FoxM1 Regulates The Sensitivity Of Nasopharyngeal Carcinoma Cells To Cisplatin Via Inhibition Of MRN-ATM-mediated DNA Repair

Objective: To investigate the effects of silencing FoxM1 on the sensitivity of nasopharyngeal carcinoma cells to cisplatin,and its possible molecular mechanisms.Methods: FoxM1 mRNA levels in NPC cell lines 5-8F,HONE-1,CNE-1,CNE-2,HNE-1 and normal cell line NP69 were tested by RT-PCR.FoxM1 protein levels following cisplatin treatment were tested by western blot.The mRNA and protein levels of 5-8F and HONE-1 cells after transfection were measured by RT-PCR,qPCR and western blot.MTT assay was adopted to test the sensitivity of 5-8F and HONE-1 cells to cisplatin.Clonogenic assay was used to detect cell proliferation ability.Apoptosis was detected by Annexin V-FITC/PI staining assay.The expression of ?H2AX foci was evaluated by Laser scanning confocal microscope.QPCR was used to detect mRNA expression of FoxM1,NBS1,MRE11 and RAD50.Western blot was used to detect protein expression of Bax,Bcl2,caspase3,cleaved-caspase3,FoxM1,NBS1,P-NBS1,MRE11,RAD50,ATM and P-ATM.Results: FoxM1 mRNA level in normal cell line NP69 was significantly low as compared with NPC cell line 5-8F,HONE-1,CNE-1,CNE-2 and HNE-1(P < 0.01).FoxM1 protein expression was increased following cisplatin treatment in 5-8F and HONE-1 cells(P < 0.01).FoxM1-siRNAs remarkably reduced FoxM1 expression at both mRNA and protein levels.Knockdown of FoxM1 increased sensitivity to cisplatin in NPC cells(P < 0.01).Cells transfected with FoxM1 siRNA resulted in fewer colonies than cells transfected with NC siRNA at different concentrations of cisplatin(0,0.5,0.75?g/ml)(P < 0.05).Cells treated with FoxM1 siRNA and cisplatin presented a higher apoptotic rate compared with cells treated with NC siRNA and cisplatin or cells treated with cisplatin only(P < 0.01).It is also worth noting that the expression of Bax and cleaved caspase-3 was significantly increased(P < 0.05)while Bcl2 expression was decreased(P < 0.01)in cells treated with both cisplatin and FoxM1 siRNA.Cells treated with FoxM1 siRNA and cisplatin exhibited a greater number of ?H2AX foci in comparison to cells treated with NC siRNA and cisplatin or cisplatin only(P < 0.01).Knockdown of FoxM1 decreased NBS1 mRNA expression(P < 0.01),while the mRNA expression of MRE11 and RAD50 remained almost unchanged.At the protein level,knockdown of FoxM1 reduced NBS1,P-NBS1,and P-ATM(P < 0.01).Conclusion: FoxM1 was overexpressed in NPC cell lines and couldbe stimulated by cisplatin.Down-regulation of FoxM1 could accumulate cisplatin-induced DNA double-strand breaks,which lead to an increasing of apoptosis,and finally result in sensitizing the NPC cells to cisplatin.Its mechanism may be through inhibiting MRN-ATM-mediated DNA repair.