In Vitro Study Of Effects And Mechanisms Of MTOR Inhibitor Rapamycin On Human T-cell Acute Lymphoblastic Leukemia Cells

T-cell acute lymphoblastic leukemia(T-cell acute lymphoblastic leukemia,T-ALL)is aclonal lymphoid malignancy with poor prognosis.Over the last two decades,theoutcome for patients with T-ALL has improved dramatically with current therapy,however,some relapsed or refractory patients often develop resistance to standardchemotherapy.The 5-year disease-free survival rate is only 28-39%.For acute leukemia,the mammalian target of rapamycin(mTOR)pathway plays animportant role in energy metabolism,the regulation of cell proliferation,and promotingleukemia cell survival.The over-activation of the mTOR pathway is also implicated inhuman leukemia resistance to conventional chemotherapy.Thus,the development ofnovel therapeutic agents override the resistance,and directly targeting this signalingnetwork is crucial.One potential class of novel therapeutics is rapamycin.Preclinicalstudies have shown that rapamycin may be effective for the treatment of prostate,smallcell lung,glioblastoma,renal cell,and breast cancer.However,the use of rapamycin islimitedly documented in hematological malignancies,especially for T-ALL.Thus in thepresent study,we used two T-ALL cell lines(Jurkat and Molt-4)and T-ALL primarycells to explore the antileukemic mechanism of rapamycin in T-ALL and evaluate the potential clinical implications in the treatment of T-ALL.There are three sections in ourresearch.Section 1:Effect of Rapamycin on Growth in T-ALL CellsFirstly,the effects of rapamycin on the cell proliferation in T-ALL cell lines(Jurkat andMolt-4)and primary cells were assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Exposureto rapamycin(0.1-100 nmol/L)resulted in a significant inhibitory effect on theproliferative activity ofT-ALL cells:an effect that was dose and time dependent.The50% inhibitory concentration(IC50)values for Jurkat,Molt-4 and primary cells at 72 hwere 16,23 and 16 nmol/L,respectively.However,for normal human bone marrowmononucleated cells(MNCs),the IC50 concentration was over 461 nmol/L,which is farbeyond the clinical usage.To determine the mechanism of growth inhibition byrapamycin in more detail,we used Jurkat cells to analyze the cell apoptosis andautophagy profile after treatment with rapamycin.The classic method,such as genomic DNA fragmentation assay and PARP cleavageimmunoblotting assay,were used to detect apoptosis qualitatively.It showed nogenomic DNA fragmentation and PARP cleavage in Jurkat cells treated with 10nmol/Lrapamycin for 72h.The cell apoptosis index was investigated quantitatively by AnnexinV/propidium iodide(AV/PI)staining followed by flow cytometry analysis.Exposure tolnmol/L,10nmol/L and 100nmol/L rapamycin for 72h caused 5.33±1.33%,5.78±2.26%,5.69±2.20 apoptotic cells,which was comparable to that of the untreated group(5.37±2.19%)(p＞0.05),indicating rapamycin could not induce apoptosis in Jurkatcells.Autophagy,another form of programmed cell death is thought to be as prevalent asapoptosis.Using electron microscope,some large autolysosome and vacuoles wereobserved in Jurkat cells treated with 100nmol/L rapamycin for 72h.Furthermore,using acridine orange staining followed by flow cytometry analysis,cell autophagy index wasinvestigated quantitatively.Exposure to 100 nmol/L rapamycin for 72h,the autophagyindex was apparently increased to 44.56±13.79%,while in the untreated control group,the autophagy index was 2.55±1.50%(p＜0.05).3-methyladenine(3-MA),whichinhibits autophagosome sequestration,could decrease the autophagy index to 6.02±3.91%,but it could not promote the apoptotic cell death.Finally,Jurkat cells treatedwith 1-100 nmol/L rapamycin showed a dose-dependently up-regulation of Beclin-1mRNA and protein expression,the classic marker for autophagy.Section 2:Effect of Rapamycin on mTOR pathway Cell Cycle and TelomeraseActivity in T-ALL CellsFirstly,we confirmed that the PI3K/Akt/mTOR signaling pathway is over-activated inJurkat and primary T-ALL cells.Rapamycin at 10nmol/L markedly reduced thephosphorylation ofp70S6K and S6 in Jurkat cells.This effect was seen as early as 12 hafter exposure to rapamycin,and the total proteins of the pan-S6 and p70S6Kmaintained stable levels.Interestingly,rapamycin could also slightly downregulate thephosphorylation of Akt,which functioned upstream ofmTOR.Since mTOR signaling relates to cell cycle progression,we therefore determine theregulation of cell cycle by rapamycin.The cell cycle profile was analyzed by flowcytometry with PI staining.As expected,G1 phase arrest was induced by rapamycin(100nmol/L)after 16 h in Jurkat and Molt-4 cells,and the ratio of G1 continued to growas treatment was prolonged to 32 h.Furthermore,we evaluated the effect of rapamycinat different concentrations on the cell cycles arrest.Jurkat cells were synchronized cellsby serum