorescence was used to detect nucleoplasmic translocation of HMGB1 in myeloma cells.Co-immunoprecipitation was performed to detect the interaction between HMGB1 and Beclin-1.A human plasmacytoma xenograft model was established to assess the anti-MM effects in vivo.The body weight and tumor volume of nude mice was recorded.The anti-MM activities of TMZ and/or Nira were evaluated by H&E staining,IHC and the TUNEL assay.[Results]When TMZ was combined with Nira,Nira significantly enhanced the inhibitory effect of TMZ on myeloma RPMI8226 and NCI-H929 cells,and significantly reduced the IC50 of TMZ in myeloma cells.Edu assay and soft agar clone formation experiments showed that TMZ and Nira co-treatment significantly inhibited the proliferation of myeloma cells.Flow cytometry revealed a significant increase of apoptosis in the combination group.Immunofluorescence demonstrated that γH2A.X was increased significantly in the combination group.The key elements of the DNA damage repair pathway including p-ATM,p-CHK2,RAD51 and γH2A.X were significantly increased in the combination group.TMZ alone treatment caused cyto-protective autophagy,leading to a significant increase in autophagy-related genes and proteins.Nira down-regulated autophagy,and when co-treatment with TMZ,it significantly inhibited autophagy caused by TMZ.The expression of cleaved caspase-3 was significantly increased in the combination group.The synergistic effect of TMZ and Nira was mainly through blocking damaged DNA repair and inhibiting protective autophagy.The results demonstrated that co-treatment with TMZ and Nira promoted DNA damage,cell cycle arrest,and apoptotic death in cultured cells.The underlying mechanisms was through suppression DDR and autophagy after inhibition of PARP1 via decreasing PAR modification and nucleoplasmic translocation of HMGB1,inhibiting the formation of HMGB1/Beclin-1 complex.The experiment in vivo showed that,compared with the TMZ or Nira alone group,apoptotic(TUNEL-and cleaved caspase-3-positive)cells in the combination group were extensively increased as well as the morphologic features of apoptosis.On the contrary,proliferative(Ki-67-positive)cells were overtly decreased,in accordance with the previously observed proliferation arrest.Moreover,we demonstrated that γH2A.X and RAD51 expression levels were starkly higher in the combination treatment group.The combination regimen showed no significant weight loss,and the animals showed good general health and activity,with no signs of discomfort,which showed a good tolerability for the Nira and TMZ combination.[Conclusion]PARP1 played a critical role during DNA damage repair and autophagy.PARP1 inhibitor Nira blocked the repair of DNA damage caused by TMZ,resulting in accumulation of lethal double-strand breaks.Nira diminished TMZ-mediated autophagy induction,repressed DDR,caused proliferation arrest and increased apoptosis following TMZ treatment in vitro and in vivo,resulting in increased susceptibility of myeloma cells to TMZ cytotoxicity.Inhibition of PARP1 sensitized genotoxic agents and represented an important therapeutic approach for MM.These findings provided preliminary evidence for combining PARP1 inhibitors with TMZ for MM treatment. |
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