Abstract
Cytokines and growth factors stimulate AML cell proliferation by activating various signaling pathways and high levels of cytokines have been associated with poor prognosis. Blocking signal transduction of cytokine-stimulated pathways may therefore inhibit AML growth and proliferation. Atiprimod is a cationic amphiphilic azaspirane. Although their mechanisms of action are not completely understood, azaspiranes were demonstrated among others to downregulate various cytokine receptors such as for interleukin (IL)-1, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α. Owing to this spectrum of activities, we hypothesized that atiprimod inhibits activation of intracellular signaling pathways in AML cells resulting in apoptosis and growth inhibition. We first studied the antiproliferative effect of atiprimod on 5 AML cell lines (K562, HL-60, KG-1, OCIM2, OCI/AML3) using the MTT assay. Cells were incubated for 72 hours without and with increasing concentrations of atiprimod (1, 2, 3, and 4 μM), then harvested and their metabolic activity and viability determined as optical density measurements. To corroborate the results, we also studied the effect of atiprimod on OCIM2 cells using a clonogenic cell line assay. Next we determined expression of Stat3 and Stat5, as well as Phospho-stat3 and phospho-stat5 in the K562 cells by Western Immunoblotting where cells were incubated in the absence and presence of increasing atiprimod concentrations (0.5, 1, 2, 3, 4 μM). We further evaluated induction of apoptosis of OCIM2 and K562 cells following incubation with atiprimod at 1 and 4 μM using the annexin V-FITC assay and finally analyzed caspase 3 and PARP cleavage in K562 cells at atiprimod concentrations of 0.5, 1, 2, 3, and 4 μM using Western Immunoblotting. To demonstrate the effect of atiprimod on marrow cells from AML patients and healthy volunteers we incubated marrow cells with atprimod at increasing concentrations and used the blast colony assay to measure inhibition of proliferation. Our results demonstrate that: 1) atiprimod inhibits proliferation of AML cell lines and AML blast proliferation from patient samples, but not significantly normal hematopoietic progenitors from samples of healthy controls; 2) atiprimod inhibits phosphorylation of Stat 3 and 5; and 3) atiprimod induces apoptosis in OCIM2 and K562 cells by cleavage of caspase 3 and PARP. In summary, our data suggest that atiprimod inhibits phopshorylation of Stat 3 and 5, induces caspase-dependent apoptosis, and blocks AML cell proliferation. Further evaluation of atiprimod in clinical trials of AML and MDS should be considered.
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