starvation.Serum stimulation resulted in the transition of cells from the G1phase to the S phase(G1:40.3%,S:39.1%).Rapamycin significantly blockedserum-induced entry to the S phase in a dose-dependent manner in Jurkat cells.The G1phase index was 41.2%,64.3% and 66.4% for lnmol/L,10nmol/L and 100nmol/L, respectively.To further determine the mechanism of G1 phase arrest by rapamycin,we investigatedthe effects of rapamycin on cell cycle related proteins,specifically CDK4,CDK6,cyclin D2,cyclin D3,p27Kipl,p21 wafl and c-Myc.Rapamycin treatment maintainedelevated levels of p27Kipl and p21 wafl in Jurkat cells,and downregulated cyclin D3without affecting the cyclin D2 protein levels.It decreased the c-Myc expression but didnot affect the protein levels of either CDK4 or CDK6.On the other hand,we investigate the regulation of telomerase by rapamycin in Jurkatcells.First,telomerase activity was measured by the TRAP assay.When the cells weretreated with rapamycin,telomerase activity was downregulated within 24 h and had areduced tendency that was dose and time dependent.Because telomerase activitycorrelates with the expression of hTERT mRNA,RT-PCR was used to examine theexpression of hTERT mRNA in Jurkat cells treated with rapamycin for 48 h.We foundthat treatment with varying concentrations of rapamycin significantly reduced thehTERT mRNA level in a dose-dependent manner.Furthermore,by Western blotting,thehTERT protein level decreased and correlated with these changes in the expression ofhTERT mRNA induced by rapamycin.Section 3:Synergistic Cytotoxic Effect of Rapamycin and Idarubicin by inhibitionof mTOR pathway in T-ALL CellsFor patients with T-ALL,the induction chemotherapy regimens usually includeanthracyclines.Thus in this section,we tried to determine the antileukemic effect ofidarubicin combined with the the anthracycline idarubicin(IDA).The IC50 of IDA forJurkat and Molt-4 was 21.8nmol/L and 2.21 nmol/L,respectively.When combined with10nmol/L rapamycin,the IC50 of IDA for those two cell lines was decreased to2.95nmol/L and 1.28nmol/L.The combine index was＜1,indicating the synergisticcytotoxic effect of IDA and rapamycin. Both electron microscope and Annexin staining flow cytometry analysis revealed thatrapamycin significantly increased apoptotic sensitivity to IDA.Treatment of Jurkat andMolt-4 cells with IDA at a low concentration for 48h resulted in 17.51±3.20% and22.78±3.54% apoptosis,respectively.However,when combined with 10nmol/Lrapamycin,the apoptosis index increased to 50.17±5.19% and 47.33±9.03%,respectively(p＜0.05).The combination of IDA with rapamycin was prone to activateCaspase3,PARP,Caspase8 and Caspase9,indicating both extrinsic and intrinsicapoptotic pathways were involved.To further investigate the mechanism of the intrinsic apoptosis,we analyzed Bcl-2family members,which regulate cytochrome c release from mitochondria.The resultsshowed rapamycin could reverse the up-regulation of Bcl-2 protein triggered by IDA.The Bax/Bcl-2 ratio is significantly higher in two-agent combination group than that inIDA or rapamycin single agent group.However,the two-agent combination did notaugment the inhibition of Survivin expression.Besides,the mTOR signaling upstream Akt and downstream S6K were activated byphosphorylation in response to IDA treatment.Rapamycin significantly inhibit mTORsignaling activation triggered by IDA.Furthermore,rapamycin alone or IDA alone didnot affect the ERK phosphorylation,but the combination of the two agents led tosynergistic inhibition of ERK phosphorylation.Thus,combination of IDA withrapamycin exerts antileukemic effect via inhibition ofmTOR and ERK signaling inT-ALL.In summary:(1)Rapamycin directly inhibits the proliferative activity of T-ALL celllines(Jurkat and Molt-4)and primary T-ALL cells in dose and time dependent manner(2)As a single agent,rapamycin could induce cell autophagy but not apoptosis in Jurkatcells,accompanied with up-regulation of Beclin-1 expression,the marker for autophagy.Inhibition of autophagy by 3-MA could not enhance the apoptotic cell death.(3) Rapamycin reduces the phosphorylation of p70S6K and S6 which belong to mTORdownstream proteins,as well as the phosphorylation of Akt,which functions upstreamof mTOR.(4)Rapamycin induces Gl phase arrest,up-regulates the protein level ofP21 wafl as well as P27kipl,and downregulates cyclinD3 and c-Myc.(5)Exposure torapamycin reduced telomerase activity,and reduced hTERT mRNA and proteinexpression.(6)Combination of IDA with rapamycin exerted a synergisticanti-poliferative effect and promoted apoptosis by both extrinsic and intrinsic apoptoticpathways.The intrinsic apoptotic pathway was triggered via regulation of Bax,Bcl-2and Survivin.(7)Rapamycin could reverse up-regulation of Bcl-2 protein andover-activation ofmTOR signaling induced by IDA.The findings provide a strongrationale for developing therapy with rapamycin for T-ALL.