The PLK1 Inhibitor BI 2536 Induces Cell Death In Neuroblastoma Through Apoptosis And Autophagy

Background:Neuroblastoma(NB)is the most common extra-cranial solid tumor in childhood with the overall 5 years' survival less than 40%.Polo-like kinase 1(PLK1)is a serine/threonine-protein kinase expressed during mitosis and over expressed in multiple cancers,including neuroblastoma.Our results show that higher PLK1 expression in NB tissue relate to poor outcome of NB patients.Methods:Cell Counting Kit-8(CCK8)assay and Colony formation assay were used to exam the neuroblastoma cell growth.Cell apoptosis and cell cycle distribution were determined by using Annexin V/PI staining and flow cytometry.LC3-GFP transfection followed by fluorescence confocal microscopy and transmission electron microscope were performed to analyze the autophagy of NB cells.Multiple apoptosis markers and autophagy markers were analyzed With real-time polymerase chain reaction(PCR)in NB cells treated with PLK1 inhibitor BI2536.Results:Our results showed that PLK1 is highly expressed in almost all of the 8 neuroblastoma cell lines except NGP cells.In addition,the status of MYCN amplified or not does not seem to affect PLK1 expression.In SEQC-498 cohorts containing 498 neuroblastoma patients' samples,high PLK1 expressionwas remarkable associated with both poor relapse free and overall survival of patients.BI2536,a small molecule inhibitor against PLK1,significantly reduced cell viability in a panel of NB cell lines,with IC50 less than 100 nM.BI 2536 treatment resulted in significant cell morphology change,appearing as cell floating and shrinkage PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells.Not surprisingly,cell cycle analysis displayed accumulation of cell populations in the G2 phase from 12.76±1.33%to 63.64±3.28%in SH-SY5Y cells in response to 5nM BI 2536 treatment for 24 hr.At same time,a decrease in the population of G1 and S phase cells was observed.Higher concentration of BI 2536 administration induced more serious mitosis disorder.In similar,the G2 population was increased from 6.06±3.66%to 18.94±7.14%,with G1 fraction decreased from 56.30±4.63%to 46.01±4.54%in SK-N-BE(2)cells exposed upon 10nM BI 2536.The proportion of apoptotic cell remarkablely increased in the BI 2536 treated group(41.33±5.45%in 5 nM treated group and 49.39±6.28%in 10 nM treated group)compared with the control group(20.08±2.01%)in SH-SY5Y cells.Realtime PCR array revealed the PLK1 inhibition related genes,such as BIRC7,TNFSF10,LGALS1 and DAD1 et al.Moreover,autophagy activity was investigated in the NB cells treated with BI 2536.BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-?expression.Formation of autophagosome induced by BI 2536 was observed by(transmission electron microscope)TEM.However,BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPK?T172 phosphorylation.Conclusion:The current study showed that PLK1 was over expressed in neuroblastoma cells.BI 2536,a specific PLK1 inhibitor,not only inhibited cell proliferation by disturbing cell cycle progress but also triggered cell apoptosis.Furthermore,BI 2536 blocked autophagic flux via suppression of AMPKa activation.This analysis provides evidence for the important role of PLK1 in neuroblastoma cell survival,and BI 2536 could be considered as a possible treatment strategy for neuroblastoma.