Abstract

NF-k B is constitutively activated in the majority of acute myelogenous leukemia (AML) specimens. Anthracyclines used in AML treatment can activate NF-k B and inhibit apoptosis through synthesis of IAPs upregulation. Because proteasome activity is necessary for NF-k B activation and p53 degradation, inhibitors of the proteasome have been proposed as pro-apoptotic agents to sensitize leukemic blast cells to anthracycline chemotherapy. Apoptosis was quantified in THP-1 cells, incubated with increasing concentrations of (IDA) alone or in association with a low concentration (0.25 μM) of MG132 as a proteasome inhibitor. A low apoptotic rate was found with either MG132 or IDA alone with a range of 5 to 20 ng/ml. The combination of both drugs induced a percentage of apoptotic cells which was significantly higher than the addition of the effects of both drugs separately (p<0.01). A low concentration of IDA (5ng/ml) induced, in the presence of MG132 as much apoptosis as 20 ng/ml of IDA alone. Above 40 ng/ml of IDA, the sensitizing effect of MG132 was no more significant. This shows that proteasome inhibition lowers the threshold of sensititvity of THP-1 cells to IDA-induced apoptosis. Such a sensitization was not found in a MG132-resistant THP-1 derived cell line. This difference was not related to the uptake of the drug since no difference in IDA incorporation by THP-1 cells was found in the presence or the absence of MG132. Western blot analysis of I-k B phosphorylation and degradation failed to show any difference between MG132 and control-treated cells. Conversely increased expression of Bax and Bim pro-apoptotic proteins was evidenced. The increase in Bim precedes the induction of apoptosis and participates in Idarubicin-induced apoptosis. Bortezomib gave the same results for apoptosis induction and synergistic effect with IDA, increased expression of Bax and Bim pro-apoptotic proteins was also evidenced. MG132 also potentiated IDA-induced apoptosis in primary blast cells from 22 AML patients while no effect of MG132 was found on the IDA-induced apoptosis normal lymphocytes, PHA-stimulated lymphocytes, normal cord blood CD34+ cells and bone marrow normal myeloid cells. These data confirm that proteasome inhibition induces a specific sensitization of leukemic cells to IDA and suggest that an increase in the pro-apoptotic potential (Bim, Bax) are involved in the process.

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