Actylation Of Apurinic/apyrimidinic Endonuclease1 (APE1) Enhances Radiation Sensitivity By Regulating Beclin1-autophagy

Part ?:Role of acetylation of apurinic/apyrimidinic endonuclease1(APE1)in autophagy induced by radiotherapy in He La cellsBackground: Autophagy,namely macroautophagy,is a highly conserved pathway for degradation and recycling of proteins and damaged organelles,which maintains organelle balance and supports cell survival under stress conditions by generating nucleotides,amino acids and other biomolecules.Autophagy is so common in eukaryotic cells,because of the wide range and abundance of stress stimuli,including oxidative stress,radiation and starvation.In cancer,autophagy may play a dual role,growth promotion as well as cell killing,because of the different kinds of stress,induced pathways,cell lines,and so on.Radiotherapy has been widely used in almost 60% of all cancer patients either alone or in combination with other therapy modalities.The cell killing effect of radiotherapy is based on induction of deleterious DNA damage on the one hand and increased production of reactive oxygen species(ROS)including H2O2,O2-and OH-,on the other,which induce further DNA damage.In He La cells,strand breaks are considered to constitute the critical radiation-induced DNA lesions,and radiation-induced autophagy has been thought to effect cell killing further.Little is undeniably known,however,on the relationship between autophagy and irradiation-induced DNA damage.In MCF-7 breast tumor cells,for example,both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental model in response to irradiation.Apurinic/apyrimidinic endonuclease1/redox factor-1(APE1/Ref-1),a key enzyme of DNA base excision repair(BER)pathway,receives particular importance in DNA damage repair.[9] Due to its function in DNA damage repair,its overexpression,in particular in the cell nucleus,is associated with resistance to anticancer radio-and chemotherapy.Acetylation of APE1(AC-APE1)does not alter endonuclease activity,but regulates its transcriptional status.AC-APE1 plays a key role in redox activity of APE1 and activates APE1 related gene transcription during oxidative stress.AC-APE1 has been broadly researched,and DNA damage is also involved in autophagy activation,the relationship between autophagy and acetylation APE1 is still not fully understood,yet.Lysine residues at positions 6 and 7(Lys 6 and Lys 7)of APE1 are important targets of acetylation,which is accomplished by histone acetyltransferase p300.We have used APE1 acetylation mutant K6r/K7 r cells and APE1-WT cells to investigate the effect of AC-APE1 on autophagy induction by irradiation and to uncover its biologic functions.Objectives: Little is undeniably known,however,on the relationship between autophagy and irradiation-induced DNA damage.And,the relationship between AC-APE1 and radiosensitivity is not much known,yet.Methods:Hela cells,APE1KR/K7 R cells and APE1 WT cells after radiation treatment,AC-APE1(APE1 acetylation)and interacetion of Beclin1/Bcl2 was detected by Co-IP(Co-Immunoprecipitation),flow cytometry analyzed Hela cells autophagy level,the LC3 accumulation was observed by Immunofluorescence,western blot measured the expression of APE1?LC3 and p62.Results: We explored the relationship between AC-APE1 and autophagy post radiotherapy by using trichostatin A and k6r/k7 r cell line to regulate AC-APE1.We found that autophagy and AC-APE1 increased in a dose-effect-and time-course-dependent manner following irradiation of He La cells.In particular,AC-APE1 promoted autophagy by dissociation of the binding between Beclin1 and Bcl2.Acetylation of APE1 and autophagy boosted the radiotherapy-induced growth inhibition of He La cells.Conclusion: Taken together,APE1 has been confirmed as a target of radiotherapy in He La cells.Irradiation increased AC-APE1 in a time-and dose-related manner,and AC-APE1 gave rise to induction of autophagy and inhibition of cell growth.In particular,AC-APE1 increased Beclin1-dependent autophagy in response to radiotherapy by dissociation of the binding between Beclin1 and Bcl2.It now will be of peculiar interest to relate the role of AC-APE1 and autophagy with resistance to radiotherapy.Part ?Genistein enhances radiation sensitivity by regulating Beclin1-autophagyBackground: Radiotherapy is an important method used to treat malignant tumors.Recently,radiotherapy has been used with increased precision to reduce the rate of tumor recurrence,avoid an insufficient radiation dose to the tumor,and excessive doses to areas of surrounding normal tissue.An adjuvant drug can be used during radiotherapy to achieve a better clinical outcome.It has been confirmed that genistein(GEN),the main isoflavone component in soybeans,significantly enhances the radiosensitivity of tumor cells,and attenuates inflammatory injuries in normal tissue caused by ionizing radiation(IR).Moreover,some studies have confirmed anti-tumor effects of GEN in both clinical cases and in vitro models of prostate cancer,breast cancer,colon cancer,gastric