We investigated the effects of a new
iron chelator,
O-Trensox (TRX), compared with
desferrioxamine (DFO), on proliferation and apoptosis in cultures of the human
hepatoblastoma HepG2 and hepatocarcinoma HBG cell lines. Our results show that TRX decreased
DNA synthesis in a time- and dose-dependent manner and with a higher efficiency than DFO. Mitotic index was also strongly decreased by TRX and, unexpectedly, DFO inhibited mitotic activity to the same extent as TRX, thus there is a discrepancy between the slight reduction in
DNA synthesis and a large decrease in mitotic index after DFO treatment. In addition, we found that TRX induced accumulation of cells in the G(1) and G(2) phases of the cell cycle whereas DFO arrested cells in G(1) and during progression through S phase. These data suggest that the partial inhibition of DNA replication observed after exposure to DFO may be due to a lower efficiency of
metal chelation and/or that it does not inhibit the G(1)/S transition but arrests cells in late S phase. The effects of both TRX and DFO on
DNA synthesis and mitotic index were reversible after removing the
chelators from the culture medium. An apoptotic effect of TRX was strongly suggested by analysis of
DNA content by flow cytometry, nuclear fragmentation and
DNA degradation in oligonucleosomes and confirmed by the induction of a high level of
caspase 3-like activity. TRX induced apoptosis in a dose- and time-dependent manner in proliferating HepG2 cells. In HBG cells, TRX induced apoptosis in proliferating and confluent cells arrested in the G(1) phase of the cell cycle, demonstrating that inhibition of proliferation and induction of apoptosis occurred independently. DFO induced
DNA alterations only at concentrations >100 microM and without induction of
caspase 3-like activity, indicating that DFO is not a strong inducer of apoptosis. Addition of Fe or Zn to the culture medium during TRX treatment led to a complete restoration of proliferation rate and inhibition of apoptosis, demonstrating that Fe/Zn-saturated TRX was not toxic in the absence of
metal depletion. These data show that TRX, at concentrations of 20-50 microM, strongly inhibits cell proliferation and induces apoptosis in proliferating and non-proliferating HepG2 and HBG cells, respectively.