Inhibition of ATP-synthase potentiates cytotoxicity of combination drug menadione/ascorbate in leukaemia lymphocytes

The combination drug menadione/ascorbate (M/A) manifests synergistic dose-dependent antiproliferative and cytotoxic effects towards cancer cells, but not towards normal cells of the same origin especially at concentrations that can be achieved in vivo by its oral and parenteral administration. It is assumed that M/A alters selectively dysfunctional cancerous mitochondria. However, the exact molecular mechanism is not clear yet. The aim of the present study was to elucidate the role of adenosine triphosphate (ATP) synthase activity and its suppression by oligomycin-A on M/A-induced cytotoxicity, mitochondrial superoxide and ATP level in leukaemic lymphocytes. Cells were treated with different concentrations of M/A in the absence and presence of oligomycin-A (100 ng/mL) for 24 h and 48 h. The cell growth and viability, steady-state ATP level and mitochondrial superoxide were analysed using conventional analytical tests. The results showed that suppression of ATP synthase activity by oligomycin-A decreased the cell growth and viability and increased the production of mitochondrial superoxide and depletion of ATP in cells treated with low/tolerable doses of M/A (up to 5/500 mu M/mu M), compared to the cells treated with M/A only. Oligomycin-A did not affect these parameters in cells treated with high doses of M/A (10/1000 and 20/2000 mu M/mu M). The inhibition of ATP synthase potentiated the cytotoxicity of M/A, particularly in leukaemic lymphocytes treated with low/tolerable doses. We assume that the cytotoxicity of M/A is tightly connected to impairment of oxidative phosphorylation, and mitochondrial ATP depletion is a crucial factor for cell death.