Antiproliferative and apoptotic effects of O-Trensox, a new synthetic iron chelator, on differentiated human hepatoma cell lines

Abstract

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 G₁ and G₂ phases of the cell cycle whereas DFO arrested cells in G₁ 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 G₁/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 G₁ phase of the cell cycle, demonstrating that inhibition of proliferation and induction of apoptosis occurred independently. DFO induced DNA alterations only at concentrations >100 μM 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 μM, strongly inhibits cell proliferation and induces apoptosis in proliferating and non-proliferating HepG2 and HBG cells, respectively.