| Background:Acute myeloid leukemia?AML?is an aggressive hematological disorder that occurs as malignant proliferation,differentiation blockage,and unregulated apoptosis of hematopoietic stem progenitor cells?HSPCs?,and harms human being health badly.The incidence of AML is increasing worldwide and associated with a 5-year overall survival of less than 50%.Up to now,great progress has been made in the chemotherapy of AML.However,most of patients still suffer from refractory and relapse because of chemo-resistance.eventually leading to treatment failure.Currently,the exact mechanisms of chemo-resistance is not fully clear,and factors affecting the chemo-resistance needs further exploration.Therefore,it is of great significance to explore chemo-resistance pathogenesis and eliminating strategy.Hydrogen sulfide?H2S?is the thirdgasotransmitterfollowingnitric oxide?NO?and carbon monoxide?CO?.It is synthesized by mammalian tissues via two cytosolic pyridoxal-5'-phosphate-dependent enzymes:cystathionine-?-synthase?CBS?and cystathionine-?-lyase?CSE?.CBS/H2S signaling is detected indigestive system,cardiovascular system,reproductive system,endocrine system,nervous system and urinary system.CBS/H2S signaling can exert various biological function such as cancer biology,neurotransmission,antioxidant,anti-inflammatory effects,insulin secretion and cardioprotection.The growing literatures see CBS-derived H2S as an important regulator of the growth and survival of tumor cells.The overexpression of CBS in tumor augments growth rate and confers resistance to chemotherapy.Nevertheless,the role and its mechanisms of CBS/H2S signaling in AML has not yet been reported.In this study,we intend to detect the expression level of CBS/H2S signaling in AML cell lines,and investigate the effect of CBS/H2S signaling on AML chemo-resistance and proliferation in vitro or in vivo.Our study is of great importance in providing a novel strategy for eliminating chemo-resistance in AML.Objective:The aim of this study was to detect the CBS expression levels and H2S production in chemo-resistance AML cell line,AML cell line and healthy bone marrow cell.To identify the roles of CBS/H2S signaling on chemo-resistance and proliferation of AML cell via upregulate or downregulate CBS/H2S signaling in AML.To further explore the effect of CBS/H2S signaling on AML chemo-resistance and progression in vivo by AML animal experiment.The study will provide new targeted therapy for refractory and relapse AML.Methods:1.Western blot analysis:Total protein of human chemo-resistance AML cell line?K562/A02?,human AML cell line?THP-1,HL60,U937,K562?and mouse AML cell line?C1498?,mouse bone marrow cell line?32D?,mouse bone marrow cells was extracted by RIPAcell lysissolution.Western blot analyzed CBS expression level.2.H2S release test:The human chemo-resistance AML cell line?K562/A02?,human AML cell line?THP-1,HL60,U937,K562?and mouse AML cell line?C1498?,mouse bone marrow cells were homogenized by Ultrasonic homogenate instrument.The homogenate was used to detect H2S production by H2S release test.3.Cell proliferation assay:The AML cell proliferation was measured using the Cell Counting Kit-8?CCK-8?.3.1.Using pharmacological inhibition?AOAA?or siRNA-mediated CBS knockdown downregulated the CBS/H2S signaling of AML cell line.Then detect the proliferation of AMLcell by CCK-8.3.2.Using pharmacological method?NaHS?upregulated the CBS/H2S signaling of AML cell line.Then detect the proliferation of AMLcell by CCK-8.3.3.Following CBS knockdown,the cells were incubated with exogenous NaHS.Then detect the proliferation of AMLcell by CCK-8.4.AML cell chemotherapy sensitivity assay:The AML cell chemotherapy sensitivity was measured using the Cell Counting Kit-8?CCK-8?.4.1.The AML cells were treated with chemotherapy drug after CBS/H2S signaling knockdown.The cell viability was analyzed with the CCK-8 assay.4.2.The AML cells were treated with chemotherapy drug after CBS/H2S signaling upregulation.The cell viability was analyzed with the CCK-8 assay.4.3.The AML cells were treated with chemotherapy drug after CBS/H2S signaling knockdown,then incubated with NaHS.The cell viability was analyzed with the CCK-8 assay.4.4.The bone marrow cells from wild mice or CBS+/-mice were treated with chemotherapy drug.The bone marrow cell viability was analyzed with the CCK-8 assay.5.AML mice model:AML mice were established by administering C1498 cells intravenously via the lateral tail vein.The photomicrographs of H&E stained formalin-fixed paraffin-embedded sections of liver,spleen and bone marrow,the weight of liver and spleen,the count of peripheral white blood cells?WBC?were tested.6.Study on the effects of CBS/H2S signaling on AML progression in vivo.6.1.Using pharmacological method?AOAA?downregulated the CBS/H2S signaling in AML mouse.Detect organomegaly and weight of liver and spleen,observe infiltration of AML cell bone marrow,liver and spleen using H&E staining,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.6.2.Using pharmacological method?GYY4137?upregulated