Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells

Abstract

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC₅₀ values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Keywords: Apoptosis; Cell cycle; Leukemia cell lines; VX-11e; Voreloxin.

Fig. 1

VX-11e (a) and voreloxin (b) inhibited leukemia cell proliferation. MOLM-14, K562, REH and MOLT-4 cells were incubated for 24 h with increasing concentrations of VX-11e (VX) or voreloxin (VOR). The percentages of proliferating cells and the IC₅₀ values of each drug were determined by the Muse Ki67 Proliferation Kit. Each value is the mean ± SD of three independent experiments

Fig. 2

Synergistic anti-proliferative effects of VX-11e and voreloxin. a MOLM-14, b K562, c REH and d MOLT-4 cells were incubated for 24 h with the constant ratio dose at the IC₅₀ values of VX-11e (VX) and voreloxin (VOR). The CI and Fa values were calculated using the Chou-Talalay method described in ″Materials and methods″. Each value is the mean ± SD of three independent experiments

Fig. 3

VX-11e alone and in combination with voreloxin decreased ERK activation. MOLM-14, K562, REH and MOLT-4 cells were incubated for 24 h with VX-11e and voreloxin alone or in combination. ERK activity was determined using the Muse MAPK Activation Dual Detection Kit. a Representative dot plots and b graph of ERK activation in MOLM-14, K562, REH and MOLT-4 cell lines. Each value is the mean ± SD of three independent experiments. *(p < 0.05), **(p < 0.01) versus control; #(p < 0.05), ^##(p < 0.01) versus VX-11e; ^$(p < 0.05), ^$$(p < 0.01) versus voreloxin

Fig. 4

Combination of VX-11e and voreloxin induced cell-cycle arrest. a MOLM-14, b K562, c REH and d MOLT-4 cells were incubated for 24 h with VX-11e (VX) and voreloxin (VOR) alone or in combination. Cell cycle distribution was determined using the Muse Cell Cycle Kit. Each value is the mean ± SD of three independent experiments. *(p < 0.05), **(p < 0.01) versus control; ^#(p < 0.05), ^##(p < 0.01) versus VX-11e; ^$(p < 0.05), ^$$(p < 0.01) versus voreloxin

Fig. 5

Combination of VX-11e and voreloxin enhanced apoptosis in leukemia cells. MOLM-14, K562, REH and MOLT-4 cells were incubated for 24 h with VX-11e (VX) and voreloxin (VOR) alone or in combinations. a Representative dot plots of Annexin V/7-AAD apoptotic assay and b graph showing the percentage of apoptotic cells. Each value is the mean ± SD of three independent experiments. *(p < 0.05), **(p < 0.01) versus control; ^#(p < 0.05), ^##(p < 0.01) versus VX-11e; ^$(p < 0.05), ^$$(p < 0.01) versus voreloxin

Fig. 6

VX-11e in combination with voreloxin increased p21 and reduced survivin and NF-κB p105/p50 protein levels. a MOLM-14, b K562, c REH and d MOLT-4 cells were incubated for 24 h with VX-11e (VX) and voreloxin (VOR) alone or in combination. The expression of p21, survivin, p50 and p105 proteins was detected by Western blot. β-actin was used as a loading control. Quantification of the proteins was performed by densitometric analysis of the blots and normalized to the internal loading control. Each value is the mean ± SD of three independent experiments. *(p < 0.05), **(p < 0.01) versus control; ^#(p < 0.05), ^##(p < 0.01) versus VX-11e; ^$(p < 0.05), ^$$(p < 0.01) versus voreloxin

Fig. 7

VX-11e in combination with voreloxin inhibited NF-κB translocation into the nucleus. a MOLM-14, b K562, c REH and d MOLT-4 cells were incubated for 24 h with VX-11e (VX) and voreloxin (VOR) alone or in combination. Cells were immunostained for NF-κB (red fluorescence) and the nuclei were stained blue with Hoechst 33342. Representative confocal images and box-and-whisker plots representing the number of NF-κB positive dots per nucleus from three independent experiments. Bar = 10 μm. The box, line and whiskers represent the quartiles, median and range of data (minimal and maximal values), respectively. *(p < 0.05), **(p < 0.01) versus control; ^#(p < 0.05), ^##(p < 0.01) versus VX-11e; ^$(p < 0.05), ^$$(p < 0.01) versus voreloxin (Color figure online)