2-HBA

2-HBA is a potent inducer of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) which can also activate caspase-3 and caspase-10.

Signaling Pathways >> Apoptosis >> Caspase

Research Areas >> Cancer

Caspase-3

Caspase-10

When L1210 cells are exposed to 0.6 μM 2-HBA (bis(2-hydroxy-benzylidene)acetone), the specific activities of NQO1 and glutathione reductase increase by 6- and 1.5-fold, respectively. The total cellular glutathione content is also coordinately induced by 2.4-fold. In a more detailed study it is found that NQO1 is induced by 2-HBA in a concentration-dependent manner. Treatments with 2-HBA cause cell cycle arrest and apoptosis in both L1210 wild type cells and their Y8 drug-resistant counterparts in a concentration-dependent manner. 2-HBA can also activate caspase-3 and caspase-10[1].

Cells (20,000 per well) are grown for 24 h in 96-well plates, then exposed to 2-HBA (bis(2-hydroxybenzylidene)acetone) for either 24 h (for glutathione determination) or 48 h (for determination of enzyme activities). At the end of the exposure period, cells are collected by centrifugation (1500×g for 15 min at 4°C), washed with DPBS, and finally lysed in 0.08% digitonin. An aliquot (25 μL) is used for protein analysis. Activity of NQO1 is determined by the Prochaska test[1].

After exposure to 2-HBA (bis(2-hydroxybenzylidene)acetone) for 24 h, duplicate aliquots of cells (1×106) are collected by centrifugation and washed with cold DPBS. Apoptosis is determined using the Annexin-V-FLUOS assay with simultaneous determination of the necrotic fraction by the uptake of propidium iodide[1].

[1]. Dinkova-Kostova AT, et al. Bis(2-hydroxybenzylidene)acetone, a potent inducer of the phase 2 response, causes apoptosis in mouse leukemia cells through a p53-independent, caspase-mediated pathway. Cancer Lett. 2007 Jan 8;245(1-2):341-9.

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