| Although the prognosis of acute promyelocytic leukemia(APL)has improved significantly,all-trans retinoic acid(ATRA)resistance in APL relapsed patients remains to be solved.In this study,the clinical achievable concentration of the staurosporine derivatives UCN-01 and enzastaurin,respectively,in combination with ATRA,induced terminal differentiation and/or apoptosis in ATRA-resistant APL cell lines,NB4-R1 and NB4-R2.UCN-01 is a PKC inhibitor and enzastaurin is a specific inhibitor of PKCβ.The combination of ATRA with other PKC selective inhibitors or another PKCβ inhibitor could not mimic the cellular effects of the co-treatment of UCN-01/enzastaurin and ATRA.Furthermore,the combination of enzastaurin and ATRA did not inhibit PKCβ.Thus,PKC/PKCβ may not be involved in the combination-induced differentiation.The combination of UCN-01 /enzastaurin with ATRA activated MEK/ERK,significantly increased the protein levels of CCAAT/enhancer binding protein β(C/EBPβ)and/or PU.1.In addition,enzastaurin synergizes with ATRA to degrade PML-RARα.U0126(a MEK-specific inhibitor)not only suppressed the combination-induced differentiation,but also restored the protein levels of C/EBPβ and/or PU.1.However,the RAF-1 inhibitor did not suppress MEK activity and the combination-induced differentiation.Therefore,the combination of UCN-01/enzastaurin and ATRA overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of C/EBPβ and/or PU.1.The combination of enzastaurin and ATRA collapsed mitochondrial transmembrane potential,but did not activate caspase-3,-6 and-7.In addition,caspase3/7 and caspase6 specific inhibitors did not suppress the combination-triggered apoptosis.Therefore,the apoptosis induced by the combination of enzastaurin and ATRA is mitochondrial dependent but caspase independent.These findings may provide a theoretical basis for further research in vivo and discovery of potential therapies for ATRA-resistant APL patients. |
