Dose-dependent regulation of superoxide anion on the proliferation, differentiation, apoptosis and necrosis of human hepatoma cells: the role of intracellular Ca2+

Dose-dependent regulation of cellular processes is one important characteristic of signaling molecules. Although recent studies suggest that reactive oxygen species (ROS) may act as in vivo signaling molecules, the dose-dependent regulation of ROS on cellular processes together in one system needs to be evaluated. After treating cells with gradually increased O(2)(-), generated by the hypoxanthine-xanthine oxidase system, it was found that: (i) the proliferation of hepatoma cells firstly increased at 1-2 microM O(2)(-), then decreased markedly as the concentration increased; (2) at 8 or 16 microM O(2)(-), re-differentiation of hepatoma cells was induced, as indicated by the indices relating to cell malignancy or differentiation, such as cell surface charge, alpha-fetoprotein, gamma-glutamyltranspeptidase, tyrosine-alpha-ketoglutarate transaminase, cAMP, and the tumor's clonogenic potential; (iii) at 16 microM O(2)(-), accompanied by the re-differentiation of cells, cell apoptosis was also simultaneously induced as indicated by the appearance of apoptotic bodies, detached cells, and other apoptotic morphological features, as well as specific DNA fragmentation; (iv) at the highest concentration of O(2)(-) (32 microM) in this study, cell necrosis was dramatically induced as shown by Trypan blue exclusion; (v), an increase of intracellular Ca(2+) ([Ca(2+)](i)) was observed at all concentrations of O(2)(-) treatment, and this [Ca(2+)](i) increase was found to be involved in the regulation of O(2)(-) on the cellular processes. In conclusion, these results indicate that O(2)(-) could dose-dependently regulate the processes of cells, where Ca(2+) is one of its molecular targets, and hence provide a direct support for the hypothesis that ROS themselves are important signaling molecules.