The Study On The Effects And Mechanism Of NLRP3 Inflammasome And Dopamine In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a common hematological malignancy.With the development of chemotherapy and hematopoietic stem cell transplantation,overall survival of the disease has been improved.But for the old age group further improvement is still urgent because of more side effects and less remission rate.The pathogenesis of AML is still unknown.Most studies focus on the gene mutation and related signal pathways.Accumulating evidences reveal that immune response is also important.Inflammation is previously thought to be the body’s defensive response to infection and cancer.NLRP3 inflammasome as a component of immune system has been found related to several cancers.Many cancers are related with chronic inflammation,for instance,gastric cancer with Helicobacter pylori-induced gastritis,hepatocarcinoma with hepatitis B virus(HBV)or hepatitis C virus(HCV)infection and colorectal cancer with ulcerative colitis.The mechanisms how inflammation induces tumor are complicated.Genomic instability is usually observed in inflammation-induced cancers.Chronic inflammation can promote carcinogenesis interfering DNA repair systems.When the DNA damage is not repaired,molecular signaling pathways are altered as well.Among these molecular carcinogenesis pathways,NF-κB and STAT3 are the important ones.NLRP3 inflammasome is composed of NLRP3,the adaptor protein ASC and pro-Caspasel.Activation of inflammasome can cleave and activate Caspasel.In the following Caspasel activates the pro-IL-1β3 and pro-IL-18.IL-1β0 and IL-18 futher regulate the inflammation through downstream molecules such as NF-κB and IL-6.NLRP3 inflammasome is important in the pathogenesis of inflammatory and autoimmune diseases including gouts,diabetes,obesity,atherosclerosis and IBD.Single nucleotide polymorphisms of the genes associated with NLRP3 inflammasome are related to several cancers.The Q705 K polymorphism in NLRP3 is quite prevalent in the healthy population and is associated with increased risk of malignant melanoma.The IL-18(rs1946518)polymorphism is related to breast,Lung and nasopharyngeal cancer.IL-1β(rs 16944)polymorphism is related to the risk of non-small cell lung cancer and cervical carcinoma.The-94 ins/del ATTG polymorphism in NF-κB promoter is related to cancer susceptibility,including gastric cancer,ovarian cancer,prostate cancer,and oral squamous cell carcinoma.The single nucleotide polymorphisms of NLRP3 inflammasome genes may be related to pathogenesis and prognosis of AML.NF-κB activity is more prominent in primitive human leukemia cells than normal primitive bone marrow cells.We have found in new diagnosed and refractory AML patients NLRP3 and caspase 1 molecular expressions and the plasma level of IL-1β and IL-18 were significantly higher than the normal.NLRP3 inflammasome is activated in the process of AML pathogenesis.We speculate that the polymorphisms of NLRP3 related genes may be associated with the onset and progression of acute myeloid leukemia.So we detect the polymorphisms of NLRP3(rs35829419),CARD8(rs2043211),IL-1β(rs 16944),IL-18(rs 1946518)and NF-kB-94 ins/del ATTG in de novo AML patients to define their roles in the susceptibility and severity of AML.NLRP3 inflammasome plays a role in the onset and progress of gastric cancer,skin cancer,breast cancer and liver tumor.The excessive expression of NLRP3 inflammasome promotes the progress of melanoma in the late stage.NLRP3 expression is strictly regulated in the resting cells.When stimulated,NLRP3 activates IL-1β and IL-18 to regulate the immune response.NLRP3 inflammasome and its products IL-1β and IL-18 have also been shown to inhibit NK and T cell mediated anti-metastasis and immune surveillance,thus promoting the occurrence and development of cancer.Currently,there are many factors that can activate NLRP3 inflammasome including ATP,toxins,K+exudation,reactive oxygen species and mitochondrial