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and the pharmacologic inhibitor CC 10 mM decreased kinase phosphorylation, as expected 37,40 . The inhibitory response was not qualitatively affected by variations in culture ALK Inhibitor medium conditions, as detailed in Section 2 final results not shown . Time course analysis revealed that AMPK inactivation was a rapid response, already detected at roughly 1 h of treatment, and maintained thereafter Inhibitor 7C and D . When analyzed, 2 DG also decreased phosphorylation in the AMPK upstream effector LKB 1, though the decrease was in general of lower intensity than in the case of AMPK Inhibitor 7D . Concerning ATO, this agent either did not modify or slightly down regulated AMPK phosphorylation, and did not normally affect the decrease created by 2 DG Inhibitor 7D .
Finally, treatment for 4 h with 2 DG did not affect AMPK phosphorylation in NB4 and THP 1 cells Inhibitor 7E , which in the case of NB4 cells is consistent with earlier observations 39 . Of note, treatment with lonidamine did not reduce, but instead stimulated LKB ALK Inhibitor 1 and AMPK phosphorylation Inhibitor 7A and B . This may well be a consequence of improved ROS production Supplementary Inhibitor 1 , considering that AMPK was characterized as an oxidative tension inducible kinase, even in the absence of ATP depletion 28,40,41 . Prolonged remedies 16 24 h with lonidamine plus ATO, and also to some extent with 2 DG plus ATO, normally decreased total and phosphorylated AMPK levels, possibly because of kinase degradation see double bands in Inhibitor 7B and D . AMPK might play pro apoptotic or pro survival roles 37,42 .
To investigate the functional consequence of 2 DG provoked AMPK inactivation in HL60 cells, we examined the effect in the kinase inhibitor CC. The results in Inhibitor 7F indicate that AG-1478 co treatment with 10 mM CC potentiated apoptosis generation by ATO albeit with lower efficacy than 2 DG , and slightly augmented apoptosis by 2 DG plus ATO. The former observation was qualitatively corroborated making use of an AMPKa directed siRNA Inhibitor 7G , though this approach was limited by the low efficacy and the toxicity in the transfection procedure. This suggests that AMPK plays a defensive role in this experimental model, and hence its inactivation by 2 DG may well in part explain the improved apoptotic efficacy of 2 DG plus ATO in HL60 cells. Of note, CC did not enhance but instead slightly attenuated apoptosis generation by ATO plus lonidamine.
Digestion Nevertheless, as indicated above lonidamine stimulated AMPK phosphorylation, AG-1478 in contrast to 2 DG. In this regard, a protective action of CC was previously observed by us making use of ATO plus the phenolic agent genistein, which activated AMPK by way of ROS production 28 Akt and ERK modulation, and effect of Akt and ERK inhibitors It was reported that 2 DG might either stimulate 43,11 or inhibit 44,45 Akt and ERK pro survival kinases. Hence, we examined the phosphorylation activation of these kinases in HL60 cells treated with 2 DG and ATO, alone and in combination. Therapy with 2 DG alone brought on a rapid stimulation 30 min of Akt and ERK phosphorylation Inhibitor 8A , to later decrease at prolonged time periods 16 or 24 h Inhibitor 8B .
When examined, 2 DG also stimulated the phosphorylation of mTOR and p70S6K downstream Akt kinases , too as of MEK1 2 upstream ERK kinases Inhibitor 8A . Interestingly, ALK Inhibitor ATO alone exerted small if any effect on Akt and ERK phosphorylation, but attenuated their stimulation by 2 DG Inhibitor 8B . Finally, 2 DG also stimulated Akt and ERK phosphorylation in NB4 and THP 1 cells, though with lower intensity than in HL60 cells Inhibitor 8C . Many reports indicate the existence of mutual inhibitory interactions among Akt and AMPK 42,46,47 . For this reason, we examined the effects of Akt and ERK inhibitors on AMPK activation. It was observed that co treatment with all the PI3K inhibitor LY294002 LY, 30 mM or and the MEK ERK inhibitor U0126 U, 5 mM not only prevented 2 DG provoked Akt or ERK phosphorylation, as expected but additionally attenuated to some extent the decrease in AMPK phosphorylation Inhibitor 8D .
Hence, AMPK inhibition by 2 DG might be in part a consequence in the improved Akt and ERK activation. To understand the relevance for apoptosis of Akt and ERK activation by 2 DG and its inhibition by ATO, we examined the effects of LY294002, U0126, and the Akt inhibitor AG-1478 triciribine AktiV, 10 mM , on 2 DG toxicity. As indicated in Inhibitor 8E, co treatment with all inhibitors improved apoptosis generation by 2 DG alone, hence mimicking the pro apoptotic effect of ATO. Taken together, these final results indicate that Akt and ERK activation by 2 DG operates as a restrain for apoptosis, and hence their inhibition by ATO might in part explain the improved apoptotic ALK Inhibitor efficacy of 2 DG plus ATO combination. We earlier reported that protein kinase activities might modulate ATO transport uptake or export mechanisms in leukemia cells AG-1478 26 . Hence, we asked no matter whether co treatment with 2 DG may well result in improved intracellular ATO accumulation