Influence of vitamin D3 metabolites on cell proliferation and cytotoxicity of adriamycin in human normal and neoplastic cells.

The common features of biological activity displayed by vitamin D family members and adriamycin suggest the possibility of synergistic effects of the combination of these compounds. Until now, the mechanisms responsible mainly for adriamycin cytotoxic action have not been indicated. Therefore, observation of the possible common cell targets for adriamycin and vitamin D metabolites could shed more light on the mechanisms of cytotoxic activity of adriamycin. In the present study, the influence of calcidiol (25-hydroxyvitamin D(3)) and calcitriol (1alpha,25-dihydroxyvitamin D(3)) on the proliferation and cytotoxicity of adriamycin was studied. The following cell lines were tested: normal human fibroblasts-CRL 1502, human melanoma cells-ME18 and its subline-ME18/R, resistant to adriamycin. As was shown, 72 h of incubation with calcidiol or calcitriol, both at 10 microM, inhibited growth (to approx. 60%) only of the ME18 cells. Dose and time dependence of this effect has been confirmed. Antiproliferative events did not correlate with an increase of adriamycin cytotoxicity. It was noted that calcidiol and calcitriol had no significant influence on the adriamycin IC(50) values in any cell lines tested. These results point to the divergent mechanisms of action of adriamycin and vitamin D(3) metabolites.

Activation of programmed cell death (apoptosis) by adriamycin in human neoplastic cells.

We have studied the occurrence of the apoptosis phenomenon in the action of adriamycin (ADR) on human melanoma cells sensitive (ME18) and resistant (ME18/R) to ADR. The study has shown that the intensity of apoptotic morphological changes noted in melanoma cells depended on the duration of the ADR treatment. We have not observed any positive correlation between the induction of apoptosis and sensitivity to ADR. We have used a fluorescence microscope and flow cytometer to evaluate apoptotic events in cells treated with ADR.

The in vitro study of influence of antioxidant enzymes level and repair capacity on cytotoxic bleomycin activity in human and mouse cells.

Bleomycin, an antitumour antibiotic was used to study the possible relationship between DNA single strand breaks repair capacity, antioxidant enzymes level and cytotoxic activity of the drug in mouse cells: AKR and BALB/c and in human cells: CRL 2088 and CRL 1307 (xeroderma pigmentosum). The BALB/c and CRL 1307 cells were used because of having defects in DNA repair capacity. A positive correlation was shown to exist between IC50 values and repair ability which suggested that DNA single strand breaks could be responsible for cytotoxic effects of bleomycin in human and mouse cells. Also antioxidant enzymes level have occurred as, at least partly, participating in bleomycin cytotoxic efficiency. About 10-fold higher resistance of AKR cells to bleomycin in comparison with the other cells, as appeared here, did not exhibit the straight correlation with antioxidant status of the cells. It prompts participation of the other mechanism in bleomycin cytotoxic action than that based on free radical generation. Also drug distribution and metabolism should be considered as a possible factor needed in bleomycin efficacy evaluation.

Studies on the mechanisms of an H2O2 adaptive dose on DNA damage in human neoplastic cells treated with adriamycin.

The possible influence of a hydrogen peroxide adaptive dose on DNA damage induced by adriamycin treatment in human melanoma cells, sensitive (ME18) and resistant (ME18/R) to adriamycin was investigated. In our earlier work, it was shown that the human melanoma resistant subline exhibited, in contrast to the parental cell line, the capacity to evoke an adaptive response provoked by low doses of hydrogen peroxide. This was observed as a diminished cytotoxic effect of adriamycin used at a toxic dose. The current work showed that an adaptive dose of hydrogen peroxide (2 microM) reduced DNA--single strand breaks, generated by a challenging dose of adriamycin in both, sensitive and resistant human melanoma cells. For better understanding of the adaptive response mechanism, exogenous free radical scavengers were used. In this study, it was shown that superoxide dismutase used at a concentration of 200 u/ml and mannitol at concentration of 100 mM modulated adriamycin--generated DNA--single strand breaks in parental melanoma cells. None of the exogenous scavengers, used in this study, influenced the cytotoxic effects of adriamycin either in sensitive or in resistant melanoma cells.

DNA repair of adriamycin induced damage in mouse and human cells differing in catalase activity.

Catalase is known to counteract cytotoxic effect of adriamycin, used as an anti-neoplastic drug. In cells with low catalase activity no repair of adriamycin induced lesions was observed up to 48 h post treatment. In cells with high catalase activity after 48 h the repair was either complete or partial depending on the human or mouse cell type used.

Studies on adaptation to adriamycin in cells pretreated with hydrogen peroxide.

Various investigations have reported the occurrence in bacterial and mammalian cells of an adaptive response to the toxic effects of oxidants or agents that cause oxidation via redox reactions. In our previous study, it was shown that several cell lines pretreated with a low dose of hydrogen peroxide (H2O2) exhibited an adaptive response to subsequent high doses of adriamycin (ADR), whereas other cell lines did not. Based on the observation that the cell lines utilized differed in their sensitivity towards adriamycin, we undertook the present investigation with the goal of evaluating possible relationships between the levels of antioxidant enzymes and sensitivity towards adriamycin. Another aim was to determine relationships between the inducibility of these enzymes and the occurrence of adaptation. We utilized African Green monkey kidney (V3), human embryo (CLV98), human melanoma (ME18), and Chinese hamster ovary (CHO) cell lines and experimentally developed adriamycin-resistant human melanoma (ME18/RN) and Chinese hamster ovary (CHO/RN) cell sublines. Cytotoxicity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and trypan blue exclusion. The levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were determined in the same kind of experiment as that revealing the occurrence of adaptation. The rank order established for catalase activities was similar to that for sensitivity towards adriamycin. Aberrant increases in the tested enzymes were demonstrated in experimental groups of all kinds of cells. We conclude that in our cell systems catalase is a major determinant of adriamycin resistance. Whether the occurrence of the adaptive response under study is dependent on the contribution of catalase, itself dependent on the degree of resistance to the drug, is discussed.