the CBS/H2S signaling in AML mouse.Detect organomegaly and weight of liver and spleen,observe infiltration of AML cell bone marrow,liver and spleen using H&E staining,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.6.3.Construct AML CBS+/-mice.Detect organomegaly and weight of liver and spleen,observe infiltration of AML cell bone marrow,liver and spleen using H&E staining,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.6.4.Take together,explore the effects of CBS/H2S signaling on AML progression.7.Study on the effects of CBS/H2S signaling on AML chemo-resistance in vivo.7.1.The AML mice were treated with chemotherapy drug after CBS/H2S signaling knockdown.Detect organomegaly and weight of liver and spleen,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.7.2.The AML mice were treated with chemotherapy drug after CBS/H2S signaling upregulation.Detect organomegaly and weight of liver and spleen,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.7.3.The AML CBS+/-mice were treated with chemotherapy drug.Detect organomegaly and weight of liver and spleen,detect the count of peripheral white blood cells,record the survival period and draw percent survival curve.7.4.Take together,explore the effects of CBS/H2S signaling on AML progression.Results:1.The results of western blot analysis and H2S release test showed that CBS/H2S signaling is upregulated in drug-resistant K562/AO2 cell line.Moreover,C1498 strikingly more highly expressed CBS/H2S signaling compared with either healthy mice bone marrow cells or mice bone marrow cell line?32D?.CBS siRNA largely reduced the production of H2S in K562/A02 cell line.2.The results of the AML cell proliferation using CCK-8 showed that AOAA?an CBS inhibitor?significantly suppressed AML cell viability via Cell Counting Kit-8 assay.Consistent with pharmacological interventions,the siRNA-mediated down-regulation of CBS significantly decreased AML cell lines proliferation rates.NaHS,the donor of H2S,significantly resulted in the stimulation of proliferation in AML cell.NaHS largely rescued either AOAA-or CBS siRNA-induced proliferation inhibition in AML cells.3.The results of the AML cell chemotherapy sensitivity using CCK-8 showed that either AOAA or CBS siRNA significantly increasedADM-induced cell death,which was reversed by NaHS.Consistently,pharmacological inhibition or genetic knockout of CBS increased the ADM-induced bone marrow cells death from CBS+/-mice or their wild-type littermates,which is reversed by NaHS.Notably,bone marrow cells from CBS+/-mice were more sensitive to ADM than wild-type mice.4.AML mice were established by administering C1498 cells intravenously via the lateral tail vein.C1498-luc tumor-bearing mice were hepatomegaly,splenomegaly and leukocytosis.Histopathological examination demonstrated massive leukemic cells infiltration in spleen,liver and bone marrow tissues in AML mice.And the AML mice died less than one month.5.The results of AML progression showed effected by CBS/H2S signaling shows that AOAA treatment alleviated AML hepatomegaly,splenomegaly and leukocytosis.In addition,AOAA-treated AML mice had a longer median survival time compared to control mice?43 vs 26d?.Consistent with the results of pharmacological inhibition by AOAA,CBS-deficient mice?CBS+/-?exhibited the mitigation of neutrophil infiltration,splenomegaly and hepatomegaly,also prolongation of lifespan?41 vs.26d?.On the contrary,administration of GYY4137?a slow-releasing H2S donor?promoted the development of AML in vivo,which exhibited the aggravation of neutrophil infiltration.splenomegaly and hepatomegaly,and shortened survival time?18 vs.26d?.6.The results of AML progression showed effected by CBS/H2S signaling shows that AOAA significantly enhanced AML sensitivity tocytosine arabinoside.Moreover,AOAA treatment prolonged survival time of AML mice?median survival time 72 vs.54d?.Consistent with the results of pharmacological inhibition by AOAA,CBS-deficient mice?CBS+/-?exhibited prolongation of lifespan?65 vs.54d?.On the contrary,administration of GYY4137 promoted the development of AML in vivo,which exhibited shortened survival time?31d?.Conclusion:1.CBS/H2S signaling was upregulated in AML cells,particularly in drug-resistant K562/A02 cell line.Overexpression of CBS/H2S signaling contribute to malignant proliferation and chemo-resistance of AML cells.2.Down-regulation of CBS significantly inhibited AML cell lines proliferation and chemo-resistance.Up-regulation of CBS significantly promoted AML cell lines proliferation and chemo-resistance.These current findings highlight the potential importance of CBS/H2S signaling in the progression and drug-resistance of AML.3.Animal experiment confirmed that inhibition of CBS/H2S signaling significantly suppressed the development and drug-resistance of AML.Overexpression of CBS/H2S signaling accelerated the progression of AML and lowered the drug-sensitivity.CBS/H2S signaling may be a new target for further therapies to combat drug-resistance in AML. |
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