The Mechanism Of Aspirin To Inhibit Glycolysis-Mediated NSCLC Proliferation And Angiogenesis

Lung cancer becomes the highest mortality and the second highest morbidity cancer in the world.Non-small cell lung cancer(NSCLC),accounting for about 80% of lung cancer,receives widely attention because of its poor prognosis and low survival rate.In NSCLC patients,the higher the glycolysis level,the larger the tumor size,which makes glycolysis a possible target for diagnosis and treatment of NSCLC.Non-steroidal anti-inflammatory drug aspirin is clinically used for treatment of pain,fever,inflammation and prevention of cardiovascular diseases.Many researchers have conducted the secondary analysis from the clinical data of aspirin in treatment of cardiovascular and cerebrovascular diseases,and have found that aspirin reduces the morbidity and mortality of various solid cancers,such as colorectal cancer,breast cancer,gastric cancer and lung cancer.A lot of experimental studies have proved that aspirin promotes apoptosis and inhibits proliferation,migration,stemness,angiogenesis and cancer-related inflammation in NSCLC cells,but the effect and mechanism of aspirin on aerobic glycolysis of NSCLC is less reported.In the present study,we investigated the inhibitory effect and mechanism of aspirin on aerobic glycolysis and proliferation in NSCLC A549 and NCI-H1299 cells and xenograft model in nude mice.First,A549 and H1299 cells were treated with 0,1,2,4,6,8 and 10 mM aspirin for 24,48 and 72 h,respectively.MTT results showed that aspirin inhibited the cell viability of A549 and H1299 in dose-and time-dependent manners.The cell viability of the two cell lines decreased by about 50% after 8 mM aspirin treated for 24 h.Therefore,NSCLC cells were treated with 8 mM aspirin for 24 h in the follow-up experiments.Then,the results of western blot and colony formation assays showed that the PCNA expression and the number of forming colonies of cancer cells were decreased after aspirin treatment,which further demonstrated that aspirin inhibited the proliferation of NSCLC cells.NSCLC cells follow the “Warburg Effect”and preferentially utilize glycolysis to generate material and energy even under aerobic conditions.Next,we detected the effect of aspirin on the production of pyruvic acid and lactic acid and biochemical results showed that aspirin treatment significantly inhibited production of them in A549 and H1299 cells.2-DG,a glycolysis inhibitor,was used to treat the cells alone or in combination with aspirin and the results showed that both 2-DG and aspirin reduced the PCNA expression and the combination of 2-DG and aspirin did not further reduce the PCNA expression in the cancer cells.These results suggested that aspirin inhibited NSCLC cells proliferation by blocking glycolysis.Hexokinase 2(HK2),which catalyzes the first step of glycolysis reaction,is usually upregulated in most cancer cells and promotes the conversion of metabolic reprogramming to aerobic glycolysis.Its localization on mitochondrial outer membrane is necessary for its catalytic activity.Impaired mitochondrial function may result in the release of HK2 from the outer membrane of mitochondria into the cytoplasm and the loss of catalytic activity.JC-1 staining,DCFH-DA fluorescent probe staining and western blot assays were performed to test the effect of aspirin on mitochondrial function.The results showed that aspirin downregulated the mitochondrial membrane potential,reactive oxygen species(ROS)level and PGC-1α expression,indicating that aspirin induced mitochondrial dysfunction in NSCLC cells.SIRT3 is a histone deacetylase in mitochondria,which plays an important role in regulating mitochondrial function.The results of western blot and SIRT3 activity assay showed that aspirin increased SIRT3 expression and also enhanced SIRT3 activity.Then SIRT3 small interfering RNA(siRNA)was used to knockdown of SIRT3 in A549 and H1299 cells to explore the role of SIRT3 in aspirin exerted to mitochondrial dysfunction and glycolysis.The results showed that knockdown of SIRT3 reversed the downregulation of ROS levels,production of pyruvic acid and lactate by aspirin treatment.These results indicated that aspirin induced mitochondrial dysfunction and inhibited