dysfunction.A study shows in bone marrow mononuclear cells of MDS patients the NLRP3 and Caspase expressions are significantly higher than the normal group.In low and high risk MDS patients the mRNA expressions of IL-1β and IL-18 are higher than the normal group.In ALL cells,higher expressions of NLRP3 and Caspasel are also observed.And the cells show the natural resistance to glucocorticoids.The NLRP3 inflammasome may induce drug-resistant in leukemia cells.Our study has improved that polymorphisms of NLRP3 related genes are associated with the outcome of AML.The NLRP3 inflammasome activation may impact on the biological characteristics of AML cells.Then we further study the expressions of NLRP3 inflammasome genes in AML cells and the chemosensitivity of AML cells.Researches show tumors are associated with mental stress obviously under the condition of inflammatory stimulation.Researchers think this is associated with the connection of the central nervous system and immune system,but the mechanism remains to be studied.Stress can activate sympathetic adrenergic system to produce the neurotransmitters such as dopamine,adrenaline,and finally promote cancer.Neurotransmitter can also regulate the immune system through the cytokines,in addition to maintain hemodynamic stability.The activation of a adrenergic receptor is related to the increase of TNFa and IL-6 in monocytes,while the activation of βadrenergic receptor system is related to the increase of TNFα,IL-6,IL-1 and IL-10.Mental stress can activate the cAMP/PKA pathway through the β2 adrenergic pathway to increase tumor angiogenesis,finally promoting the growth of malignant tumor cells.Bone marrow microenvironment is formed by a complex network of vessel,sympathetic nerve fibers and stromal cells.It plays an important role in normal hematopoiesis including proliferation,renewal,and differentiation of hematopoietic stem cells.Hematopoietic microenvironment changes after chemotherapy.The sympathetic nervous system signal is abated,affecting the normal hematopoietic stem cell survival and proliferation.Changes in the bone marrow microenvironment play an important role in AML.Leukemia stem cells(LSCs),which are produced by mutant HSC or directed progenitor cells,maintain their ability to self-renew and proliferate in the bone marrow microenvironment that can lead to disease recurrence after treatment.Chronic stress can promote the progress of ALL through β adrenergic signaling pathway by changing the bone marrow microenvironment in the mouse model.In the MLL-AF9 AML model,neuropathy of the sympathetic nervous system(SNS)promotes leukemic bone marrow infiltration.Development of AML disrupts SNS nerves and induces proliferation of phenotypic MSPCs primed for osteoblastic differentiation.Sympathetic nerve lesions are accompanied with AML,which may in turn promote leukemic progression.After being treated with DR1/5 specific dopamine receptor antagonist,the diffusion and colony forming ability of MLL-AF9 THP1 AML cells are reduced,the process of cell cycle slows down,new DNA synthesis and cell migration rate are reduced.In the early experiments we found that the NLRP3 inflammasome was activated in the AML cells.One study showed in the bone marrow derived macrophages dopamine could inhibit NLRP3 inflammasome through the DR1 pathway leading to cAMP accumulation.DA could promote ubiquitin of NLRP3 by binding to it to inhibit the NLRP3 inflammasome.We further study the effects and mechanisms of neurotransmitter DA in AML cell proliferation and NLRP3 inflammasome and their connection.Objective:1.383 AML cases and 300 randomly selected healthy individuals were detected for the polymorphisms and expressions of NLRP3 related genes.2.The mRNA expressions of NLRP3 inflammasome-related genes were detected in AML cell lines before or after LPS treatment.3.The effects of up-regulated and down-regulated NLRP3 on the sensitivity of AML cell lines to chemotherapy drugs were studied.4.The