Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cells

Abstract

Differentiation therapy with all-trans retinoic acid (ATRA) is well established for acute promyelocytic leukemia (APL). However, the narrow application and tolerance development of ATRA remain to be improved. A number of kinase inhibitors have been reported to induce cell differentiation. In this study, we investigated several combinations of these kinase inhibitors. Recently, we revealed that the Akt inhibitor triciribine (TCN) efficiently induces differentiation of NB4 APL cells and acute myeloid leukemia (AML) M2-derived HL-60 cells through activation of the ERK/MAPK pathway. In the present study, we found that the p38 MAPK inhibitor PD169316 had profoundly enhanced the TCN effect for differentiation of NB4 and HL-60 cells. Morphologically, the combination of these two agents efficiently reduced the nuclear-to-cytoplasmic ratio and induced the expression of myelomonocytic markers (CD11b, CD11c) and some ectopic markers (erythroid glycophorin A, lymphoid CD7 and CD20), as determined by PCR and flow cytometry analyses. Western blotting analysis revealed that these agents efficiently induced phosphorylation of ERK. To clarify the molecular mechanisms involved in the TCN and PD169316-induced differentiation, we performed microarray analyses using NB4 cells. Pathway analysis using DAVID software indicated that "viral protein interaction with cytokine and cytokine receptor" and "cytokine-cytokine receptor interaction" were enriched with high significance. Real-time PCR analysis demonstrated that genes for components of these pathways, including chemokines like CCL1, CCL2, CCL3, CCL5, and CXCL8 as well as cytokines and receptors like CSF1, IL-10, IL-10RA, IL-10RB, IL-1β, and TNFSF10, were upregulated in NB4 and HL-60 cells during TCN and PD169316-induced differentiation.

Fig 1. Effects of combinations of TCN and p38 MAPK inhibitors on the expression of differentiation markers in NB4 cells.

(A) Percentages of CD11b-positive NB4 cells analyzed by flow cytometry after addition of the indicated kinase inhibitors. Representative histograms of at least three independent experiments are shown. (B–E) Effects of combinations of TCN and p38 MAPK inhibitors (PD169316, CAY10571, SB203580) on the differentiation of NB4 and HL-60 cells examined by real-time PCR analysis for several markers. The expressions of (B) CD11b, (C) CD11c, (D) CD14, and (E) MPO were examined. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed using different batches of cDNA. Statistical significance was determined by two-way ANOVA followed by a Tukey multiple comparisons test (***p<0.001 vs. ctrl).

Fig 2. Flow cytometry analyses of differentiation markers in NB4, HL-60, THP-1, and U937 cells.

(A–D) Effects of combinations of TCN and PD169316 on the differentiation of (A) NB4, (B) HL-60, (C) THP-1, and (D) U937 cells examined by flow cytometry. In NB4 (A) and HL-60 (B) cells, ATRA was added for a comparison. The expressions of 18 differentiation markers were examined. The heat maps show the expression levels of the leukocyte cell surface markers in terms of the geometric mean fluorescence intensity. The data presented were obtained from three independent flow cytometry analyses, and each box shows the mean geometric mean fluorescence intensity of three analyses.

Fig 3. Morphologic analyses of NB4 cells and HL-60 cells in the presence or absence of TCN and/or PD169316.

(A) NB4 cells were collected and subjected to Wright–Giemsa staining. Budding of the cells is indicated by arrows. The magnification of the images shown in the panels is ×400. (B) Violin plot analysis of the N/C ratio percentages in individual NB4 cells. The N/C ratio was calculated from the cytoplasm and nuclear areas using Image J software. Significance was analyzed (***p<0.001). The mean values and 25 percentiles are shown as thick and thin lines, respectively. (C) HL-60 cells were collected and subjected to Wright–Giemsa staining. Budding of the cells is indicated by arrows. The magnification of the images shown in the panels is ×400. (D) Violin plot analysis of the N/C ratio percentages in individual HL-60 cells. The data were analyzed as described for (B).

Fig 4. Effects of TCN and PD169316 on signaling pathways in NB4 and HL-60 cells.

Cells were cultured in the presence or absence of TCN and/or PD169316 for the indicated periods, and analyzed for ERK and p38 MAPK activity by western blotting. The membranes were probed with an anti-phospho-ERK rabbit polyclonal antibody or anti-phospho-p38 MAPK mouse monoclonal antibody. The membranes were stripped and re-probed with an anti-total-ERK mouse monoclonal antibody or anti-total-p38 MAPK mouse monoclonal antibody to verify equal protein loading. The indicated numbers are the relative densities calculated by Image J 1.54 software, and were based on the densities of the bands for phospho-ERK or phospho-p38 MAPK divided by total-ERK or total-p38 MAPK, respectively.

Fig 5. Results of the microarray analyses.

(A) Gene expression profiles of NB4 cells treated with TCN versus control cells. The results of this comparison were already presented in a previous study [11]. (B) Gene expression profile of NB4 cells treated with PD169316 versus control cells. (C) Gene expression profile of NB4 cells treated TCN+PD169316 versus control cells. (D) Results of the pathway analysis using DAVID software (https://david.ncifcrf.gov/home.jsp) for 510 genes. The X-axis shows the number of the negative log base 10 p-values. Higher numbers indicate higher significance.

Fig 6. Validation of the microarray data for selected genes in NB4 and HL-60 cells.

(A–O) Total RNAs extracted from NB4 cells cultured in the presence or absence of TCN and PD169316 for 72 h were analyzed by real-time PCR with specific primers for (A) CCL1, (B) CCL2, (C) CCL3, (D) CCL5, (E) CSF1, (F) CSF1R, (G) CSF2RA, (H) CXCL8, (I) IL-10, (J) IL-10RA, (K) IL-10RB, (L) IL-1β, (M) IL-2RB, (N) TNFSF10, and (O) TNFSF15. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed using different batches of cDNA. Statistical significance was determined by two-way ANOVA followed by a Tukey multiple comparisons test (***p<0.001 vs. ctrl).