| BACKGROUND:Lung cancer is leading cause in incidence and mortality among all types of tumors and remains the most prevalent malignancy among Chinese men,and second only to breast cancer among women.There are approximately 787,000 newly diagnosed lung cancer patients each year in China,nearly 2,100 are diagnosed daily.Lung cancer accounts for 20% of all cancer deaths in China while about 85% of these cases are diagnosed as non-small cell lung cancer(NSCLC),unfortunately,up to 84% of NSCLC patients are progressing to advanced cancer.The regular chemotherapy treatment protocol in recent years is platinum-based combined with another chemotherapeutic agent(gemcitabine,pemetrexed,etc.),but the clinical treatment effect is poor and some patients are prone to chemotherapy resistance.Therefore,drugs for lung cancer treatment have become a research hotspot,and with the appearance of more and more targeted drugs such as receptor tyrosine kinase(RTK)and vascular endothelial factor growth(VEGF)inhibitors,the survival rate of lung cancer patients has been improved.Targeted drugs have become resistant in clinical use,and some patients have developed into various types of genetic mutations that have led to drug resistance problems and subsequently reduced the effectiveness,so there is an urgent clinical need to develop new targeted drugs to solve the treatment problems.Adenosine monophosphate activated protein kinase(AMPK)is a class of highly conserved eukaryotic protein kinases that is a key protein in the regulation of energy in cells and serves as an energy receptor.It is activated when intracellular energy is reduced,thereby stimulating the entry of sugars and fats into the cytosol for oxidative processes,and is generally activated by AMP binding or by phosphorylation of the 172-position threonine(Thr)of AMPK.In lung cancer,mutations in LKB1 are also associated with lung cancer,and heterozygous or pure loss of LKB1 is found in the majority of non-small cell lung cancers.The aim of this study was to investigate whether activation of adenosine monophosphate-dependent protein kinase(AMPK)could exhibit anticancer effects in lung cancer.This study was aimed to observe the inhibitory effect of small molecule ASP4132 as an activator of AMPK on lung cancer cell lines in vivo and in vitro and to investigate its mechanism of action.The study is divided into the following parts:Part I: Effects of ASP4132 on proliferation,migrations and cycles of lung cancer cell lines;Part II: Exploration of the mechanism of ASP4132 on NSCLC cells.Part III:Validation of ASP4132 in NSCLC xenograft growth in SCID mice.In summary,ASP4132 exhibits tumor inhibition in vivo and in vitro by activating AMPKα.Part 1: Effects of ASP4132 on proliferation,migrations and cycles of lung cancer cell linesObjective:Investigating the anticancer effect of ASP4132 on lung cancer cell lines in vitroMethods:1.Cell culture: Lung cancer cell lines A549 and H1944 were incubated in a cell culture incubator at 37℃ with 5% CO2 concentration until the cells fused to about80% of the culture dish and propagated once.The cells were tested for mycoplasma and pathogenic microorganisms every two months to prevent contamination from affecting the experimental results.2.ASP4132 powder was dissolved in DMSO to form a 100 m M liquid for storage,which was diluted using PBS solution and added to the cell culture medium together with the cells prior to the cell experiments.3.CCK-8 method was used to detect the effect of ASP4132 on the growth of A549 cell line at different concentrations: The experiment was divided into two groups,and ASP4132 was diluted into different gradient concentrations(0.1u M,0.3u M,1u M,3u M)in the experimental group and placed in the incubator for 24 h,48h,72 h,96h.The inhibition rate was calculated for different concentrations as well as to observe the time-dependent effect of the drug.4.For clone colony formation assay:The experiment was divided into 2 groups,the experimental group added to complete medium with ASP4132 was diluted into different concentration gradients(0.1u M,0.3u M,1u M,3u M),the control group without ASP4132.Cell plate clonal colony formation was observed after 10 days.5.To detect cell proliferation by Edu method: different concentrations of gradient drugs(0.1u M,0.3u M,1u M,3u M)were added and treated for 48 h.Fluorescence microscope detection,no less than 1200 nuclei in five random fields under 1×100 microscope,calculate the percentage of Edu-positive nuclei.6.Cell cycle detection by flow cytometry: add PI 20 u L to a final concentration of 50ug/m L tin foil and stain at 4 degrees for 30 minutes,mix the cells well,filter with a 200 mesh filter,and assay on the machine within 24 h.7.Transwell cell migration assay: cells were starved by adding serum-free medium for 12 h.The cells were incubated for 24 h,removed from the chambers,air-dried and stained using crystal violet,and photographed on the microscope to analyze the results.The procedure