Pyridoxine Selectively Induces Monocyte-macrophages Death As Specific Treatment Of Acute Myeloid Leukemia

Background:Acute myelocytic leukemia(AML)is an aggressive hematological malignancy with less than 30% of 5-year overall survival rate,highlighting the requirment of improved therapeutic intervention.AML is a highly heterogeneous disease group.Based on cell morphology and histochemical characteristics,FAB divides AML into different types from M0 to M7.At present,in addition to all-trans retinoic acid which plays a revolutionary role in the treatment of promyelocytic leukemia by inducing cell differentiation,cytarabine and daunorubicin(or similar compounds)combined chemotherapy is the major treatment for other types of AML.However,due to the resistance of leukemia cells to chemotherapeutic drugs,most patients eventually relapse resulting in a low survival rate,especially in elderly patients and patients with high-risk genetic characteristics.Secondly,due to the non-specific cytotoxicity,while killing leukemia cells,chemotherapeutic drugs could also selectively damage normal hematopoietic stem cells and tissue cells.Therefore,our goal is to find natural small molecules with lower toxicity and high selectivity to target AML.Objective:This paper examined the molecular mechanism of selective killing of mononuclear-macrophages by pyridoxine,and evaluated the delay of tumor invasion in mice,providing a theoretical basis for targeted treatment of acute myeloid leukemia.Methods:1.Different cell lines were stimulated with pyridoxine;live cell workstation was used to observe the typical death morphology of monocyte-macrophage after pyridoxine stimulation.Dose response and time course reaction of pyridoxine-induced monocyte-macrophage death was detected through annexin V-PI staining and analysis,to comprehensively investigated the killing effect of pyridoxine on different cells;2.To elucidate the molecular pathway of pyridoxine-induced monocyte-macrophage death,western bloting analysis was used to detect the expression of major proteins that regulate the programmed cell death pathway,including apoptosis,necroptosis,and pyroptosis;3.We used lentiviral system infection and flow screening to obtain THP-1 cell lines that stably transformed firefly luciferase.Through the injection of tail vein,THP-1-luciferase cells were injected into severely immunodeficient mice(B-NDG mice)to construct an acute myeloid leukemia mouse model.After being treated with pyridoxine,the biochemical conditions in mice were detected with a small animal imager to evaluate the delayed effect of pyridoxine on tumor invasion in mice;4.We treated normal C57/BL mice with different doses of pyridoxine,recorded the changes in body weight of the mice,analyzed the blood routine and blood biochemical indicators of the mice,and measured the changes in the ratio of T / B cells in the spleen by flow cytometry.To evaluate the toxic and side effects of pyridoxine on normal mice;5.Peripheral blood from healthy volunteers was obtained,and mononuclear cells were extracted.After pyridoxine stimulation,the changes of cell viability were measured to evaluate the killing effect of pyridoxine on normal human mononuclear cells;6.Mononuclear cells were extracted from the peripheral blood of patients diagnosed with acute myeloid leukemia.After being stimulated with pyridoxine,the staining level of Annexin V-PI was detected by flow cytometry to evaluate the killing effect of pyridoxine on primary AML cells.To detect the expression of cleaved caspase-3/8 in cells by western blot,and analyze the molecular pathway of pyridoxine acting on primary AML cells.Results:1.Pyridoxine can selectively induce programmed death of monocyte-macrophage,includingapoptosis in U937 and pyroptosis in THP-1.It has no killing effect on other cell lines;2.After blocking caspase activity,the pyridoxine induced cell death switched to necroptosis,and accompanied with the expression of a large number of inflammatory factors;3.Pyridoxine can delay tumor invasion and prolong the survival time of AML model mice;4.At comparable doses,pyridoxine could not affect the body weight,blood routine and blood biochemistry of normal mice,and the ratio of T / B cells in the spleen;5.Pyridoxine can activate caspase-3 / 8 in primary AML cells,induce cell death of primary AML,without significant effect on normal human mononuclear cells.Conclusion:Pyridoxine can selectively induce monocyte-macrophage programmed death,delay tumor invasion in mice,and induce primary AML cell death in vitro,providing a theoretical basis for the application of pyridoxine in the treatment of acute myeloid leukemia.