| Objective: Acute promyelocytic leukemia (APL) is an abnormal hemato-poietic stem cell clonal malignant disease, which is one of the special type ofacute myeloid leukemia. PML gene on the15th and chromosome translocationchromosome genes RARa on the17th cause characteristic cytogenetic abno-rmalities t (15;17)(q22,q21), and form PML-RARa fusion gene. And confi-rmed promyelocytic leukemia/retinoic acid receptor fusion gene is the mole-cular basis of acute promyelocytic leukemia. The encoded fusion protein hasthe activity of a tyrosine kinase, and the cell-associated pathway is furtheractivated, which promote cell proliferation and anti-apoptosis cells. Currentlyacute promyelocytic leukemia in basic clinical disease can be cured by retinoicacid (ATRA) and arsenic trioxide(ATO) to induce differentiation therapy. Youcan get85%-90%complete remission rate, but tretinoin application is proneto retinoic acid syndrome, for long-term use can be drug-resistent, and somepatients with refractory complex are not easy to induce remission or relapseafter remission.Rapamycin (RAPA)which belongs to macrolide, in essence, is an anti-biotic, mTOR pathway inhibitor. Rapamycin as immunosuppressants, byblo-cking the signal transduction to inhibit the growth and proliferation of immunecells, inhibit the synthesis of IL-2and other immune molecules exert immuno-suppressive effects while suppressing the generation of IgG in transplantpatients and the occurrence of specific immunity. Because its strong immunesuppression has been approved by FDA for organ transplant anti-rejection.Compared with the traditional immunosuppressant, rapamycin hashigh efficiency and small drug side effects, especially small renal toxicity,witha small dosage. Rapamycin can reduce immune rejection in rheumatologicalsystematic diseases and organtrans plant recipients. It also has significant treatment effect on the kidney function restoration and other diseases.Research shows that rapamycin not only has immunosuppressive, but also hasdual roles in anti-tumor It inhibits cell cycle transition,and cellular DNAreplication, mainly by inhibiting the mTOR signaling pathway and causes thefailure of normal mitosis. Rapamycin can induce tumor cell apoptosis andinhibit angiogenesis, tumor invasion and vascular endothelial proliferation toexert its anti-tumor effect. Confirmed in laboratory studies, rapamycin hasbeen applied to the treatment of certain solid tumors in clinical trial, and caninhibit mTOR signaling pathway. More and more researches proved thatleukemiais related to excessive activation of signaling pathways, so the effectof rapamycin in the treatment of hematopoietic malignancies are concernedand researched by more and more people.Currently RATA and ATO as first-line drugs to treat acute promyelocyticleukemia clinically have significant effect, but there are still some patientswith drug problems, and it should be valued. Pathogenesis of acutepromyelocytic leukemia is due to PML-RARa fusion protein. The fusionprotein has atyrosine kinase activity, thereby blocking cell differentiation,leading to the occurrence of the disease.Therefore targeted degradation of thefusion protein can restore the original function of the gene, so that blockingcell differentiation and maturation,which is therapeutic drug mechanisms.Butonce patients enhanced tyrosine kinase activity, and excessively expression ofmultidrug resistance gene, anti-apoptoticability of leukemic cells increased,which reduced patients’ reaction to chemotherapy drugs. Studies have shownthat patients may be drug-resistant to the combined result of a variety ofmechanisms. The process is complex and its exact mechanism has yet to bestudied. For patients drug-resistant occur, we can try to study other new drugsto treat acute promyelocytic leukemia.Now a number of studies have shown that acute promyelocytic leukemiaphosphatidylinositol3-kinase (PI3K)/protein kinase B (Akt)/mammaliantarget of rapamycin (mTOR) signaling pathway exists abnormal activation,which plays an important role in tumor cell proliferation, apoptosis, different- tiation, and drug resistance. The experiment is on the basis of long-termstudies