for invasion experiment was the same,except that a layer of Matrigel basement membrane was spread in the chambers in advance.50mg/L of Matrigel 1:8 was diluted and added into the membrane at the bottom of the chambers,and air-dried at 4°C,taking care not to leave air bubbles.8.CCK-8 method was used to detect the effect of drug on the growth of normal bronchial epithelial cells BEAS-2B at 1u M concentration: The experiment was divided into two groups,in which ASP4132 was diluted to 1u M and placed in the incubator for 72 h.Ten replicate wells were set,and the control group was added with medium not containing ASP4132.Results:1.CCK-8 results showed that the growth of lung cancer cell lines was inhibited after treatment with different concentration gradients of the drug(0.1u M,0.3u M,1u M,3u M),and 0.1u M had no effect on cell inhibition.0.3u M-3u M drug produced significant growth inhibition in all lung cancer cell lines and was statistically different.The growth inhibitory effect of the drug gradually increased with the duration of drug action on the cell lines.96 h 3u M concentration of the drug had the strongest inhibitiory effects.2.The clonal colony formation assay showed that,similar to the CCK-8 assay,there was no statistically significant reduction in clonal colonies after the 0.1u M drug was applied to the cell lines.0.3u M-3u M drug exhibited a more significant reduction in clonal colonies,confirming that 0.3u M-3u M drug could produce a significant growth inhibitory effect on lung cancer in vitro.Combining the results of clonal colony and CCK-8 experiments,we chose 1u M 24 h as the experimental concentration for follow-up.3.Edu assay showed that: 0.1u M concentration of the drug changed few Edu positive cells in the cell line,and there was no statistical difference with the control group.0.3u M-3u M significantly reduced the number of Edu positive cells and inhibited the proliferation of cells in vitro.4.Cycle detection by flow cytometry: After 1u M drug treatment of the cell line,the assay revealed an increase in G1 phase cells and a decrease in S phase cells after treatment with the drug,and the cell line showed a cycle block in G1 phase after the drug treatment,which was consistent with results of cell growth and proliferation.5.Migration and invasion assay: the number of invading and migrating cells was significantly reduced in the drug addition group compared to the control group,and the drug inhibited the migration and invasion phenotype of lung cancer cells in vitro.6.For normal bronchial epithelial cells,ASP4132 did not produce significant drug toxicity at effective concentrations and did not inhibit the growth of the normal epithelial cells.Conclusion:ASP4132 inhibited the proliferation and growth of lung cancer cells in vitro along with cell cycle arrest in the G1 phase,and reduced the proliferation and migration ability of lung cancer cells.Part 2: Exploration of the mechanism of ASP4132 on NSCLC cells.Objective:To explore the mechanism of the effect of ASP4132 on non-small cell lung cancer cell linesMethods:1.Caspase-3 activity assay and protein assay: Caspase-3 reaction buffer containing fluorescent substrate was added to both wells,cell lysate was added to the blank wells,and cell protein extract was added to the sample wells.The " increase in fold" of fluorescence intensity was used as the activity of Caspase-3,and three replicate wells were made for each well.Western blots were performed to detect changes in the expression of caspase-3,cleave-caspase-3 and cleave-caspase-9 using the extracted total cellular proteins.2.Single-stranded DNA antibody ELISA assay: The sample was divided into sample wells to be tested and blank wells,sealed and incubated at 37°C for 30 minutes,followed by washing and enzyme addition and other operations followed by machine detection.3.Td T-mediated d UTP Nick-End Labeling(TUNEL)method and Hoechst3342co-staining to detect apoptosis:.Add Hoechst 3342 stain,Td T mixture,FITC and DAPI dye to stain the cells sequentially,observe under the microscope(×100),select a random field of view for each group and count the apoptotic cells and the total number of cells(not less than 1200 cells).4.Annexin V assay for apoptosis: The dosing group was treated with ASP41321 u M for 48 h and divided into negative control group,PI dye group,FITC dye group,and FITV-PI dye group.5.Z-DEVD-FMK and Z-VAD-FMK drug inhibition experiments: Divided into experimental and control groups,each group was further divided into experimental groups with the treatment of three drugs,DMSO,DEVD and VAD,and the nuclear percentage of apoptotic cells was detected by Hoechst fluorescence color detection.The number of viable cells was counted using Trypan Blue staining and the effect of caspase inhibitor addition on cell growth was detected by CCK-8.6.Mitochondrial protein extraction and immunoprecipitation assay: The mitochondrial proteins were extracted and incubated with Cy PD antibody,followed