Abstract
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). In the following study, we have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents which target the plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV4-11 cell lines, as well as primary leukemia cells. The drug blocked AML cells in G2/M phase of the cell cycle and, at longer incubation times (24 hr), decreased survival and induced cell death by apoptosis, as demonstrated by MTT assays and flow cytometric analysis of annexin V-FITC/propidium iodide stained samples. The IC50 for perifosine with THP-1 cells was 15.8 microM, while MV4-11 cells were more sensitive to the drug, as the IC50 was 2.7 microM. In THP-1 cells, perifosine caused complete Akt dephosphorylation on Ser 473 and Thr 308, without affecting total Akt expression levels. Perifosine treatment also resulted in dephosphorylation of the Akt downstream target p70S6 kinase. Perifosine did not affect phosphorylation and activity of the Akt upstream kinase, PDK1 nor altered formation or equilibrium of mTORC1 and mTORC2 complexes. Perifosine induced activation of apical caspases-2, -8, -9, and of executioner caspases-3, -6-, and -7, as well as cleavage of PARP and Bid. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on Fas/FasL interactions, as blocking antibodies to either Fas or FasL significantly reduced perifosine mediated cytotoxicity. Perifosine also activated JNK, and a JNK selective inhibitor (SP600125) markedly reduced perifosine-dependent apoptosis. In MV4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine was cytotoxic for AML blasts with activated Akt (IC50 range: 5.6–7.8 microM). Perifosine also reduced the clonogenic activity of CD34+ cells from AML patients displaying constitutive Akt upregulation, but not the clonogenic activity of CD34+ cells from AML patients without Akt activation, or from healthy donors. The inhibitor markedly increased blast cell sensitivity to etoposide. Taken together, our findings indicate that perifosine, either alone or in combination with existing drugs, is a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K/Akt survival pathway. Furthermore this novel Akt inhibitor perifosine appeared not to affect normal human hematopoietic stem cells, further documenting its therapeutic potential.
Alteration of the proliferative rate of acute myelogenous leukemia cells in vivo in patients