over leukemia in signal transduction mechanisms, and aims to explo-ring whether rapamycin can inhibit the growth of leukemia cell lines byblocking the activity of mTOR targets, and how the mTOR pathway inhibitor,rapamycin affect acute promyelocytic leukemia cell line HL-60cell, itsproliferation, apoptosis and downstream gene biology and protein expression,and to exploring new methods and strategies for the treatment of acutepromyelocytic leukemia in clinical practice.Methods:1Determined by MTT method to detect different concentrations ofrapamycin and the application effect on HL-60cell proliferation.2Determined by NADH cell stained method to detect different concentr-ations of rapamycin and the application effect on HL-60cell mitochondria，indirect reaction apoptosis.3RT-PCR to detect the effects of different concentrations of rapamycinon HL-60cells mRNA of PI3K/Akt/mTOR pathway downstream.4Western Blot method effects on the expression of different concent-rations of rapamycin on HL-60cells PI3K/Akt/mTOR pathway downstream ofprotein expression.5Statistical processing.Results:1Different concentrations of rapamycin on HL-60cell proliferationWith different concentrations of rapamycin in HL-60cells after24h andfound rapamycin inhibited HL-60cell proliferation, and dose dependence,20,40,80,160,320,640nmol/L HL-60cell inhibited rate of treatment groupwere significantly higher than the solvent control group (P<0.05). Rangingfrom20to640nmol/L concentration, with the concentration of rapamycinincreasing, HL-60cell inhibited rate were significantly higher. And theconcentration of5nmol/l,10nmol/L,20nmol/L rapamycin-treated group had nosignificant effect on the HL-60cell viability(P>0.05),with an IC50of211.9nmol/L 2Different concentrations of rapamycin on HL-60cell mitochondriaWith different concentrations of rapamycin in HL-60cells after24hfollowed by NADH staining found that compared to the overall trend of thecells into groups to reduce40,80nmol/L HL-60cells treated with the controlgroup, cell distributed and the mitochondria began to turn to clumps orplaques from the crescent-shape with incomplete membrane and nuclearmembrane, even cell disintegration. With concentration increasing, thisphenomenon is more obvious.3Effects of rapamycin on mRNA expression of HL-60cells PI3K/Akt/mTORdownstream factorWith different concentrations of rapamycin in HL-60cells after24h,treatment with40nmol/L,80nmol/L,160nmol/L,using real-time quantitativePCR results showed that after treatment, elF4E binding proteins(4E-BP1),P70S6K1, vascular endothelial growth factor(VEGF), hypoxia-induciblefactor-a(HIF-a) gene expression level than the control group with a reductionof the expression(P<0.05), and with increasing concentrations of rapamycinand its expression was reduced(P<0.05).4Effects of rapamycin on the HL-60cells PI3K/Akt/mTOR downstreameffector proteinsAfter treatment with different concentrations of rapamycin HL-60cells ata concentration of40nmol/L,80nmol/L,160nmol/L.Western blot resultsshowed that the drug-treated group compared with the control group in theHL-60cells mTOR downstream factor.4E-BP1, VEGF, HIF-a proteinexpression decreased(P<0.05),and with the concentration of rapamycinincreased expression of these proteins is reduced(P<0.05).Conclusion:1Rapamycin treatment with HL-60cells in a certain range of concent-rations can inhibit the proliferation and concentration-dependent manner. Thehigher concentration is, the weaker the capablity of the cell proliferationbecome. Moreover, it can reduce the number of mitochondria and affect itsquality, promote apoptosis of HL-60cells. 2Rapamycin within a certain range of concentrations within the HL-60cells can reduce PI3K/Akt/mTOR pathway downstream of4E-BP1, P70S6K1,VEGF, HIF-a gene expression level, you can make4E-BP1, P70S6K1, VEGF,HIF-a protein levels decrease, and in a dose-dependent manner. Rapamycinable to treat acute promyelocytic leukemia from a certain effect, you can try inacute promyelocytic therapeutic application of rapamycin, providing newavenues for treatment for leukemia gene therapy table to provide a scientificbasis... |
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