The Role Of Azelaic Acid And Its Derivatives In The Treatment Of Acute Myeloid Leukemia

Part 1:The antileukemic effect of Azelaic acidObjective The current main treatment of acute myeloid leukemia(AML)is still chemotherapy.The adverse reactions and complications after chemotherapy made some patients intolerant,additionally,the drug resistance may appear after multiple chemotherapy and often accompanied by poor prognosis.Azelaic acid(AZA)is a nine-carbon straight chain saturated acid with a small molecular weight,which has been proved to have an obvious inhibitory effect on a variety of malignant tumors in previous studies,but its effect on AML has not been clarified.The purpose of this study is to develop more effective and less toxic small molecule agents and provide new sights for the clinical treatment of acute myeloid leukemia.Methods The bone marrow of patients who were newly clinically diagnosed with acute myeloid leukemia were collected and the primary AML patient cells were isolated by Ficoll centrifugation,AML cell lines HL60,U937,THP-1 and Molm-13 were cultured in our lab.The killing effect of azelaic acid(AZA)at different concentrations on AML cell lines and AML-PC were analyzed by CCK-8 method.AZA-BDP,a fluorescent azelaic acid compound,was synthesized by attaching fluorescent groups to azelaic acid.The changes of cell morphology after AZA treatment were observed under fluorescence confocal microscope.The cytotoxicity effects of AZA on normal cell lines such as PBMC,293T,AML12,h FOB1.19 and MC3t3-E1 were detected by CCK-8 and flow cytometry analysis to determine the biological safety of AZA.JC-1 and Annexin V-FITC/PI labeled cells analysis were used to detect the apoptosis rate and cell cycle.Results AML cells and AML-PC were treated with AZA at different concentrations for 24,48 and 72 hours,respectively.The results showed that AZA significantly inhibited the proliferation of AML cell lines and AML-PC and the median IC₅₀ of 24h was about 5 m M.The synthesized AZA-BDP can emit green light when excited by blue light.According to the intensity of green fluorescence,it can be found that AZA can enter cells quickly,the cells gradually swell and begin to show the phenomenon of cytoplasmic vacuolization.AZA can induce the apoptosis of AML cells,but the same concentration of AZA has no significant effect on normal cells.Conclusion AZA can significantly inhibit the proliferation of AML cells and induce the apoptosis of AML cells.Part 2: The anti-leukemia effect of AZA by decreasing the intercellular ROS levelObjective AML patients had a significantly higher ROS level than that in healthy people.Elevated ROS levels were closely related to the progression,relapse and drug resistance of AML.The purpose of this study was to explore whether azelaic acid can exert anti-AML effects by reducing ROS levels and oxidative stress injury,thus inducing apoptosis in AML cells.Methods AML cell lines U937,HL60,and Molm-13 cells were cultured in our lab,and the bone marrow of newly diagnosed AML patients was collected and primary AML patient cells(AML-PC)were isolated.Flow cytometry analysis was used to detect the changes in ROS levels and oxidative damage indicators such as SOD,MDA,GSH,and T-AOC after AZA treatment;CCK-8 was used to detect the effect of AZA on AML cell lines and AML-PC cell proliferation.Molm-13 cells which were labeled with GFP were treated with AZA and the changes in fluorescence intensity were observed under fluorescence microscope.By cooperating with the School of Physics and Science and Technology of Wuhan University,we designed a microfluidic chip that can trap AML cells,the changes in cell morphology and structure after AZA acts on AML cells at a single cell level were captured by confocal microscopy;flow cytometry analysis was used to detect the changes in mitochondrial membrane potential,apoptosis rate and cell cycle after AZA treatment;differential expressed proteins(DEPs)before and after AZA treatment in THP-1 cells were determined by LC-MS,and the biological function of DEPs were analyzed by WEGO analysis;patient-derived xenograft AML model was used to detect the disease remission and antioxidant indexes in AML mice after AZA treatment.Results AZA can significantly decrease intercellular ROS levels and increase the antioxidant capacity in AML cells;AZA can inhibit the proliferation rate of AML cells and PC-AML cells.Furthermore,single-cell microfluidic experiments suggested that AML cells gradually swelled,apoptotic bodies appeared and eventually lysed to death;CCK-8 and flow cytometry analysis showed that cell viability was inhibited and early apoptosis increased after AZA treatment;proteomic analysis showed that the antioxidant protein Peroxiredoxin(Prdx)significantly increased after AZA treatment and STRING analysis results indicated that Prdx was closely related to a variety of antioxidant indicators;WB,PCR and ICH results suggested that the levels of antioxidant indexes and Prdx expression in AZA group were also increased in vivo,the spleen infiltration was relieved and the survival was significantly extended..Conclusion AZA promoted AML cell apoptosis by decreasing ROS levels.Analysis of the mechanism found that AZA can increase Prdx expression.In vivo experiments suggested that AZA exert S anti-leukemia effects by regulating redox.Part 3: The immunoregulatory effect of Azelaic acid against AMLObjective Immunotherapy has become a hot topic in the field of tumor therapy.The rise of immune checkpoints,Car-T cell therapy,tumor vaccine and other therapeutic methods had benefited more patients from immunotherapy.The purpose of this study was to investigate the immunomodulatory effects of AZA and its role in AML mouse models.Methods Peripheral blood were collected from healthy volunteers for the isolation of PBMC,PBMC were further purified and isolated to NK and T cells.NK and T cell proliferation rates were detected by CCK-8 method.AML cell lines,NK and T cells were co-cultured at ratio of 1:3,the killing effect before and after AZA treatment were detected by LDH release assay.The immune cell activation markers were analyzed by flow cytometry.The cytokines levels were measured by enzyme-linked immunosorbent assay(ELISA).Microfluidic chips were prepared to trap NK cells and tumor cells,and