Study On The Effects And Mechanisms Of Ouabain Regulating Metabolism Of Acute Myeloid Leukemia Cells

Background Acute myeloid leukemia(AML)is the most common type of leukemia in adults,among which the M2 subtype is often seen in clinical practice.There are huge differences between AML and normal cells in the metabolic level,which has been proved an effective target for AML treatment.Ouabain,which belongs to cardiac steroids,has been paid more attention in recent years because of its antitumor effect.Previous studies in our laboratory have found that cardiac steroids such as ouabain can inhibit proliferation and induce apoptosis of tumor cells in various hematological malignancies.However,no research until now has been found on the role of ouabain in regulating intracellular metabolism in AML.The nonsense-mediated m RNA decay(NMD)pathway is an m RNA surveillance mechanism in eukaryotic cells.NMD plays a pivotal role in cell proliferation and apoptosis.Recent studies indicate that ouabain has the ability to inhibit NMD pathway.However,little is known about the molecular mechanism.Objective This study focuses on metabolomics to find out potential metabolic biomarkers in patients with AML-M2,and to explore the regulation of ouabain on the level of metabolites in AML cells and elucidate the mechanism of ouabain in treating AML-M2 from the perspective of metabonomics.Methods 1.Metabolomics analysis was performed to detect disturbed metabolites and enriched pathway,and to screen metabolic biomarkers which were related to the prognosis of AML patients.2.The effects and mechanisms of ouabain on metabonomics of AML cell line kasumi-1 were detected.Results 1.M2-subtype AML was distinguished from healthy people based on Metabolomics features.1)The most changed metabolism in peripheral blood and bone marrow supernatants is amino acids,as well as fatty acids.2)The abundance of lysine,methionine and serine in plasma and bone marrow was significantly down-regulated than in healthy control.Lysine has high specificity and accuracy in assisting the diagnosis of acute myeloid leukemia.The abundance of lysine in plasma can reflect prognosis of AML patients.2.The effects and mechanisms of ouabain on kasumi-1 cells.1)Ouabain can inhibit the proliferation and induce apoptosis of Kasumi-1 cells.2)Lysine can promote the proliferation of Kasumi-1 cells and regulate the activity of NMD pathway.3)Ouabain can regulate intracellular metabolism and affect lysine metabolism.4)Transcriptome sequencing showed that ouabain could regulate the activity of NMD pathway.Ouabain can regulate the signal transduction function of Na+/K+-ATPase,induce endoplasmic reticulum stress,increase intracellular calcium concentration,and inhibit NF-?B activity.Conclusion We found that the metabolism of amino acids in plasma and bone marrow changed significantly.Lysine is an essential amino acid for leukemia cell proliferation.The plasma lysine abundance may be a potential metabolic biomarker for diagnosis and prognosis of AML patients.Lysine is essential for AML cell proliferation.Ouabain can repress the metabolism of lysine through regulation of h CAT(lysine transporter),resulting in inhibition of proliferation of AML cells.The mechanisms comprise inducing endoplasmic reticulum stress through signal transduction of Na+/K+-ATPase,up-regulating intracellular calcium concentration,blocking the activity of NF-?B,and ultimately decreasing NMD activity.In this study,the anti-AML mechanism of ouabain was described in detail,which provided a theoretical basis for further study.