Background Deoxyspergualin (DSG) is a novel immunosuppressive drug currently undergoing clinical trials for treatment of transplant rejection or autoimmune diseases. The polyamine analogue DSG is a synthetic derivate of spergualin containing a spermidine moiety. Polyamines are known to induce apoptosis in several mesenchymal cell lines in presence of fetal calf serum (FCS).
Objectives The aims of the present study were
to test whether DSG alone or in combination with other immunomodulating drugs induces apoptosis or necrosis in immunocompetent cells (fresh isolated cells, PHA generated lymphoblasts) and whether this effect depends on the presence of FCS;
to analyse which cellular mechanisms are involved in the execution of the cell death program induced by DSG.
Methods Freshly isolated quiescent or activated human PBMCs, PHA generated lymphoblasts and several tumour cell lines (Jurkat, SKW-3, U937, RS4.11., MV4.11.) were incubated with different concentrations of DSG in presence of FCS or human AB serum (hABS). The rate of apoptotic cells was analysed after Propidium-Iodide/AnnexinV (PI/AxV) and PI/Triton staining by flow cytometric analysis.
Mitochondrial-membrane-potential (ΔψM) was measured by flow cytometric analysis after staining with Dihexyloxocarbocyaniniodide (DiOC6).
Results By means of PI/Triton and PI/AxV staining we found an induction of apoptosis by DSG in presence of FCS in quiescent PBMCs, PHA generated lymphoblasts and the tumour cell lines Jurkat, SKW-3, U937. In presence of hABS no induction of apoptosis was found. Necrosis was excluded in any of the experiments.
DSG was also capable to induce apoptosis in the tumour cell lines RS4.11., MV4.11. which are refractory to conventional chemotherapeutic agents operating through the induction of apoptosis. In lymphoblasts coactivation with IL-4, IL-7 or IL-15 did not prevent DSG-induced apoptosis, whereas IL-2 had various effects.
A decrease of ΔψM was seen after treatment of cells with DSG in presence of FCS. DNA degradation measured by PI Triton staining was abolished by adding the caspase inhibitor zVAD FMK.
Combination of DSG with Cycloheximid (CHX), a protein synthesis inhibitor which is described to interact with the regulation of cellular polyamine synthesis, lead to a remarkable increase in the induction of apoptosis by DSG, while CHX alone showed no effects.
Similar effects as described above for DSG were achieved if spermine, a physiological polyamine, was added to the culture.
Conclusion These results show, that DSG in presence of FCS is able to induce apoptosis in several cell systems. Interestingly induction of apoptosis was seen in resting cells and two cell lines, that are resistant to conventional chemotherapeutic agents. Our findings point out that induction of apoptosis by DSG in presence of FCS requires activation of caspases and is associated with a decrease in ΔψM. The structural conjunction of DSG with polyamines and the additive effects in combination with CHX suggest, that alterations of the cellular polyamine contents might be involved in the induction of apoptosis by DSG.
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