glycolysis through upregulation of SIRT3.Cyclophilin D(CypD)in the mitochondrial matrix probably involves in SIRT3-regulated HK2 localization.Thus,we supposed that aspirin inhibited glycolysis by SIRT3/CypD/HK2 signaling.By immunoprecipitation assay,we demonstrated that aspirin inhibited the acetylation of CypD,and knockdown of SIRT3 impeded the inhibition of CypD deacetylation in A549 and H1299 cells.Mitochondria were isolated from cytoplasm with mitochondrial separation kit to detect the effect of aspirin on HK2 expression in mitochondria of the cancer cells.The western blot results showed that both total HK2 expression and the HK2 expression in mitochondria was downregulated,and the HK2 expression in cytoplasm was upregulated in A549 and H1299 cells after aspirin treatment.After knockdown of SIRT3,the aspirin-induced above changes of HK2 localization between mitochondria and cytoplasm were disappeared in H1299 cell.These results suggested that aspirin inhibited cell proliferation by blocking SIRT3/CypD/HK2-mediated glycolysis in NSCLC cells.AMPK,an energy sensor,is an important anti-cancer target of aspirin.Our western blot results showed that the expression of p-AMPK(Thr172)and p-ACC(Ser79)were upregulated after 8 mM aspirin treatment in A549 and H1299 cells for 24 h.By using the inhibitor of AMPK activity,compound C(CC),and SIRT3 siRNA,we detected the regulatory relationship between AMPK and SIRT3 under the action of aspirin.The results of western blot and SIRT3 activity assay showed that CC reversed the upregulation of SIRT3 induced by aspirin,but after knockdown of SIRT3,aspirin still increased the expression of p-AMPK and p-ACC in cancer cells.These indicated that aspirin upregulated SIRT3 expression and activity via activating AMPK signaling and activated the AMPK/SIRT3 pathway.Next,cancer cells were treated with CC or SIRT3 siRNA alone or in combination with aspirin to investigate the role of AMPK/SIRT3 during aspirin-inhibited glycolysis and proliferation of NSCLC cells.Biochemical and western blot results showed that CC or SIRT3 siRNA weakened the inhibitory effects of aspirin on the production of pyruvic acid and lactate and the expression of PCNA,suggesting that aspirin-induced inhibition on cellular glycolysis and proliferation were dependent on the activated AMPK/SIRT3 pathway in NSCLC cells.Based on the above results in vitro,we employed xenograft model in BALB/C nude mice using A549 cells and the results showed that,the volume and weight of xenograft in nude mice were downregulated by 100 mg/kg aspirin treatment for 24 days.Meanwhile,aspirin upregulated the protein expression of p-AMPK,p-ACC,SIRT3 and downregulated the protein level of HK2 in the xenograft tumors.So,aspirin inhibited NSCLC cell proliferation in vitro and xenograft growth in vivo by blocking AMPK/SIRT3-mediated glycolysis.Angiogenesis is the process by which new blood vessels sprout from existing ones.Tumor cells secreted various pro-angiogenic factors into the tumor microenvironment,and these factors bind to the corresponding receptors in endothelial cells and activate proliferation,migration and tube formation pathway to promote the angiogenesis of endothelial cells.Angiogenesis is essential for the growth of solid tumor because it provides oxygen and nutrients for tumor growth and channels for tumor metastasis.Impaired angiogenesis leads to dormancy or necrosis of tumor cells.Therefore,inhibition of tumor angiogenesis is an important strategy for anti-tumor therapy.There are few studies involving the effect and mechanism of aspirin on angiogenesis of endothelial cells,and extensive and in-depth studies are still needed.In the present study,we investigated the effects of aspirin on angiogenesis in vitro and in vivo by using human umbilical vein endothelial cells(HUVECs),chicken embryo allantoic membrane(CAM)model and xenograft model in BALB/C nude mice.HUVECs were treated with 0,0.1,0.25,0.5,1,2,4 and 8 mM aspirin for 24 h,and MTT results showed that aspirin with a concentration greater than or equal to 1 mM significantly inhibited cell viability.After treated with 4 mM aspirin for 24 h,the cell viability was close to 50% of that of the control group.Therefore,HUVECs were treated with 4 mM aspirin