The Effect And Mechanism Of MTOR Pathway Inhibition Combined With Chemotherapy In Vitro And In Vivo On Acute Myeloid Leukemia

ObjectiveTo investigate the effects of mTOR signaling pathway inhibitor rapamycin in combination with chemotherapy on acute myeloid leukemia cells in vivo and in vitro and the mechanisms.Methods1.Human acute monocytic leukemia cell line SHI-1 was treated by 0.5 uM doxorubicin+0.5 uM cytarabine(chemotherapy group),5 uM rapamycin(rapamycin group),and doxorubicin+0.5 uM cytarabine+5 uM rapamycin(combined group).The proliferation and apoptosis of SHI-1 cells in different groups was detected by CCK8 and flow cytometry.The proteins which involved with autophagy,apoptosis and mTOR signal pathway,and Caveolin-1 protein were detected by western blot.2.1×10~7 or 5×10~7 SHI-1 cells were inoculated into NPG mice via the tail vein,and NPG rats were randomly sacrificed every week from the second week after inoculation until natural death occurred in NPG mice.Nested RT-PCR and peripheral blood smear,flow cytometry,histopathology and immunohistochemistry were used to detect the proliferation and infiltration of SHI-1 cells in NPG mice.3.1×10~7 SHI-1 cells were inoculated into NPG mice via tail vein.W hen the CD45 and 33 double positive cells in peripheral blood of NPG mice were greater than1%,the NPG mice were randomly divided into 3 groups for treatment.11 NPG mice in the chemotherapy group were treated with doxorubicin(1 mg/kg/day for 3 days)and cytarabine(100 mg/kg/day for 5days).12 NPG mice in the rapamycin group were treated with rapamycin(10mg/kg/day for 5 days),12 NPG mice in the combined chemotherapy group were treated by doxorubicin,cytarabine and rapamycin.The drug was injected intraperitoneally.10 NPG mice without any treatment were control group.One group was randomly sacrificed on the first day after treatment,and the proliferation and infiltration of SHI-1 cells in NPG mice were detected by gross anatomy,flow cytometry and histopathology.Results1.Rapamycin and chemotherapy could effectively inhibit the proliferation and induce apoptosis in SHI-1 cells.In the combination group,rapamycin could significantly enhance the inhibition apoptosis rate of SHI-1 cell induced by chemotherapy(P<0.05).When SHI-1 cells were treated by rapamycin or chemotherapy,the proteins of CAV1,Bax,P53,ATG5,Beclin-1 and LC3-II of SHI-1cell were increased,and these proteins were significantly increased than that treated by rapamycin or chemotherapy when SHI-1 cells were treated by rapamycin plus chemotherapy.Howerer,the proteins of Bcl-2,caspase3,mTOR,p-mTOR,4EBP1,p-4EBP1,p70S6K1,and p-P70S6K1 were decreased than control group when treated by rapamycin or chemotherapy,and these proteins were significantly decreased than that treated rapamycin or chemotherapy group when SHI-1 cells were treated by rapamycin plus chemotherapy.2.After inoculated subcutaneous,SHI-1 cells were grown and formed solid tumor in the NPG mice.When inoculated via tail vein,the media survival time of NPG mice inoculated with 1×10~7 or 5×10~7 SHI-1 cells were 30 and 33 days respectively.The specific MLL-AF6 fuse gene of SHI-1 cell was amplified in the spleen and bone marrow at 14 days after inoculation.A few human CD45+CD33+cells were detected in the peripheral blood by flow cytometer at 21 days after inoculation.At the day 28 after inoculation and when NPG mice were dead,massive CD45+CD33+cells were detected in the peripheral blood,bone marrow and spleen;SHI-1 cells were growth as a solid tumor in the organs such as kidney,liver,spleen,stomach,heart,lymph node and the soft tissues;immunohistochemical staining had shown that the infiltrated cells were human CD45+positive.The grade of infiltration was positively correlated with cells numbers of inoculation and the survival time of NPG mice.3.The median survival time of the control group,the chemotherapy group,the rapamycin group and the combined group were 30.5,35.0,34.0,and 39.0 days,respectively.The survival time of each treatment group were significantly prolonged than the control group.The survival time of NPG mice in the combined group was significantly longer than that of chemotherapy and rapamycin group(P<0.05),but there was no significant difference between chemotherapy group and rapamycin group(P>0.05).On the first day after the treatment were finished,the spleen weights of the control group,the chemotherapy group,the rapamycin group and the combined group were 78.6 mg,16.1 mg,48.9 mg,and 11.7 mg,respectively,and human CD33+45+cells in the peripheral blood,bone marrow,and spleen were 20.2%,23.2%,16.8%in the mice of control group,13.6%,14.2%,5.50%in the mice of chemotherapy group,2.02%,2.68%,3.31%in the mice of rapamycin group,and309%,2.49%,1.22%in the mice of combination group.histopathology showed that the degree of leukemia infiltration in the mice of chemotherapy,rapamycin and combination groups was significantly lower than that in the control group on the first day after chemotherapy.When NPG mice were dead,more green tumor were found in multiple organs(kidney,liver,stomach,mesentery,mediastinum,bladder,spine,subcutaneous,posterior,neck)of the mice of control group.However,less green tumor were found in the organs of mice in chemotherapy group,rapamycin group and the combination group.Conclusions1.SHI-1 cells could proliferate and form multi-organs infiltrated leukemia in the body of NPG mice.This animal model may be a useful tool for the study of acute leukemia.2.Rapamycin could inhibit SHI-1 cell proliferation,induces apoptosis,promotes autophagy,and synergizes the cytotoxicity of chemotherapy by inhibiting the mTOR signaling pathway,promotes autophagy and enhances CAV1 and P53protein expression.3.Rapamycin combined with chemotherapy could inhibit the proliferation of SHI-1 cells in NPG leukemia mice,and enhance the cytotoxicity of chemotherapy,delay the infiltration of leukemia cells in NPG mice,and prolong the survival of leukemia mice.