Hydroxychloroquine (Hcq) Induces apoptosis in b-chronic lymphocytic leukemia cells (B-cll) Via activation of CASPASE-3 and down-regulation of BCL-2

Abstract Recently, it has been observed that HCQ, an antirheumatic drug, induces apoptosis in peripheral blood T lymphocytes but the mechanism of this induction remains unclear. A fortuitous observation in a patient with B-CLL led us to study the in vitro activity of HCQ against B-CLL cells and potential mechanisms of action. HCQ induced a decrease in cell viability in a dose- and time-dependant manner. The mean IC50 (concentration of drug required to reduce the cell viability to 50%) for 20 B-CLL patients evaluated was 32±7 μg/ml (range, 10–70 μg/ml) for 24 hours of treatment. This cytotoxic effect was due to apoptosis as demonstrated by morphological and DNA fragmentation studies, propidium iodide labeling and also annexin/PI assay. HCQ (100 μg/ml) produced apoptosis as early as 5 hours post incubation (28±4% versus 8± 3% for untreated cells) which increased to 82±4% at 18 hours post-treatment. Caspase-3 protease activity, measured by the colorimetric assay, was increased in HCQ-treated cells and correlated with the level of apoptosis. Moreover, pretreatment of B-CLL cells with the caspase inhibitor Z-VAD.fmk prevented the activation of Caspase-3 and the cytotoxic effect of HCQ. The amount of Bcl-2 was significantly reduced during apoptosis as observed by western blot and flow cytometry. Compared with levels before treatment, the decrease of Bcl-2 was 22±2% (range, 15–32%, p