Actinomycin D synergistically enhances the efficacy of CDDP by activating P53-PUMA pathway via downregulating P53-MDM2 complex on KB cells

OBJECTIVE Low dose of actinomycin D(LDAct D)was reported as a potent P53 activator and protected normal proliferating cells during anti-mitotic chemotherapy.However,the mechanism of LDAct D on P53 activation is still undetermined.In this study,the mechanism of LDAct D on the synergistic antitumor effect for cisplatin(CDDP)and P53 reactivation in KB cells was studied in detail.METHODS Cell viability was determined by MTT and LDH release.Apoptosis was determined by AnnexinⅤ-FITC/PI staining.Mitochondrial membrane potential(MMP)was detected by JC-1 stain-ing.Expression of P53,PARP,BAX,BCL-XL,PUMA,MDM2 and MDMX was detected by Western blotting(WB)and/or immunofluorescence(IF).P53-MDM2 complex was detected by ELISA.Molecular docking of receptor MDM2 and MDMX with actinomycin D(ACTD)was analyzed by Discovery Studio.RESULTS Compared with CDDP alone,P53 expression and the cytotoxicity on KB cells was significantly increased by the combination therapy.P53 regulatory proteins were increased while MMP was decreased.Meanwhile,knockdown of PUMA(P53 upregulated modulator of apoptosis)efficiently blocked the synergistic effect of LDAct D to CDDP.P53 activation was found to be accompanied with the increase of MDMX but not MDM2.Meanwhile,MDM2-P53 complex in KB cells was significantly decreased by LDAct D.Docking of both receptor MDM2 and MDMX with ACTD exhibited well established bonds with nearby amino acid residues.CONCLUSION LDAct D was probably an inhibitor of both MDM2 and MDMX.The synergistic effects of LDAct D for CDDP on KB cells depended on its effect on reactivating P53 and PUMA mediated mitochondrial apoptosis.