Abstract
The PI3K/Akt pathway is dysregulated is some acute lymphoblastic leukemias (ALL) and might therefore serve as therapeutic target. Indolylmaleimides exhibit inhibitory potencies against different protein kinases -like glycogen synthase kinase 3 (GSK3β) or protein kinase c- influencing thereby several cellular processes. Recently, it was demonstrated that PDA-66, a newly synthesized indolylmalemide based on the well known GSK3β inhibitor SB-216763, hinders microtubule polymerization in human neuronal progenitor and neuroblastoma cells. GSK3β is a downstream substrate of the PI3K/Akt and Wnt pathways and is often deregulated in tumor tissues. Herein, we investigated the effects of PDA-66 and its derivates (PDA-66E und PDA-377) on B and T-lymphoblastic leukemia cells.
B- and T-ALL cell lines (SEM, RS4;11, REH, Jurkat, MOLT-4 and CEM) were incubated for 72 h with increasing concentrations (0.1 µM-5.0 µM) of PDA-66, PDA-66E, PDA-377 and comparatively analyzed to SB-216763. To evaluate the effect of each substance WST-1 assay, cell proliferation, cell cycle analyses as well as apoptosis rates were determined. Activities of indolylmalemides were analyzed by GSK3β kinase assay. Detection of key molecules of Wnt and PI3K/Akt signaling pathway was performed using Western blot.
PDA-66 and derivates inhibited proliferation and metabolism of ALL cells significantly in a dose dependent manner. Interestingly, all PDA derivates showed a stronger inhibitory effect on proliferation than SB-216763 but the inhibitory effect on GSK3β kinase was lower than SB-216763. Antiproliferative effects of PDA-66 were studied in more detail. The incubation of 1 µM PDA-66 led to condensation of chromatin in the nucleus, karyorrhexis and an increasing amount of vacuoles after 48 h of treatment. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT-4 cells were arrested in G2 phase, SEM cells remained increasingly in G0/1 phase. After 48 h all PDA-66 treated cell lines showed a significant increase in apoptosis compared to control cells (SEM: 2.1 ± 0.9 % to 10.5 ± 1.3 %; RS4;11: 2.5 ± 0.7 % to 7.4 ± 1.1 %; Jurkat: 3.8 ± 0.6 % to 8.3 ± 1.9 %; MOLT-4: 3.7 ± 1.2 % to 16.3 ± 5.1 %). Apoptosis of ALL cells was initiated by cleavage of caspase 3 and 7 and Poly (ADP-ribose) polymerase (PARP). Furthermore, with increasing concentration of PDA-66 a decrease of pGSK3βSer9 was observed after 4 h in SEM cells. However, no influence on the total form of β-catenin was detectable. Nevertheless, there was an influence of PDA-66 on the expression of 4EBP-1 and p4EBP-1Ser65. SEM, RS4;11 and Jurkat cells showed a decrease of the phosphorylated as well as the total form of 4EBP-1 after an incubation of 4 and 24 h.
In conclusion, our results demonstrate that the newly synthesized indolymalemides have pronounced antiproliferative effects in ALL cells. In particular, the indolymalemide PDA-66 should be further investigated concerning it’s clinical efficiency as well as well as it's intracellular ways of action.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