by incubation with G-Sepharose beads,and finally the bead protein complexes were sampled for western blots to detect P53 and adenine nucleotide transport protein 1(ANT1)proteins.7.JC-1 mitochondrial membrane spot staining: JC-1 dye(5ug/m L)was added and incubated for 12 min away from light,and the fluorescence intensity was detected on the machine at 550 nm.The green fluorescence and red fluorescence were combined to count the mitochondrial membrane potential changes.jc-1 dye was obtained from Sigma-Aldrich Chemicals JC-1 Mitochondrial Membrane Spotting Kit.8.Lactate dehydrogenase assay(LDH): 1u M ASP4132 was used to quantify the LDH content in the culture medium and normalize the total LDH level after 72 h treatment with LDH assay kit(Takara,Tokyo,Japan).9.Cyclosporine A and Cy PD knockdown: Cy PD sh RNA was constructed,and after intracellular verification of sh RNA knockdown efficiency,Cy PD inhibitor cyclosporine A(Cs A)5u M was added to the cells for 24 h,as well as Cy PD sh RNA and Cy PD sh RNA-Control,respectively,and then divided into control and experimental groups,and cell viability was detected by CCK-8 after ASP4132 1u M treatment for 72 h,and the number of live cells was detected by Trypan Blue staining.10.Western Blots and in vitro activity assay of AMPKα: Total protein was extracted and the expression of intracellular protein was detected by relevant antibodies.Activity was measured by flash counter after in vitro incubation with AMP-[γ-32P] ATP mixture and the substrate SAMS peptide.11.Ribosomal S6 protein kinase 1(S6K1)as well as epidermal growth factor receptor(EGFR),platelet-derived growth factor receptor(PDGFR),protein kinase B(Akt)protein and m RNA expression: divided into drug-treated and control groups,total cell protein was extracted for Western Blots to detect p-S6K1 as well as total S6K1,EGFR,PDGFR and EGFR downstream Akt expression,respectively.12.Autophagy assay: LC3 B fluorescence-stabilized cell lines,which had been prepared in the laboratory,were used for this experiment,and total cell protein was extracted after the control and dosing groups.13.sh-AMPK treatment of lung cancer cells: Divided into a dosing group and a control group,where within each group is divided into a knockdown RNA group with the addition of sh-AMPK lentivirus wraps,and a control group.Cell viability was assayed.14.AMPKα dormant assay: A non-activating AMPKα1(T172A,dn AMPKα1)adenovirus plasmid was constructed,After the cells were cultured using flow sorting of GFP-positive cells,lung cancer cells stably expressing dn AMPKα1 were screened for experiments by adding medium containing puromycin afterwards.Cell growth and apoptosis were detected using CCK-8 and other previously described experiments.15.AMPKα1 sustained activation experiment: AMPKα1(T172D,ca AMPKα1)plasmid was received from the Soochow University laboratory,which was continuously activated AMPKα1,and the same experimental protocol as that of dormant bodies was adopted.Results:1.Caspase-3 activity and protein expression assay: compared with the control group,the activity of caspase-3 in the experimental group increased significantly,and western blots suggested that the expression of cle-caspase-3 increased significantly,and cle-PARP also showed increased protein expression.2.Single-stranded DNA antibody ELISA assay: The intracellular single-stranded DNA antibody was significantly increased in the dosing group,suggesting that the cells showed more serious apoptosis,and ASP4132 inhibited the DNA replication of lung cancer cell lines,verifying the inhibitory effect of ASP4132 on cell proliferation and growth.3.Td T-mediated d UTP Nick-End Labeling(TUNEL)method and Hoechst3342co-staining:In the experimental group treated with ASP4132,there was an obvious increase in apoptotic cells,and the nuclei appeared to be sequestered and fragmented,showing blue fluorescence,and the nuclei of such stained cells were also TUNEL-positive cells.Compared with the control group,apoptosis was significantly induced by the addition of the drug in the experimental group.4.Annexin V assay for apoptosis: compared with the control group,the treatment group significantly increased early apoptosis and late apoptosis phase of lung cancer cell lines,and ASP4132 had a significant effect of increasing apoptosis of lung cancer cells in vitro.5.Z-DEVD-FMK and Z-VAD-FMK drug inhibition assay: The experimental results showed that there was no significant change in apoptosis after adding the inhibitor without ASP4132.In ASP4132 treatment group,there was a significant decrease in apoptosis induction in lung cancer cell lines with the addition of caspase-3 and caspase family inhibitors,and Typan blue staining also showed that the number of viable cells in the ASP4132 drug-treated group increased significantly after the addition of caspase family inhibitors compared with the group without inhibitors.However,it did not completely reverse the ASP4132-induced apoptosis.In conclusion,the