The Molecular Mechanisms Of Angelica Sinensis Polysaccharide On Human-derived Leukemia Stem Cell Senescence

ObjectiveAcute myelogenous leukemia represents a disease of hematopoieticstem cell disorder, in which a small subpopulation of leukemia stem cellswith potential of self-renewal are responsible for the accumulation of largenumbers of immature myeloblasts. In addition to their crucial roles inleukemia initiation and progression, LSCs are also responsible for the highfrequency of relapse and chemoresistent that is characteristic of currentAML therapies.Chinese angelica traditionally has been used to enrich and invigorateblood for centuries. Angelica sinensis polysaccharide (ASP), an acetoneextract polysaccharose found as the major active component in Dong quai(Chinese angelica sinensis), is a promising agent that has been found tohave several properties, including antitumor activity, anti-radiation,immune-enhancement and pro-hemopoiesis. Our previous studies showthat ASP has bidirectional effects of leukemia cells proliferation inhibitionand HSCs ageing alleviation. But the effects of ASP on leukemia stem cells remain unclear. This study focused on the effects of ASP on leukemia stemcell and the underlying molecular mechanisms used by a serial of modernbiological methods, which will be beneficial for exploring new approachesfor leukemia therapies.MethodsSeparation and identification of CD34＋CD38－LSCs: The leukemiabone marrow were obtained from acute myelogenous leukemia patient inaseptic condition. bone marrow mononuclear cells were harvested bydensity gradient centrifugation, magnetic activated cell sorting for CD34＋CD38－LSCs cells, flow cytometry for the purify of CD34＋CD38－LSCsafter separation, colony forming assay for self-renewal of CD34＋CD38－LSCs cells.Effects of ASP on CD34＋CD38－LSCs proliferation: The effects ofASP on CD34+CD38－LSCs in vitro was investigated by adding variousdose(0~80μg/ml) of ASP for48h coculture. The inhibition of ASP onCD34+CD38－subpopulation proliferation was detected by CCK-8assay.Detection of CD34＋CD38－LSCs senescence associated biomarkers:The cell cycle distribution of CD34+CD38－LSCs after ASP exposuredetected by flow cytometry. The percentage of senescent cells was detectedby SA-β-Gal staining. The colony-formed ability were detected byColony-Assay. Detection of senescence associated molecular biological markers:Cells from two groups were harvested for detection：1) The levels ofsenescence associated regulated gene p16,Rb expressions were performedby quantitative RT-PCR;2) The changes of telomere length were tested bysouthern blotting assay;3) The protein expressions of senescenceassociated regulation proteins P16、CDK4and Cyclin E were detected byWestern Blotting;4)The telomerase activities of cells were detected byTRAP-PCR, the changes of telomere length were detected by SouthernBlotting.Establishment of AML mice model：The NOD/SCID mice wereinjected with500μL cell suspension containing2×107LSCs via tail veinto reconstruct AML disease for4weeks. Each mouse was intraperitoneallyinjected with4mg cyclophosphamide (2mg per day for two day) beforecells transplantation. Negative control mice were given with normal salineas the same parameters. The living condition, hemogram, white bloodcounting, liver index and kidney index were calculated for transplantationefficacies.Detection of ASP on AML mice leukemia syndrome alleviation：The NOD/SCID mice with established AML disease were intraperitoneallyinjected with200mg/kg ASP for14d, dose of2.5mg/kg Ara-c to parallelmice as control group,200mg/kg ASP and2.5mg/kg Ara-c as combinedgroup, negative control with normal saline as the same parameters. A serial of senescence associated parameters were analyzed to investigate theeffects ofASP onAML disease in NOD/SCID mice.ResultsThe CD34+CD38－cells subpopulation can effectively be isolated byMACS; cells shows features of well-stacked morphology, hightransparency, well refraction under inverted phase contrast microscope. Thepurity of CD34+CD38－cells population is up to91.15±2.41%; Trypan bluestaining showed the viable of cells was up to95.42±3.52%(P＜0.05); anobvious increase in colony forming ability after separation.ASP had an remarkable dose and time-dependent inhibition on CD34+CD38－LSCs proliferation in vitro culture. The number of SA-β-Galstaining positive cells had been increased compared to control group cells;changes after ASP co-culture including an decrease in colony-formingabilities; Changes including chromatin converge into cell membraneboundary; senescence associated heterochromatin formation(SAHF);Mitochondria aggregation and swell edema of Golgi complex after ASPco-cultureMolecular biological changes including an decrease level on p16, Rbgene and telomerase activities; an shorter length on telomere of CD34+CD38－cells; an decrease in CDK4and Cyclin E protein expressions; an increase in P21and P16protein expressions after40μg/ml ASP co-culturefor48h;NOD/SCID mice shows a bad living condition and remarkableleukemia syndrome including drooping spirits; loss of weight; Abdominalmass; an increase of white blood cells counting in peripheral blood; Activebone marrow hyperplasia; tumor cells liver infiltration; lots of humanderived leukemia cells in mice bone marrow presented byimmunofluorescence after LSCs transplantation.After ASP offering, AML mice show some remarkable changescompared to control mice including an decrease of white blood cellscounting in peripheral blood; clear grains of hepatic lobule; alleviation ontumor cells liver infiltration; an increase on the ratio of ageing BMNC cells;but decrease on colony forming ability; cell cycle arrest at the phase ofG0/G1; increase on P16and Rb proteins; decrease on cell cycle regulatedproteins CDK4and Cyclin D1expressions.ConclusionThe CD34+CD38－LSCs subpopulation can effectively be isolated byMACS.ASP can effectively induce senescence of human derived leukemiabone marrow CD34+CD38－LSCs. The molecular mechanisms of ASP on CD34+CD38－LSCs are relatedwith cell telomere system and p16-Rb signaling pathways in vitroco-culture.NOD/SCID mice were successfully established with AML by LSCstransplantation.The leukemia syndromes of NOD/SCID AML mice had beenalleviated by APS, which is related with CD34+CD38－LSCs agingregulation.