p70S6 kinase is a target of the novel proteasome inhibitor 3,3'-diamino-4'-methoxyflavone during apoptosis in human myeloid tumor cells

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce Apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3'-diamino-4'-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser(136)), activation of caspases -8, -9, and -3 and cleavage of the Caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr(389)-phosphorylation and (ii) protein levels of the Caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent Apoptosis and P70S6K degradation were simultaneously prevented by the Caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the Proteasome Inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and Apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated Proteasome inhibition and Apoptosis of leukemia cells.