induction of apoptosis in lung cancer cells by ASP4132 is partly dependent on the activation of caspase family.6.Mitochondrial protein extraction and immunoprecipitation assay: compared to the control group,the addition of the drug did not affect the protein expression of Cy PD-P53-ANT1 in mitochondria.in the ASP4132 drug group,there was an interaction of Cy PD-P53-ANT1 protein,a complex that is an important complex in the activation process of programmed necrosis,suggesting the occurrence of intracellular necrosis.7.JC-1 mitochondrial membrane staining: JC-1 aggregates in the matrix of mitochondria and forms a polymer,which can produce red fluorescence;when the mitochondrial membrane potential is low,JC-1 can not aggregate in the matrix of mitochondria,when JC-1 is a monomer and can produce green fluorescence.Compared with the control group,the experimental group obviously showed a change from red fluorescence to green fluorescence,which indicated that the mitochondrial membrane potential showed depolarization and also indicated early apoptosis of the cells.8.LDH assay: Compared to the control group,LDH in the cell culture medium of the experimental group was significantly increased,suggesting the possibility of programmed necrosis of cells.9.Cyclosporin A and Cy PD knockdown: ASP4132-induced cell growth inhibition could be partially reversed by Cs A as well as sh Cy PD,demonstrating that ASP4132 is dependent on Cy PD-induced programmed necrosis to exert inhibitory effects on cell growth.10.Western Blots and in vitro activity assay of AMPKα: The expression of p-AMPKα1 was significantly increased in the drug group with the addition of ASP4132 and was accompanied by increased expression of p-ACC.In vitro activity assay,AMPKα1 activity was significantly increased in the drug group with the addition of ASP4132.ASP4132 clearly activated AMPKα1 in vitro.11.Ribosomal S6 protein kinase 1(S6K1)and epidermal growth factor receptor(EGFR),platelet-derived growth factor receptor(PDGFR),and protein kinase B(Akt)protein and m RNA expression: p-S6K1 expression was reduced and total S6K1 was not significantly changed;EGFR as well as PDGFR appeared significantly reduced,and downstream activated Akt also appeared reduced.q-PCR detected no significant changes in messenger RNA of EGFR and PDGFR.ASP4132 action was reflected at the protein level,not in the process of gene transcription.12.Autophagy assay: LC3B-II was significantly increased,autophagy was induced,fluorescence microscopy showed a significant increase in autophagic spots,and ASP4132 induced autophagy in lung cancer cells in vitro.13.sh-AMPK and ko-AMPK treatment of lung cancer cells: the inhibitory effect of ASP4132 on lung cancer cells was restored after AMPK knockdown or knockout,and the inhibitory effect of ASP4132 on cancer cell proliferation was dependent on the expression of AMPK.14.AMPKα dormant: The growth and apoptosis of AMPKα1 dormant lung cancer cells that could not be activated were reversed in the treatment group compared to the control group.activation of AMPKα1 was required for ASP4132 to exert its anticancer effects.15.AMPKα1 sustained activation assay: Sustained activation of AMPKα1significantly inhibited the growth and proliferation of lung cancer and promoted apoptosis.The treatment of ASP4132 did not increase the inhibition of cell growth and promotion of apoptosis.Once again,ASP4132 was shown to be dependent on AMPKα1 for its in vitro anti-cancer effects.Conclusion:ASP4132 exhibited anti-cancer effects in vitro dependent on the activation of AMPKα1 and is able to inhibit the proliferation,growth and migration,and invasion of lung cancer cells by regulating various pathways such as programmed necrosis,induction of autophagy,and increased Cy PD-P53-ANT1.Part 3: Effects of ASP4132 in vivo.Objective:To use a xenograft mouse tumor model to validate the anticancer effect of ASP4132 in vitroMethods:5 weeks of age-Severely combined immunodeficient mice(half male,half female)weighing 18.5 g-19.5 g were used.3×106 lung cancer cells were grown in the peritoneal cavity of each mice,and the tumors grew to approximately 100 mm3 in size around 3 weeks and were labeled as day 0.The mice were randomly assigned into control and drug-treated groups of 10 mice each.Tumor size was measured every 6 days and tumor volume was calculated(V = length×width×height×0.5236),and one tumor in each group was selected on day 7 and day 14 for tissue protein extraction and protein expression detection by Western Blots.All mice were weighed and counted after execution on day 42 for subsequent calculations.Results:ASP4132 orally treatment group,the tumor volume was significantly smaller than that of the control group;compared with the control group,the tumor weight of the treatmentConclusion:ASP4132 produced significant inhibition of tumor growth in mice fed orally in vivo,and no significant adverse effects were observed in mice. |
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