The Efficacy Of Nutlin3 On Cell Death Of Acute Myeloid Leukemia Cell Lines And Its Mechanism

Background:Approximately 80%Acute Myeloid leukemia(AML)carry wild type p53,but the activity of p53 pathway is frequently inhibited by MDM2(murine double minute 2).MDM2 is a p53-specific E3 ubiquitin ligase,which has been observed to be overexpressed in approximately half of leukemia.The inacitivation of p53 by MDM2 protects leukemia cells from apoptosis,leading to chemoresistance and refractory disease.Selective small molecule antagonist of MDM2 Nutlins is potent in reactivating and stabilizing p53 through releasing p53 from MDM2-p53 binding.As a p53 target gene.MDM2 is stimulated by increased p53,which,in turn,mediates the degradation of p53.Such process may abrogate the anticancer effect of Nutlin3 in a long term,and even induce new clones resistant to chemotherapy.Triptolide(TPL),a diterpenoid epoxide,originally purified from the medicinal plant Tripterygium wilfordii Hook F,has been regarded as potent candidates for treating tumors.Recent studies show that anti-cancer effects of TPL through multiple RNA polymerase-mediated transcription inhibiton by targeting a subunit of the transcription factor TFIIH,which leads to downregulation of certain short-lived mRNA.Objective:This study aims to investigate the anti-leukemia effects of Nutlin3,a selective MDM2-p53 binding inhibitor in wild-type p53 acute myeloid leukemia cells in vitro and to investigate whether combination with TPL would contribute to synergistic effect as well as its molecular mechanisms.Method:1.Proliferation and apoptosis:We detected the inhibition of cell proliferation of Nutin3 treatment with OCI-AML3 and MOLM13 cells at different time points by CountBright Absolute Counting Beads kit as well as Cck-8 kit.By DAPI staining and Annexin V/PI using flow cytometry we detected the induction of apoptosis.2.JC-1 staining was used to determine the mitochondrial membrane potential of OCI-AML3 and MOLM13 cells by JC-1 probe.3.OCI-AML3 p53 shRNA and control cells were used to determine apoptotic rates after treatment with Nutlin3 and/or Triptolide by flow cytometry,4.2-hour pretreatment with Z-VAD-fmk,a pan-inhibitor of the caspase family,OCI-AML3 and MOLM13 cells preceded treating with drugs,then the apoptotic rate was determined by Annexin-V/PI assay.5.Quantitative Real time-PCR:The fold change of MDM2、XIAP level in OCI-AML3 after treatment with Tritptolide or DMSO were performed using TransStart tip green qPCR Supermix(Transgene,China).The human housekeeping gene β-actin was used as the RNA loading control.6.Western blotting:The expression of MDM2,p53 and proteins related to mitochondrial apoptosis in OCI-AML3 and MOLM13 cells after treating with Nutlin3 and Triptolide.Results:1.Nutlin3 were effective against the proliferation of OCI-AML3 and MOLM13 cells.Nutlin3 inhibitied growth of OCI-AML3 and MOL13 cells effeicently in a time course.The IC50 for Nutlin3 were(5.53±2.86)μmol/L and(2.03±0.23)μmoI/L,respectively.The IC50 for Triptolide were(18.46±4.71)μmol/L and(23.60±8.92)μmol/L,respectively.Anti proliferation effect of combination treatment were 0.24 and 0.9,caculated by Chou-Tataray methods,indicating synergistic effect of both drugs in suppressing proliferation in AML cells.2.After Nutlin3 treatment for 48hrs,condensed chromatin and apoptosis body were shown in nucleus with DAPI staining under fluorescence microscope.3.AnnexinV/PI assay showed that Nutlin3 with various concentration(0.625、1.25、2.5、5、10μmol/L)induced apoptosis of OCI-AML3 cells with apoptotic rates(1.79±1.86)%、(1.29±1.74)%、(4.19±2.45)%、(10.21±3.65)%、(30.03±5.26)%respectively and the difference between Nutlin3 and control was significant(χ2=15.38,P=0.009).0.625、1.25、2.5μmol/L Nutlin3 induced concentration-dependent apoptosis in MOLM13 cells,with apoptotic rates(0.93±0.17)%、(2.38±0.26)%、(20.40±4.03)%respectively and the difference measured by Kruskal-Wallis has statistic significance(χ2=58.37,P<0.001).Apoptosis was more efficient after combined with Triptolide with CI=0.6 and 0.45,respectively.4.Nutlin3 induced apoptosis of OCI-AML3 cells in p53-dependent manner.P53 knocking down abrogated the effect of Nutlin3(t=2.655,P=0.025),while Triptolide partially relied on p53 pathway to induce apoptosis in OCI-AML3 cells.However,combination treatment showed agonism in OCI-AML3 carrying p53 shRNA on the contrary to synergism in control cells.5.Z-VAD-fmk could suppressed Nutlin3 and Triptolide-induced apoptosis.The results suggested that both drugs induced p53 wild type AML cell apoptosis in Caspase-dependent manner.6.JC-1 probe detected that the decreased mitochondrial membrane potential after treatment with Nutlin or Triptolide.The results of nonparametric test showed that the mitochondrial membrane potential combination treatments could be down-regulated more efficiently by combination drugs than single agent.(t=14.42,P<0.001 and t=12.28,P<0.001)7.Quantitative Real time-PCR showed that Triptolide inhibited the transcription of MDM2 and XIAP in a time manner.Western blotting showed that Nutlin3 and Triptolide stimulated the activation of mitochondrial apoptosis pathway in leukemia cells with wild-type p53 with increased activation of Caspase3 accompanied by PARP degradation,especially markable after co-treatment.The expression of p53 and its targets,es.PUMA and Bax was upregulated significantly while a robust decrease in MDM2 and anti-apoptotic protein Mcl-1 expression was seen in combination group.Conclusion:1.Nutlin3 markedly inhibited proliferation and induced apoptosis of OCI-AML3 and MOLM13 cells,in time-dependent manner.2.Cell death of OCI-AML3 and MOLM13 by Nutlin3 closely related to p53 pathway triggering mitochondrial apoptosis machine and inducing OCI-AML3 and MOLM13 cells apoptosis.3.Nutlin3 and Triptolide significantly induce apoptosis and inhibited proliferation in p53 wild type AML cell lines in a synergistic manner.4.Nutlin3 and Triptolide-induced apotosis were mediated by Caspase activation.Nutlin3 induced p53-dependent apoptosis,while TPL induced both p53-dependent and p53-independent apoptosis.5.The molecular mechanism of Nutlin3-induced apoptosis in leukemia cells with wild-type p53 was related to the activation of mitochondrial apoptosis pathway after p53 activation.The decrease in MDM2 and anti-apoptotic factors by Triptolide enhanced p53 activity,contributing to synergistic effect of mitochondrial-mediated apoptosis induction.