Page 132 - Haematologica April 2020

H. Kumar et al.
lated kinase (ERK) activation.15,16 Interestingly, mitogen- activated protein kinase (MAPK) kinase inhibitor U0126 failed to inhibit clofazimine but not phorbol myristate acetate (PMA)-induced CD41 expression (Figure 2A, Online Supplementary Figure S5A). Consistently, PMA but not clofazimine induced ERK phosphorylation in K562 cells (Figure 2B). Apart from ERK, ROS induces megakary- ocytic differentiation17-19 and determination of cellular ROS revealed that clofazimine significantly enhanced ROS pro- duction from 12 h onwards (Figure 2C, Online Supplementary Figure S5B). Clofazimine also enhanced mitochondrial superoxide (Figure 2D, E) and H2O2 (Figure 2F) and caused mitochondrial membrane depolarization (Figure 2G, Online Supplementary Figure S5C). Co-treat- ment with ROS scavengers and inhibitors revealed that α-
tocopherol, which interacts with superoxides20 and also blocks H2O2 and peroxynitrite-mediated toxicity,21-23 com- pletely abrogated clofazimine-induced cell death while the ROS scavenger N-acetyl-L-cysteine and H2O2 decom- poser catalase had lesser but significant effects, and the mitochondrial complex inhibitors rotenone and antimycin and the NADPH oxidase inhibitor diphenyleneiodonium were ineffective (Figure 2H). α-tocopherol also inhibited clofazimine-induced CD41 and CD61 expression (Figure 2I, Online Supplementary Figure S5D-F). These results indi- cate that clofazimine induced ROS-dependent cell death and differentiation.
Cancer cells, including cancer stem cells, display high levels of ROS coupled with increased antioxidants that help them detoxify ROS.24-27 We thus investigated whether
IJK
LM
N
OP
Figure 1. (continued from previous page) (I, J) CFZ induces apoptosis in CP-CML CD34+ cells. (I) CD34+ cells from imatinib-resistant patients were isolated using a CD34 microbead kit (Miltenyi Biotech) and were treated with 5 μM CFZ or salinomycin for 48 h. Cells were then divided into two groups. One group was assessed by immunos- taining for CD34 and the other group was stained with annexin V/PI and the cells were then analyzed by flow cytometry (dot plots in Online Supplementary Figure S2B). (J) The CD34+ population from imatinib-resistant CP-CML cells was treated with CFZ (2.5 μM) for 96 h, stained with anti-CD34 and anti-CD38 antibodies and assessed for apoptosis. (K, L) CFZ does not affect viability of hematopoietic progenitors from healthy controls. (K) Cell viability, determined by a CellTiter-Glo assay. (L) Apoptosis by annexin V staining (dot plots in Online Supplementary Figure S2D). (M) CFZ (1 μM) induces CD61 and CD41 in CP-CML cells (dot plots in Online Supplementary Figure S4E, F). (N) CFZ induces monocyte-like morphology in CD34+ cells (cropped images shown; corresponding original images in Online Supplementary Figure S4G). (O, P) CFZ (1 μM) induces CD11b (O) and CD61 (P) in CD34+ CP-CML cells (histograms in Online Supplementary Figure S4H). Graphs illustrate the mean ± standard error mean. *P<0.05, **P<0.01, ***P<0.001; one-way analysis of variance followed by the Bonferroni post-test (except J, O, P; unpaired two-tailed t-test, H; Kruskal-Wallis test followed by the Dunn test, M; left panel, as indicated and right panel; Mann-Whitney U test). *Vehicle vs. treatment, #imatinib vs. other treatments, $dasatinib vs. CFZ. Microscopic images; n=3 (Leica DMI6000B, 30 fields/group). Blots are representative of three independent experiments (all full blots in Online Supplementary Figure S14). A letter P followed by a number (in N and subsequent figures) designates the patient’s identity. PARP: poly (ADP-ribose) polymerase; Cyt C: cytochrome C; CC; cleaved caspase; V: vehicle; IMT; imatinib; Dasa; dasatinib; FD; freshly diagnosed, Resp; imatinib-responder, Res; imatinib-resistant.
974
haematologica | 2020; 105(4)