Staurosporine And Its Derivative Enhances ATRA-Induced Granulocytic Differentiation In Non-APL Acute Myelocytic Leukemia Cells

Although all-trans retinoic acid(ATRA)therapy in acute promyelocytic leukemia(APL)achieved great success,it is not effective for the treatment of other subtypes of acute myeloid leukemia(AML).In our previous study,we showed that the combination of staurosporine and ATRA synergized to induced granulocytic differentiation in ATRA-resistant APL cell lines NB4-R1 and NB4-R2.The combination did not modulate the protein level of APL pathogen,PML-RAR? fusion protein.It was suggested that the synergic differentiation-inducing effect of staurosporine and ATRA might be independent of PML-RAR? fusion protein.Therefore,the combination of staurosporine and ATRA might also be effective in non-APL AML cells.Monocytic leukemia cell line U937,erythroleukemia cell line K562 and AML-M2 b cell line Kasumi were used as in vitro models.The effect of the combination of staurosporine and ATRA as well as the molecular mechanisms was investigated.In the present study,2nmol/l staurosporine exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells.As a potent PKC pan-inhibitor,the role of PKC in the combination of staurosporine and ATRA-induced differentiation was surveyed.2nmol/l staurosporine or the combined treatment did not affect PKC activity in U937 cells while staurosporine did inhibit PKC activity in U937 cells only in the concentration of 10 nM or higher.Moreover,other selective PKC inhibitors which covered all the PKC isoforms expressed in U937 cells,such as UCN-01,Go6976 or rottlerin failed to enhance ATRA-induced granulocytic differentiation in U937 cells.Therefore,staurosporine enhanced ATRA-induced granulocytic differentiation in U937 cells might be independent of PKC.To further investigate the molecular mechanisms of the enhanced effect of staurosporine on ATRA-induced differentiation in U937 cells,we focused on certain proteins or signaling pathways involving in granulocytic differentiation.The induction of CCAAT/enhancer binding protein b(C/EBP?)and C/EBP? expression is implicated in the later stage of granulocytic differentiation.Moreover,C/EBP? and C/EBP? were demonstrated to be required for ATRA-mediated differentiation in APL cells.Staurosporine enhanced ATRA-promoted up-regulation of the protein level of C/EBP? and C/EBP? in U937 cells.Activation of MEK/ERK was demonstrated to be required for some cytokine-induced myeloid differentiation as well as ATRA-triggered granulocytic differentiation in APL cells.Staurosporine and the combined treatment activated MEK and ERK.Furthermore,blockade of MEK activation by its specific inhibitor,U0126,suppressed staurosporine-enhanced differentiation as well as the elevated protein level of C/EBPs.Taken together,we concluded that staurosporine enhanced ATRA-induced granulocytic differentiation only in poor ATRA sensitive non-APL AML cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.In our previous work,we found that PKC non-specific inhibitor staurosporine could enhance ATRA-induced U937 cell granulocytic differention.Neither staurosporine nor the combined treatment affected the activity of PKC.Moreover,other PKC inhibitors could not mimic the effect of staurosporine.Therefore,the biological effect of staurosporine might be associated with its specific structure.Staurosporine is not only a PKC unspecific inhibitor but also the inhibitor of other kinases(such as myosin light chain kinase,Ca M kinase and protein kinase G).So far,it can not be used clinically.Enzastaurin is an acyclic bisindolylmaleimide derived from staurosporine.It was demonstrated to be safe and non-toxic in Phase I trials and showed antitumor activities in Phase ? and Phase ? trials.More importantly,enzastaurin is similar with staurosporine structurely and functionly.In this work,enzastaurin exhibited the similar enhancing effect of staurosporine on ATRA-induced differentiation in U937.Meanwhile,enzastaurin also significantly enhanced ATRA-promoted up-regulation of the protein level of C/EBP? and C/EBP? in U937 cells.Trametinb,the potent specific inhibitor of MEK,suppressed enzastaurin-enhanced differentiation as well as the elevated protein level of C/EBP?.b.In conclusion,similar to staurosporine,enzastaurin enhanced ATRA-induced granulocytic differentiation in U937 cells via MEK/ERK pathways-mediated modulation of the protein level of C/EBP?.