Aclacinomycin A sensitizes K562 chronic myeloid leukemia cells to imatinib through p38MAPK-mediated erythroid differentiation

Abstract

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.

Figure 1. Determination of the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis.

(A) Cells were treated with or without ACM (5, 10, 15 or 20 nM) for 24 to 72 hours. Cell viability was detected by MTT assay. (B) Intracellular hemoglobin was detected by benzidine staining assay. Apoptotic cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. Values are mean ± SEM from three experiments. ^*, p<0.05, versus untreated control (A and B). (C) K562 cells were treated without (control) or with 10 nM ACM for 3 days and then analyzed with benzidine staining assay.

Figure 2. Sequential treatment with ACM and imatinib strongly induced growth inhibition and apoptosis in K562 cells.

(A) Treatment scheme for ACM and imatinib in K562 cells. Sequential treatment (SE): cells were treated with 10 nM ACM for 3 days and then with 200 nM imatinib for additional 3 days (ACM-IM). ACM alone treatment: cells were treated with 10 nM ACM for 6 days. Co-treatment (CO): cells were simultaneously treated with 10 nM ACM and 200 nM imatinib for 3 days (ACM+IM). Imatinib alone treatment (IM): cells were treated with 200 nM imatinib for 3 days. (B) K562 cells (1.25×10⁴ cells/ml) were seeded in 96 well plate (200 µl per well) and treated as described above. Cell viability was analyzed by MTT assay. (C) Cells (1.25×10⁴ cells/ml) were seeded in 6 well plate (3 ml per well) and treated as described in Figure 2A. Apoptotic ells were stained with annexin V-FITC and PI and analyzed by flow cytometry. Values are mean ± SEM from four experiments. ^*, p<0.05 versus untreated control. ^#, p<0.05 (B and C). (D) Flow cytometry data show representative results from one of four independent experiments.

Figure 3. Sequential treatment with ACM and imatinib down-regulated Bcr-Abl, Mcl-1 and Bcl-xL, and activated caspase cascade.

(A and B) Cells were treated as described in Figure 2A. Cells were harvested and lysed, and the proteins were subjected to Western blot analysis using specific antibodies against Bcr-Abl, cytochrome c, VDAC, procaspase-9, procaspase-3, cleaved caspase-3 (C-caspase-3), PARP, Mcl-1 and Bcl-xL. β-actin was used as loading control. (C) K562 cells we treated with 5 nM ACM for three days. Subsequently they were treated for an additional 3 days with either: a. 200 nM imatinib (ACM-IM), b. 200 nM imatinib and 50 µM caspase-3 inhibitor (ACM-IM+z-DEVD-fmk), or c. 200 nM imatinib and 100 µM caspase-9 inhibitor (ACM-IM+z-LEHD-fmk). Apoptotic ells were stained with annexin V-FITC and PI and analyzed by flow cytometry. Values are mean ± SEM from three experiments. ^*, p<0.05 versus untreated control. ^#, p<0.05 versus ACM-IM group.

Figure 4. Sequential treatment with ACM and imatinib did not affect the Fas receptor system.

(A) Cells were treated as described in Figure 2A. Cells were harvested and lysed, and the proteins were subjected to Western blot analysis using specific antibodies against Fas, Fas ligand (FasL) and procaspase-8. β-actin was used as loading control. (B) Cells were co-treated with AA and U0126 for 24 h. Western blot analyses of Fas and Fas ligand in ACM-IM-treated cells and AA+U0126-treated cells.

Figure 5. ACM induced erythroid differentiation of K562 cells through p38MAPK pathway.

(A) K562 cells were treated with or without (control) 10 nM ACM, 2 µM SB202190 (SB), or ACM plus SB202190 for 3 days. Cell lysates were immunoprecipitated with anti-phospho-p38MAPK antibody. The immunoprecipitates were then subjected to in vitro kinase assay for p38MAPK described in ‘‘Materials and Methods’’. Phospho-ATF2 (p-ATF2) is the product of the kinase reaction determined by Western blotting using anti-p-ATF2 antibody. P38MAPK and β-actin were used as loading controls. Experiments were repeated three times independently. (B) Values of fold increase in p-ATF2 signal are means ± SEM. ^*, p<0.05 versus untreated control. ^#, p<0.05 versus ACM treatment. (C) K562 cells were treated as described in panel A. Hemoglobin production was detected by benzidine staining assay. (D) K562/mock and K562/p38α(AF)1 cells were treated with or without (control) 10 nM ACM for 3 days. Activation of p38MAPK was measured by in vitro kinase assay. The expression of p38α(AF) was confirmed by Western blotting using anti-Flag antibody. β-actin was used as loading control. Experiments were repeated three times independently. (E) Values are means ± SEM. ^*, p<0.05 versus K562/mock-control. ^#, p<0.05 versus K562/mock + ACM. (F) K562 cells were treated as described in panel D. Intracellular hemoglobin was detected by benzidine staining assay.

Figure 6. Sequential treatment with ACM and imatinib-induced growth inhibition and apoptosis were regulated by p38MAPK pathway.

(A) K562 cells were pretreated with 10 nM ACM in combination without or with 2 µM SB202190 (SB) for 3 days and then with 200 nM imatinib for additional 3 days (ACM-IM or ACM/SB-IM). (B) Cells were treated as described in Figure 6A. (C) Flow cytometry data (Fig. 6B) show representative results from one of four independent experiments. (D) K562/mock and K562/p38α(AF)1 cells were treated per sequential treatment scheme as described in Figure 2A. (E) Cells were treated as described in Figure 6D. (F) Flow cytometry data (Fig. 6E) show representative results from one of four independent experiments. (A and D) Cell viability was analyzed by MTT assay. (B and E) Apoptotic ells were stained with annexin V-FITC and PI and analyzed by flow cytometry. Values are mean ± SEM from four experiments. ^*, p<0.05 (A, B, D and E).

Figure 7. The knockdown of p38MAPK and reduction of erythroid differentiation decreased K562 cell sensitivity to imatinib.

The cells were transfected with a negative control shRNA (ctrl-shRNA) or a shRNA targeting p38α (p38α-shRNA) for 3 days. (A) The protein level of p38MAPK was analyzed by Western blotting (upper panel). Immunoblots are representative of three experiments, which are presented as the mean ± SEM (lower panel). ^*, p<0.05 versus control-shRNA. (B) K562 cells were transfected with shRNA plasmids and treated without (control) or with ACM (5 nM). Cells were stained with benzidine to determine hemoglobin synthesis at 72 h. (C) The shRNA-transfected cells were treated with ACM (5 nM) for 3 days and then with 200 nM imatinib for additional 3 days (ACM-IM). Cell viability was analyzed by MTT assay. (D) The same experiments as described above were performed. Apoptotic cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. Values are mean ± SEM from three experiments. ^*, p<0.05 versus untreated control. ^#, p<0.05 versus ctrl-shRNA/ACM-IM (C and D).