for 24 h in the follow-up experiments.The results of scratch assay showed that aspirin treatment reduced the migration area of endothelial cells to scratch region,and transwell assay was performed to further prove that aspirin inhibited the migration of endothelial cells.The results of tube formation assay showed that the number of forming tube of endothelial cells was reduced after aspirin treatment.To explore the mechanism of aspirin on inhibiting angiogenesis of HUVECs,transcriptome sequencing and bioinformatics analysis were performed.By comparing the transcriptome gene expression of aspirin treated group and control group after HUVECs treated with 4 mM aspirin for 24 h,913 differentially expressed genes(DEGs)were screened out.GO functional enrichment analysis identified 313 items and KEGG signaling pathway analysis identified 20 KEGG pathways.We screened out 44 DEGs related to cell proliferation,migration and angiogenesis from the 913 DEGs,including the transcription factor GATA3 and the co-receptor NRP1.Similar to epithelial-mesenchymal transition(EMT),endothelium-mesenchymal transformation(EndMT)refers to cells lose their endothelial polarity and markers,then acquire mesenchymal markers and characteristics of invasion and migration.Transcription factor GATA3 is reported to inhibit the EMT in tumor cells,but the effect of GATA3 in HUVECs is less studied.We found that aspirin treatment upregulated the expression of GATA3 in the screened DEGs,and quantitative PCR and western blot assays verified that the mRNA and protein expression of GATA3 in HUVECs were increased after aspirin treatment.HUVECs were treated with GATA3 siRNA to explore the role of GATA3 in aspirin-inhibited migration of endothelial cells.Western blot results showed that knockdown of GATA3 reduced the expression of endothelial marker VE-cadherin and enhanced the expression of mesenchymal marker Snai1.Transwell results showed that knockdown of GATA3 eliminated the inhibitory effect of aspirin on HUVECs migration.These results suggested that aspirin restricted cell migration by GATA3-inhibited EndMT process in HUVECs.In the screened DEGs and protein-protein interaction(PPI)analysis,NRP1 expression was significantly downregulated in aspirin treated group,which was confirmed by quantitative PCR and western blot assays.As a co-receptor,NRP1 forms complex with a variety of ligand-receptor couples both in endothelial cells and tumor cells to promote angiogenesis.We used NRP1 siRNA to explore the role of NRP1 in aspirin inhibited angiogenesis.The result of tube formation assay showed that aspirin or NRP1 siRNA treated alone reduced the number of forming tube of HUVECs,and aspirin combined with NRP1 siRNA treated did not further reduce the number of forming tube compared with the two alone.These results suggested that aspirin inhibited the tube formation of HUVECs through the NRP1 signaling.We supposed that transcription factor SP1 might involve in the downregulation of NRP1 by aspirin in HUVECs according to the transcription factor family analysis.To verify the assumption,western blot was performed to detect the expression of SP1 and dual luciferase assay was used to evaluate the transcriptional activity of NRP1 promoter in HUVECs.The results showed that aspirin treatment reduced the SP1 expression and also inhibited NRP1 promoter activity in endothelial cells,indicating that aspirin inhibited NRP1 expression may through downregulating SP1.The CAM model and xenograft model were used to examine the effect of aspirin on angiogenesis in vivo.The results showed that aspirin decreased the number of new blood vessels between pre-existing ones in CAM and inhibited the expression of vascular marker CD31 in xenograft tissues,suggesting that aspirin inhibited angiogenesis in vivo.In conclusion,aspirin inhibited the acetylation of CypD by activating AMPK/SIRT3 signaling pathway,then blocked glycolysis via promoting the release of HK2 from the mitochondrial outer membrane to the cytoplasm,thus inhibited the proliferation of NSCLC cells and the xenograft growth of nude mice.In addition,aspirin inhibited migration through GATA3 mediated EndMT process,impeded tube formation through NRP1 signaling in HUVECs and inhibited angiogenesis in vivo.