The Effects And Mechanism Of Rapamycin On K562Cells PI3K/AKT/mTOR Signaling Pathway

Objective:Rapamycin was a macrolide immunosuppressant, inhibited theproliferation and the invasion of the tumor cells, induced cells apoptosis. Therapamycin combined with the immunoaffinity protein (FKBP12), foemedRAPA-FKBP12binding compound. The RAPA-FKBP12binding compoundblocked the mammalian target of rapamycin (mTOR), thereby reducing theexpression of downstream molecular4EBP-1、 P70S6KB、 VEGF、HIF1α，which affected the cells cycle、protein synthesis and reducedinvasiveness and metastasis of tumor cells.Chronic myeloid leukemia is a myeloproliferative neoplasms,95%ofCML patients with BCR/ABL fusion gene positive. Although TKIsapplications enable the treatment of CML achieved a major breakthrough, butits existence drug problem, it needs to find other treatments.The study was to determine the change of rapamycin on K562cellsPI3K/AKT/mTOR pathway downstream molecules4EBP1、 P70S6BK、VEGF、HIF1αand to understand the K562cell morphology changes byelectron microscopy, to study the changes of K562cells lysosomes andmitochondria through enzymatic staining. Provide a theoretical foundationand basis for rapamycin treatment of chronic myeloid leukemia.Methods:The K562cells was cultured in vitro, followed by intervention in variousconcentrations of rapamycin for24h. The cell inhibition rates weredetermined by CCK8assay. According to the results of CCK8choose0nmol/L,80nmol/L,160nmol/L,320nmol/L concentration of rapamycin treatedK562cells24h. The change in mRNA and protein leves of4EBP1、P70S6KB、VEGF、HIF1α、BCR/ABL、SHIP were determined by western blot and real time PCR. We used electron microscopy、ACP staining andNADH staining to observe the change of cell morphology and mitochondria,lysosomes.Results:1With the increased of concentration of rapamycin,the inhibition ratesof K562cells were increased significantly.2The lower expressions of4EBP1, VEGF in protein levels with thehigher concentration of rapamycin(P<0.05).3With the increased of concentration of rapamycin, the expressions of4EBP1、P70S6KB、VEGF、HIF1αin mRNA levels decreased(P<0.05).4The damages of mitochondria ang lysosomes increased significantlywith the increased concentration of rapamycin. The degree of apoptosisincreased with the concentration of rapamycin in the electron microscope.5Rapamycin could not influent the expressions of SHIP gene and BCR/ABL.Conclusion:1Rapamycin could inhibit the growth of K562cells and promote theK562cells apoptosis.2Inhibition of mTOR by rapamycin and reduce its downstreammolecules4EBP1P70S6BK VEGF HIF1αexpression, and inhibit the proteinsynthesis and gene expression to achieve antitumor effect.3Rapamycin can damage mitochondria and lysosomes, which lead tothe tumor cells apoptosis and necrosis.4Rapamycin could not affect the SHIP gene and BCR/ABL fusion geneof K562cells.