effects and mechanisms of dopamine on the acute myeloid leukemia U937 cells,the effects and pathway of dopamine on NLRP3 inflammasome in AML cells and the relationship between them were investigated.Methods:1.PCR was used to detect the polymorphisms of NLRP3(rs35829419),CARD8(rs2043211),IL-1β(rs16944),IL-18(rs1946518)and NF-κB-94 ins/del ATTG genes in patients with AML and healthy controls.2.RT-PCR was used to detect mRNA expressions of IL-1β and IL-18 of AML patients.3.ELISA was used to detect the supernatant level of IL-1β and IL-18 of AML patients.4.RT-PCR was used to examine the mR:NA of NLRP3 inflammasome-related genes in AML cell lines and the mRNA expressions of IL-1β after LPS treatment.5.Lentivirus interference was used to up-regulated and down-regulated the NLRP3 gene.6.CCK8 was used to detect OD values of AML cells after chemotherapy.7.CCK8 was used to detect the effects of dopamine,dopamine receptor agonist,dopamine plus dopamine receptor inhibitor and dopamine plus anti-IL-(3 antibody on the proliferation of U937 cells.8.RT-PCR was used to detect the mRNA expressions of NLRP3,ASC,Caspasel,9.IL-1p?11-18?NF-rcB,TP53,bax,c-myc,bcl-2 in U937 cells after treated with dopamine.10.The IL-ip levels in the supernatant secreted by AML cells after treated with dopamine,dopamine receptor agonist and dopamine receptor inhibitor plus dopamine were detected by ELISA.11.Western Blot was used to detect the protein expressions of NLRP3,Cleaved Caspase1,leaved IL-1β,bcl-2 and c-myc in U937 cells after dopamine,dopamine receptor agonist,dopamine plus dopamine receptor inhibitor and dopamine plus anti-IL-1β antibody treatments.Results:1.Polymorphisms of NLRP3(rs35829419),CARD8(rs2043211),IL-1β(rs 16944),IL-18(rs1946518)and NF-KB-94ins/del ATTG genes were not associated with the susceptibility of AML.2.The polymorphisms of IL-1β(rs16944)in different cytogenetic risk subgroups were found statistically different,with more GA genotype in favorable-risk cytogenetic group.3.AML patients with IL-18(rs1946518)TT genotype showed higher IL-18 in supernatant than those with GG genotype.4.The GT genotype of IL-18(rs 1946518)was related to statistically poorer AML-specific survival.5.The activation of NLRP3 inflammasome in AML cells was observed.The mRNA expressions of Il-1β,I1-18,NLRP3,Caspacel and ASC were detected in acute myeloid leukemia cell lines(kasumi-1,NB4,K562,THP1 and U937).And the expressions in THP1 and U937 cells were higher than other cell lines.6.LPS could activate NLRP3 inflammasome in AML cell lines inducing the increased mRNA expression of Il-1β in dose-dependent and time-dependent manners.7.Low dose of doxorubicin and cytosine arabinoside could promote the relative mRNA expression of IL-1β,indicating the possible activation of NLRP3 inflammasome.8.Down-regulation of NLRP3 gene could increase the sensitivity of AML cells to chemotherapy,and up-regulation of NLRP3 gene expression could reduce the sensitivity of AML cells to chemotherapy.9.DA promoted proliferation of U937 AML cells.10.DA could activate the NLRP3 inflammasome in U937 cells,increasing mRNA expressions of related genes.11.Anti-IL-1β antibody partly inhibited the proliferation of U937 cells by DA.12.The mRNA expression of DR5 in U937 cells was significantly higher than other DA receptors.13.DA promoted proliferation of U937 AML cells and activated NLRP3 inflammasome through DR1/5 receptor pathwayConclusions:1.IL-1p(rs 16944)and IL-18(rs 1946518)could be served as potential predictors for AML.2.NLRP3 inflammasome activation was observed in all AML cell lines.3.Activation of NLRP3 inflammasome induced chemotherapy resistance of AML cells.4.Dopamine could promote the proliferation of U937 cells through the DR 1/5 receptor pathway.5.Dopamine could activate NLRP3 inflammasome in U937 cells through the DR1/5 receptor pathway to increase the IL-1β expression and promote proliferation partly,and there were still other pathways that promote the proliferation of AML cells.