Synergistic effect of inhibiting translation initiation in combination with cytotoxic agents in acute myelogenous leukemia cells

Abstract

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X(L) and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations.

Fig. 1

Sensitivity of AML cells to silvestrol. (A) U937 cells (35 000 cells/well) or HL-60, NB4, OCI/AML2 and OCI/AML3 cells (50 000 cells/well) were seeded in 96-well plates in the presence of increasing concentrations of silvestrol. Forty eight hours later, cell growth was measured using the MTS assay and plotted relative to vehicle (DMSO) controls. Experiments were performed in triplicates and the error of the mean is shown. The inset of the middle panel shows NB4 cells treated for 48 h with 7.5 nM Sil and labeled for the last 15 min. Rel. Incorp. denotes the Relative [³⁵S]-Met Incorporation compared to DMSO control. The experiment was performed in triplicate (B) Silvestrol induces apoptosis in AML cells. NB4 cells, 200 000 cells/well, were seeded in 6-well plates in the presence of increasing concentrations of silvestrol. Forty eight hours later, cells were harvested in RIPA buffer and Western blot analysis performed on equal amounts of proteins fractionated on 10% polyacrylamide gels. (C) Plots showing combination index (CI) values for NB4 cells receiving combinations of silvestrol with etoposide, Ara-C, or daunorubicin at the indicated ratios. 1-CI values are plotted on the X-axis with values greater than 0.2 being synergistic, lower than −0.2 antagonistic and ranging between −0.2 and +0.2 being additive. The average of 2 experiments, each performed in triplicates, is presented. The error of the mean is shown.

Fig. 2

The synergistic effect of silvestrol correlates with its potency as an inhibitor of translation initiation. (A) Chemical structures of silvestrol and Flavagline 61. (B) NB4 cells, 50 000 cells/well, were seeded in 96-well plates in the presence of increasing concentrations of silvestrol or Flavagline 61. Forty eight hours later, cell growth was measured using the MTS assay and plotted relative to vehicle (DMSO) controls. Experiments were performed in triplicates and the error of the mean is shown. (C) Inhibition of protein synthesis in NB4 cells following exposure to 50 nM silvestrol or Flavagline 61. The results are the average of triplicates with the error of the mean shown. Values are standardized against total protein content and plotted relative to DMSO controls, which were ~100 000 cpm/μg. (D) NB4 cells were treated with combinations of Flavagline 61 with etoposide, Ara-C or daunorubicin for 48 h at the indicated ratios. 1-CI values are plotted on the X-axis with values greater than 0.2 being synergistic, lower than −0.2 antagonistic and values between −0.2 and +0.2 being additive. The average of 3 experiments, each performed in triplicates, is presented. The error of the mean is shown.

Fig. 3

Effect of silvestrol on protein synthesis in AML cells. (A) Inhibition of protein synthesis in AML cells following exposure to 50 nM silvestrol. The results are the average of duplicates with the error of the mean shown. Values are standardized against total protein content and plotted relative to DMSO controls, which were ~100 000 cpm/μg for all cell lines. (B) Western blot analysis probing levels of Mcl-1 in AML lines following exposure to silvestrol. Extracts prepared from cells exposed to 50 nM silvestrol for the indicated periods of time were resolved on 10% SDS-polyacrylamide gels. Western blotting was performed to investigate protein levels after treatment with silvestrol. (C) Polysome profiling analysis on NB4 cells following treatment with silvestrol. NB4 cells were harvested after 1 h treatment with either 1% DMSO or 10 nM silvestrol. Extracts were resolved on 10–50% sucrose gradients by centrifugation in an SW40 rotor at 35 000 rpm for 2 h. (D) Semi-quantitative RT-PCR analysis on fractions collected from extracts of DMSO or silvestrol treated cells. Fractions were pooled into unbound and polysome (poly)-bound fractions and monitored for distribution of Mcl-1 and β-actin mRNA.

Fig. 4

Inhibition of protein synthesis synergizes with ABT-737 on AML cells. (A) NB4 cells (50 000 cells/well) were seeded in 96-well plates in the presence of increasing concentrations of ABT-737. Forty eight hours later, cell growth was measured using the MTS assay and plotted relative to vehicle (DMSO) controls. Experiments were performed in triplicates and the error of the mean is shown. (B) Plots showing combination index (CI) values against NB4 cells for combinations of silvestrol + ABT-737, silvestrol + daunorubicin, ABT-737 + daunorubicin or silvestrol + ABT-737 + daunorubicin at the indicated ratios. 1-CI values are plotted on the X-axis with values greater than 0.2 being synergistic, lower than −0.2 antagonistic and values between −0.2 and +0.2 being additive. The average of 3 experiments, each performed in triplicates, is presented. The error of the mean is shown.