cancer,lung cancer,pancreatic cancer,and lymphoma.GEN improves the effectiveness of radiotherapy by enhancing the apoptosis rate of cells exposed to ionizing radiation(IR).However,the mechanism by which GEN elevates the rates of apoptosis and autophagy induced by oncotherapy remains unclear.Autophagy,also known as ‘self-eating',is an important area of study in tumor therapy.Autophagy involves a lysosomal degradation pathway that provides nutrients necessary for cell survival,differentiation,development,and homeostasis by degrading superfluous or damaged organelles,misfolded proteins,and invading microorganisms.A low level of autophagy makes cells susceptibility to death when confronted with stressful conditions or agents such as certain metabolites,cytotoxins,and IR.On the other hand,elevated levels of autophagy can deplete materials essential for survival and result in cell death.Under normal conditions,stress activates autophagy to assist in resisting the damaging effects of cancer.However,excessive autophagy stimulates the program death of cancer cells,and is one effect of many anti-tumor therapies.Apoptosis is also a desired effect of anti-tumor therapy,and the relationship between autophagy and apoptosis may depend on the biological context in which these events occur.The dysregulation of apoptosis is a common phenomenon in cancer cells,and is one mechanism by which cancer cells can resist therapy.Bcl-xl,an anti-apoptosis protein,is an important member of the Bcl-2 protein family,whose other members include Bcl-2,caspase protein,and cytochrome C.Within this family of proteins,overexpression of anti-protein Bcl-2,Bcl-xl,and Mcl-1 plays an important role in development of apoptotic resistance induced by radio-and chemo-therapy.Bcl-xl,a member of the Bcl-2 family,is often overexpressed in both non-small cell and small cell lung cancers and exerts an anti-apoptotic effect.Bcl-xl prevents apoptosis by a mechanism similar to that described for Bcl-2.Bcl-xl regulates mitochondrial membrane potential and volume,inhibits the release of cytochrome c,and promotes the release of apoptosis inhibitory factors into the cell cytoplasm.Bcl-xl expression is associated with resistance against cancer therapy,Bcl-xl and expression protects cells against UV radiation–induced apoptosis.The combined use of IR and a Bcl-xl inhibitor exerts a synergistic effect by activating the bak-apoptosis pathway in cell lines resistant to apoptosis.This mechanism represents a potential novel therapeutic strategy.Bcl-xl also regulates levels of cellular autophagy level by interacting with beclin1 Beclin1 to halt the initiation of autophagy.Blocking Bcl-xl expression with a specific si RNAs can activate autophagy and promote cell death,suggesting that Bcl-xl plays an important role in crosstalk between autophagy and apoptosis.Thus we believe that Bcl-xl is a key molecular target for cancer therapy.Objectives: In this part,we investigated the effect of differential distribution of Bcl-xl induced by GEN on cell death by examining the activation of autophagy and apoptosis in non-small cell lung cancer cell.Results: We initially verified the differentiation of cytoplasmic Bcl-xl in GEN-treated A549 cells.By examining the level of cytoplasmic Bcl-xl in H1975,A549,Calu,and 293 T cells,we found that high level of cytoplasmic Bcl-xl was associated with a significant resistance to the cell death caused by ionizing radiation.Furthermore,the rates of 0,1+,2+,and 3+ intensity scores for cytoplasmic expression of Bcl-xl in biopsies of human non-small cell lung cancer tissue were 26.7,43.3,26.7,and 3.3%,respectively,clearly showing that tumor tissue with high level of cytoplasmic Bcl-xl had a low objective response rate(ORR)to radiotherapy.Interestingly,we also found that combined treatment with GEN and IR inhibited level of cytoplasmic Bcl-xl and increased the autophagy-associated cell death rate by promoting dissociation of the Bcl-xl/Beclin1 complex.Additionally,this combined treatment significantly inhibited the repair of damaged DNA and contributed to oligomerization of Bax and activation of cleaved caspase-3 and PARP1.Conclusion: our present study showed that a reduction in cytoplasmic Bcl-xl levels in A549 cells after GEN treatment is beneficial,because it contributes to cell death induced by radiation therapy.We also showed that cell death was stimulated by activating cellular apoptosis and autophagy pathways,and inhibiting the repair of damaged DNA.Additional,we confirmed the correlation between cytoplasmic Bcl-xl expression and the response of tumor cells to radiotherapy.Treatment with GEN and radiation induced apoptosis partially by affecting the autophagy pathway and inhibiting cytoplasmic bcl-xl expression.That treatment also induced autophagy by inhibiting the interaction between bcl-xl and Beclin1.Our results suggest a new scenario in which the subcellular distribution of Bcl-xl after GEN treatment affects cell death induced by IR,and also DNA repair,mitochondrial maintenance,and regulation of autophagy.