| In adults,Acute lymphoblastic leukemia comprises approximately 20%-30%of all forms of acute leukemia.Although the remission rate has achieved>80%in patients with newly-diagnosed adult acute lymphoblastic leukemia(ALL)with standard induction regimens,a majority of the responding patients eventually become refractory to initial therapy.The outcome of patients with relapsed or refractory(R/R)adult ALL remains very poor.Therefore,discovery and development of new drugs to overcome multidrug resistance is urgently needed in treatment of R/R ALL patients.Objective:we first established an arac resistant ALL cell line(NALM-6/R)by exposure of parental drug-naive NALM-6 cells to stepwise increasing concentrations of cytarabine(arac),which also displayed cross-resistance towards doxorubicin(ADM).We then investigate whether low dose TPL could re-sensitize NALM-6/R cells to arac as well as doxorubicin(ADM)in vitro and in vivo,and its relationship with interfering DNA damage and repair network.Methods:1.By exposure of parental drug-naive NALM-6 cells to stepwise increasing concentrations of cytarabine(arac),we first established an arac resistant ALL cell line(NALM-6/R),which also displayed cross-resistance towards doxorubicin(ADM).The cytotoxic and apoptotic effect of arac or ADM plus with TPL or not on NALM-6/R cells and primary relapsed or refractory ALL cells were investigated.2.To generate a mouse model of arac-resistant ALL,we subcutaneously injected NALM-6/R cells(5 × 105)into the angular veins of sublethally irradiated adult NSI(NOD/SCID IL2rg-/-)mice(20-30 g body weight;2-3 months of age).Ten days later,16 tumor-bearing mice were divided into 4 groups and injected intraperitoneally with 100 μl with ddH2O,arac(10 mg/kg,half of maximum tolerated dose in SCID mice),TPL(0.5 mg/kg,25%of lethal concentration),or TPL in combination with arac for 5 days,respectively.We evaluated the responses to this treatment by measuring the overall survival times,the weights of the spleens and the percentages of human CD45/CD19 double-positive cells in the bone marrow.3.Using a JC-1 fluorescent probe kit and Cellular Reactive Oxygen Species Detection Assay Kit,mitochondrial membrane potential assessment and intracellular reactive oxygen species(ROS)levels were assessed after different treatments for 48,respectively.Using Western blotting,proteins expression of p-y-H2AX,p-CHK1,p-CHK2 and caspase-9 in NALM-6/R cells were analyzed after different treatments for 48 h.Results1.We first established a drug-resistant cell line(designated NALM-6/R)by exposure of the human ALL NALM-6 cells to stepwise increasing concentrations of arac,after which the established NALM-6/R cell line was routinely maintained in the medium containing 5μM arac.Meanwhile,NALM-6/R cells displayed marked cross-resistance towards 0.5uM ADM.Neither arac within a concentration range of 0-5 μM nor ADM within a concentration range of 0-0.5 μM exhibited significant proliferation inhibition in NALM-6/R cells after 48h exposure.However,in the presence of the IC20 concentration(i.e.,10 nM)of TPL,the IC50 of arac and ADM against NALM-6/R cells were reduced by 20 and 15 times,respectively.NALM-6/R cells were exposed to the highest non-cytotoxic concentrations of arac(5 μM)or ADM(0.5 μM)in the presence or absence of low dose(IC20 concentration,10 nM)TPL for 48 h.Notably,co-administration of low dose TPL significantly increased apoptosis induced by arac or ADM from 10.21 ± 0.07%and 5.56 ± 0.04%to 52.40 ± 4.45%and 24.60 ± 3.23%(P<0.01 for each case),respectively.Consistently,combined treatment with 10 nM TPL and sub-toxic concentrations of arac or ADM significantly increased apoptosis in primary cells isolated from R/R ALL patients(n = 12;P<0.01 for each case,compared arac or ADM as single agent).Notably,the regimens combining TPL with arac or ADM were more effective to induce apoptosis in primary R/R B-ALL cells from patients with white blood cell counts>100 × 109/L than those with<100 × 109/L(P<0.05).2.mice treated with TPL plus arac showed a substantial reduction of tumor burden,manifested by a marked decrease inCD45/CD 19 double-positive cells in bone marrow,compared to mice receiving each single agent.Consistently,histopathology revealed a remarkable reduction of leukemia cell infiltration in bone marrow of mice receiving treatment with TPL plus arac.Further,average spleen weight of mice treated with TPL plus arac(0.17 ± 0.26 g)were significantly lower than those of mice in the control group(0.31 ± 0.23 g),as well as each single agent group(0.28 ± 0.01 g for TPL alone and 0.33 ± 0.56 g for arac alone;P<0.0001 for each case).Kaplan-Meier analysis showed that the combination of TPL with arac significantly prolonged animal survival,compared to TPL or arac alone(P<0.01).3.The combination of arac or ADM with TPL induces mitochondrial injury in ALL cells:exposure to arac(5 μM)or ADM(0.5 μM)resulted in a modest decrease in JC-1 aggregates(15.2 ± 0.76%and 20.5 ± 0.37%,respectively),co-administration of 10 nM TPL with either of these agents sharply reduced JC-1 aggregates(53.8 ± 1.23%and 51.8 ± 0.79%,respectively)reflecting loss of MMP(or mitochondrial depolarization)in NALM-6/R cells.The combination of arac or ADM with TPL triggers reactive oxygen species(ROS)production in ALL cells:Compared to treatment with each single agent,the combination of TPL with either arac or ADM significantly increased ROS generation by approximately 9 and 5 folds in NALM-6/R cells,respectively.Notably,2h pre-treatment with the ROS scavenger NAC(30 mM)dramatically prevented ROS production induced by TPL plus arac or ADM.Combined treatment with TPL and arac or ADM disrupts DNA damage checkpoint,resulting in robust DNA damage in NALM-6/R cells:As yH2A.X is commonly used as a marker for DNA damage,we next examined expression of yH2A.X to monitor the effects of TPL on DNA damage induced by arac or ADM.While TPL did not significantly affected the levels of yH2A.X,the combination of TPL with arac or ADM at the indicated doses resulted in a rightward shift of yH2A.X-FITC fluorescent peak in FACS histograms,indicating increased yH2A.X expression.Western blot was performed to assess the effects of TPL and arac or ADM alone or in combination on DNA damage checkpoint by monitoring phosphorylation of Chk1and Chk2,which reflects cytoprotective activation of cell cycle checkpoints in response to DNA damage.Notably,co-treatment with TPL markedly diminished phosphorylation of Chkl and/or Chk2 triggered by arac and ADM,accompanied by a robust increase in yH2A.X expression,consistent with the results of flow cytometric analysis and cleavage/activation of caspase 9.Conclusion:our study demonstrated that low dose triptolide could reverse arac and ADM resistance and in NALM-6/R cells as well as primary cells from patients with relapsed or refractory(R/R)ALL.Mechanistically,Co-treatment with TPL and arac or ADM upregulated pro-apoptotic caspase-9 protein,inhibited checkpoint kinase 1(Chk1)and 2(Chk2)phosphorylation,and induced γ-H2A.X.these findings provide preclinical evidence for repurposing use of TPL in combination with chemotherapeutic agents to treat R/R ALL as an alternative salvage regimen. |
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