Page 133 - Haematologica April 2020

Clofazimine inhibits chronic myeloid leukemia
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Figure 2. Clofazimine downregulates PRDX1 expression, which leads to reactive oxygen species-dependent differentiation and apoptosis. (A) Phorbol myristate acetate (10 ng/mL) but not clofazimine (CFZ) (2.5 μM)-induced CD41 expression in K562 cells is blocked by U0126 (10 μM; 30 min pre-treatment). Representative histograms are shown in Online Supplementary Figure S5A. (B) CFZ does not induce ERK phosphorylation. (C-F) CFZ induces the production of cellular reactive oxy- gen species (ROS). Total ROS (C; representative histograms in Online Supplementary Figure S5B), total superoxide (D), mitochondrial superoxide (E), and H2O2 (F). (G) CFZ induces mitochondrial membrane depolarization (dot plot in Online Supplementary Figure S5C). (H) α-tocopherol completely blocks CFZ-induced loss of K562 viability. (I) α-tocopherol (200 μM) blocks CFZ (2.5 μM)-induced CD41 expression in K562 cells (representative histograms in Online Supplementary Figure S5D). (J) CFZ reduces peroxiredoxin 1 (PRDX1) protein level within 12 h. (K) CFZ reduces PRDX1 mRNA within 6 h in K562 cells. (L) CFZ reduces a PRDX1 (-1096- +83) promoter-driven luciferase reporter activity in HEK-293 cells. (M) CFZ reduces PRDX1 protein in cells from patients with imatinib-resistant chronic phase chronic myeloid leukemia. Immunoblots are representative of three independent experiments. Graphs illustrate the mean ± standard error of mean. **P<0.01, ***P<0.001 (A,C,F,H,I,K; one-way analysis of variance followed by the Bonferroni post-test. D,E,G,M; unpaired two-tailed Student t-test). V: vehicle; PMA: phorbol myristate acetate, ERK: extracellular signal-regulated kinase; MW: molecular weight; DHE: dihydroethidium; MMP: matrix metalloproteinase; DPI: diphenyleneiodonium; NAC: N-acetyl- L-cysteine; SOD: superoxide dismutase; NFE2L2: Nuclear factor erythroid 2 like 2; CP-CML: chronic phase chronic myeloid leukemia.
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