Mechanisms of Apoptosis Induction by Khat Alkaloids in Leukaemia Cells

Apoptosis is a physiological form of programmed cell death that is initiated by means of receptor activation or intracellular stress. Molecular configurations of the khat alkaloids cathine and cathinone are similar to those observed in their physiological analogue norepinephrine, which has been shown to have apoptosis inducing effects. OBJECTIVES: To investigate possible pathways of initiation and progression in apoptosis induced by khat alkaloids in human acute myeloid leukemia (AML) cell lines. METHODS: Khat alkaloids cathinone and cathine were used to elucidate apoptosis induction in a panel of AML cell lines with varying levels of Bcl-2 expression. Cells were pre-treated with a panel of receptor antagonists before exposure to khat alkaloids. Assessment of cell death was conducted by means of morphological studies, flow cytometry (Annexin V and propidium iodide) and Western blot analysis of caspase activation. RESULTS: Cathinone- and cathine-induced apoptosis exhibited similar morphological phenotypes. In HL-60 cells pre-treated with 10 µM of the D₂ (dopamine receptor 2) antagonist haloperidol, alkaloid induced apoptotic cell death was completely inhibited for upto 48 hours. Submicromolar concentrations (≥8 x 10^-7M) of the pan-caspase inhibitor Z-VAD-fmk, as well as inhibitors of caspase-1 (Z-YVAD-fmk) and caspase-8 (ZIETD-fmk) completely blocked cathinone toxicity for up to 8 hours. Susceptibility to apoptosis in the selected cell lines was inversely correlated with endogenous expression of the anti-apoptotic protein Bcl-2. Stable or transient transfection with Bcl-2 inhibited apoptosis by 20 – 50%, and apoptosis thresholds were lowered by blocking Bcl-2 synthesis with small interfering RNA. CONCLUSIONS: The observed caspase-mediated cell death induced by khat alkaloids is consistent with previous research on apoptosis induced by whole khat extract. These data also suggest D₂ ligand activation and Bcl-2 modulation as possible mechanistic pathways through which cathinone- and cathine-induced apoptosis may occur.