the morphological changes of AML cells were captured under fluorescence microscope.AML mouse model was constructed for detecting the changes in the percentage of NK and T cells in spleen and cytokines in peripheral blood in AZA-treated group.Results Low concentration of AZA can promote the proliferation of NK and T cells.The results indicated that the optimal concentration was 10 micro Mole at 24 h,and the proliferation rates of NK and T cells was about 152% and 210%.The results of coculture indicated that NK and T cells were more active after the treatment of AZA.Flow cytometric analysis indicated that the proportion of NK cell activation marker CD107 a and TRAIL and T cell activation marker CD25 and CD69 were significantly increased by AZA.The results of microfluidic chip suggested that NK cells treated with AZA can kill tumor cells more efficient.Compared to saline group,the proportion of CD3-CD56+NK cells and CD4+CD8+T cells in spleen and the levels of TNF-? and IFN-? in plasma were significantly increased in AZA group,the symptoms were relieved and the survival was significantly prolonged after the treatment of AZA in mice.Conclusion AZA can promote the proliferation and activation of NK and T cells,increase the cytotoxic effect of immune effector cells and the release of cytokines.AZA exhibited a robust anti-leukemia effect by regulating immunity.Part 4:AZA exerts anti-leukemia effect by activation of Notch signaling pathwayObjective Notch and its downstream targets are downregulated in AML cell lines and AML patient cells,reactivation of Notch can inhibit the proliferation of AML cells.In addition,Notch signaling pathway regulates the differentiation,activation and cytotoxicity of immune effector cells.This study aims to explore the exact molecular mechanism how AZA regulate the AML cell activity and immunity,and further explore its relationship with Notch signaling pathway.Methods Protein mass spectrometry analysis(MS)was used to analyse the differential proteins between DMSO and AZA-treat THP-1 cells.The biological function of differential proteins was analysed by WEGO analysis.The changes of signal pathway activation after the treatment of AZA were detected by dual luciferase assay.Western blotting and RT-PCR were used to determine the expression changes of Notch and its downstream target.Notch inhibitor RO4929094 was applied to inhibit Notch,then the apoptosis rate and the cytotoxic of NK and T cells after the treatment of AZA were detected.Results Protein MS analysis indicated that there were 525 differential proteins after the treatment of AZA.10 of them are related to the immunity by WEGO analysis.Notch1 and Notch2 belong to the two differential proteins which are up-regulated.Notch signaling pathway was activated when treated with AZA,while it was inhibited when combined with inhibitor RO4929097.When Notch signaling pathway was inhibited by RO4929097,the killing effect of AZA on AML cells was significantly weakened,and the TNF-? and IFN-? levels secreted by NK and T cells were also significant reduced.Conclusion AZA can activate Notch signaling pathway.After the activation of Notch signaling pathway,on the one hand,AZA inhibited the proliferation of AML cells;on the other hand,it played an immunomodulatory role to promote the proliferation and differentiation of immune cells,increase the killing effect of immune effector cells and exerted anti-leukemia effect.Part 5:A new strategy to target thioredoxin reductase against AMLObjective Arsenicals has been widely used in the treatment of AML-M3.Studies have confirmed that both arsenic and AZA are thioredoxin reductase(Trx R)inhibitors.In this study,we combined AZA and arsenic to synthesis novel Trx R inhibitors with stronger targeting ability,high efficiency and low toxicity,and the anti-leukemia effects and mechanism of the novel compound were further investigated.Methods Our group cooperated with School of Chemistry and Molecular of Wuhan University and constructed a series of organic arsine for pro-drug synthesis.The targeted compounds were fabricated through amidation and esterification by binding AZA and organic arsenicals.AML cell lines were cultured in our lab,CCK-8 method was used to detect the cell proliferation rate of the synthesized small-molecule compounds so as to preliminarily screen the candidates.DTNB method,endpoint insulin assay,Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry and other methods were used to detect the Trx system activity.Apoptosis and cell cycle were detected by flow cytometry analysis and caspase-related experiments.The molecular mechanism was validated by Western blotting,RT-PCR and IHC.Plasma stability analysis and pharmacokinetics analysis further confirmed the application of the targeted compound in vivo experiments.Finally,the antileukemic effect was detected in a PDX AML mouse model.Results We synthesized a series of small molecular compounds binding AZA to organic arsenicals and screened the best one,A-Z2,which has the strongest inhibition on AML cell viability and Trx R activity.A-Z2 significantly inhibited the proliferation of AML cells compared with AZA and organic arsine Z2 at the same concentration.AZ2 has strong Trx selectivity,but has no significant effect on GSH system.A-z2 can promote apoptosis and arrest cell cycle in G2/M phase.Analysis of its mechanism showed that A-Z2 could induce mitochondrial pathway apoptosis by directly targeting Trx R to promote the release of cytochrome c and the activity of Caspase 9 and Caspase 3,and indirectly inhibit NF-KB activity to promote the release of Caspase8 and TNFa,thus enhancing its anti-leukemia effect.A-Z2 has a good plasma stability and pharmacokinetics suggest that intravenous administration can quickly reach higher blood concentration.In vivo experiments demonstrated that A-Z2 could significantly prolong the survival of AML mice.Flow cytometry analysis and immunohistochemistry results indicated that bone marrow and spleen infiltration of mice in the A-Z2 group were significantly relieved,Trx R and Trx expression levels were decreased.Conclusion The novel Trx R inhibitor A-Z2 can exert antileukemia effects by targeting Trx R to induce intrinsic apoptosis.Targeting Trx R is expected to provide new ideas for the clinical treatment of AML.