| Objective:To study the role of PLK1 inhibitors in autophagy regulation in acute myeloid leukemia.To explore the mechanism of PLK1 inhibitors in autophagy regulation in acute myeloid leukemia.Preliminary analysis of the clinical value of PLK1 in children AML.Methods:AML cells were treated with polo-like kinase 1(PLK1)inhibitors RO3280 and BI2536.AML NB4 cells overexpressing microtubule-related protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors,and their autophagy activity was analyzed.Western blot analysis of LC3(lc3-ii)cleaved during autophagy and phosphorylation of mammalian target protein of rapamycin(mTOR),adenosine monophosphate-activated protein kinase,and unc-51-like kinase 1.Autophagosomes were detected by transmission electron microscopy.Apoptosis was detected manually using BD Annexin V staining kit.The relative expression of p-mtor/mTOR was analyzed by ImageJ software.The expression of PLK1 gene was detected by quantitative real-time PCR.Patients and samples:140 children with acute myeloid leukemia who were admitted to the hematology and oncology department of children's hospital affiliated to soochow university from 2008 to 2013 received bone marrow samples at the time of diagnosis during routine clinical evaluation.Results:PLK1 is always highly expressed in leukemia cell lines.Compared with normal progenitor cells,PLK1 is overexpressed in most samples of aml patients.Several inhibitors have good autophagy induction effects.To confirm that PLK1 inhibitor induces autophagy,we treated NB4 cells with 200 nM and 500 nM RO3280 or BI2536 for 4 hours before analysis,and found that the autophagy activity level increased(500 nM BI2536:9.73gy activity was analy0.45).PLK1 inhibitor induces autophagy in AML cells.In NB4 cells treated by RO3280 and BI2536,Western blotting showed more 1c3-ii.More Ic3-ii was found in groups RO3280 with 100-500 nM and BI2536 with 200-500 nM.Beclin-1 up-regulation and SQSTM1/p62 down-regulation were also observed in the PLK1 inhibitor treatment group.Autophagosomes of cells treated with RO3280 were observed by transmission electron microscope.Compared with DMSO control group,there were more autophagosomes in NB4 cells treated with RO3280.These results are consistent with the autophagy activity analysis of lc3-gfp.PLK1 inhibitors can induce autophagy in AML cells.RNA interference inhibited autophagy in AML cells induced by PLK1.Inhibitors often have multiple candidate targets and their molecular functions are affected by other candidate targets,especially homologous molecules.The autophagy activity was calculated as follows,NB4 cells,si-plk1:11.23i-plk1:11.23roup.Autophagosomes of cells treated with ro3280 were observed by transmission electron microscope,H1-60 cells,si-plkl:6.73±0.90;Si-Nc:0.40+/-0.10(p=0.002).Inhibition of PLK1 by RNA interference can significantly induce autophagy in AML cells.PLK1 inhibits autophagy in AML cells by mTOR dephosphorylation.PLK1 is an excellent anticancer target for pediatric acute myeloid leukemia.Inhibition of PLK1 can induce apoptosis of various tumor cells.PLK1 inhibition significantly inhibited the proliferation of NB4 cells.The results showed that PLK1 expression was related to FAB subtype and minimal residual disease(MRD).Kaplan-meier survival analysis showed that patients with high PLK1 expression in tumors had a shorter survival period(p=0.007).cars only Multivariate analysis showed that PLK1 expression was a near-independent prognostic factor in pediatric AML patients(p=0.066).ROC curve analysis showed that the area under cytogenetics,MRD and PLK1 expression curves were 0.695,0.763 and 0.613,respectively.Conclusions:PLK1 inhibited autophagy in AML cells.Its inhibitory effect induces autophagy in AML cells and causes mTOR dephosphorylation in AML cells.Inhibition of PLK1 can induce apoptosis of various tumor cells.PLK1 inhibition significantly inhibited the proliferation of NB4 cells.PLK1 is a good anticancer target in pediatric AML. |
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