Search

Six bipyridyl complexes of platinum(II) with thiourea, with different substituents on thiourea moiety [Pt(bipy)(R,R'NCSNR'',R''')(2)]Cl(2) (bipy=2,2'-bipyridine: R=R'=R''=R''' =H; R=Me, R'=R''=R'''=H; R=n-Bu, R'=R''=R'''=H; R=Et, R'=H, R''=Et, R'''=H; R=p-tolyl, R'=R''=R'''=H; R=phenyl, R'=H, R''=phenyl, R'''=H), rationally designed to intercalate into DNA, have been tested against a cisplatin (cDDP)-sensitive human ovarian carcinoma cell line (2008) and its -resistant variant (C13( *)). We show here that the anti-proliferative efficacy of these drugs was dependent on molecular structure, since it increased with ancillary ligand bulkiness and hydrophobicity of substituents on thiourea moiety. In particular, the presence of two phenyl groups on thiourea moiety confers an outstanding cytotoxicity. The increasing cell growth inhibition along the series of complexes partially paralleled with drug accumulation, particularly in resistant cells, but not with drug intercalation into DNA since all compounds exerted comparable ethidium bromide displacement ability. The cDDP-resistant phenotype seems, at least in part, to be involved in the action of these compounds, since the level of cross-resistance established for most complexes appeared to be in agreement with the observed impairment of drug accumulation in the resistant subline. These findings indicate that resistance to alkylating agents such as cDDP confers low level of cross-resistance to this class of DNA intercalators, which, however, depending on substituents on thiourea moiety may present remarkable cell growth inhibition even of resistant cells.

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy-d-ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. d-Rib-treated cells with normal polyamine levels, progressing through the G(1) into the S and G(2)/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G(1) phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.

Cisplatin (DDP)-resistance confers a deficient expression of spermidine/spermine N(1)-acetyltransferase (SSAT) gene in response to the spermine analog N(1),N(12)-bis(ethyl)spermine (BESpm) in the DDP-resistant human ovarian carcinoma cell line (C13*), compared with their parental DDP-sensitive 2008 cells. This SSAT gene deficiency is correlated with a reduced growth sensitivity to spermine analogs. This study was performed to determine whether SSAT gene expression of resistant cells was kept suppressed by labile repressor proteins developed during resistance selection. We show here that inhibitory concentrations of cycloheximide (CHX) and anisomycin (ANISO) differentially affect BESpm-induced SSAT activity in 2008 and in C13* cells in a concentration-dependent manner and allow resistant cells to reach activation levels comparable to those of the sensitive cells. Northern blot analysis revealed that both CHX and ANISO in combination with BESpm caused a synergistic BESpm-mediated accumulation of SSAT mRNA in C13* cells, with respect to each drug alone, while in 2008 cells only a slight increase was observed. The more pronounced effect of inhibitors on the SSAT activity induced by BESpm in the resistant cells was also the result of a more prolonged stabilization of SSAT mRNA and enzyme protein. By contrast, sub-inhibitory concentrations of CHX and ANISO did not significantly stimulate BESpm-induced SSAT transcription and activity. These results suggest that labile repressor proteins, related to DDP-resistance phenotype, play a regulatory role in SSAT gene expression, and further indicate that by overcoming this inhibitory control it is possible to recover BESpm response.

The presence of polyamines is required for the apoptotic program triggered by 2-deoxy-D-ribose (dRib) in HL-60 cells, but their oxidative metabolites does not appear to be involved in the oxidative stress caused by the sugar. The present study points to a relationship between spermidine-induced G1 to S phase transition and the onset of dRib-induced apoptosis. Conversely, the G1 block induced by alpha-difluoromethylornithine (DFMO) is associated with a protective effect against dRib-induced cell suicide. Replenishment of the intracellular spermidine pool by exogenous putrescine and spermidine induces cell cycle progression and restores apoptotic levels. The present data indicate that the induction of cell cycle progression by spermidine is a condition facilitating the activation of the apoptotic process by dRib., (Copyright 1999 Academic Press.)

We investigated the involvement of natural polyamines in HL-60 cell death triggered by exposure to 2-deoxy-D-ribose (dRib). In contrast to previous studies, exogenous polyamines failed to protect HL-60 cells against apoptosis caused by dRib. Moreover, in our experimental conditions, depletion of intracellular levels of putrescine and spermidine by alpha-difluoromethylornithine (DFMO) delayed the onset of apoptosis by at least a day or so. Exogenous polyamines reversed the beneficial effect of DFMO and restored the apoptotic levels observed in dRib-treated cells. We suggested that polyamines, especially putrescine and spermidine, act as facilitating factors in the induction of apoptosis triggered by dRib in HL-60 cells.

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine- or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G1 to S phase transition.

We have previously reported that polyamines interfere with protein kinase C-membrane interactions. With the aim of clarifying the influence of the relationship between calcium and polyamines on this process we have investigated the effect of spermine on the formation of active protein kinase C-membrane complexes as a function of Ca++ concentrations. Protein kinase C, purified from rat brain, was allowed to interact with phospholipid vesicles of defined composition. The active complex protein kinase C-liposomes was determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The results show that, at Ca++ levels below 0.1 microM, spermine inhibits the formation of complexes between protein kinase C and membranes. At higher Ca++ concentrations, spermine does not prevent the association process but does influence the ratio between the enzyme molecules irreversibly inserted into the membrane and those reversibly associated with it. We have also demonstrated that spermine, by reducing the density of acidic component of liposomes, influences the calcium requirement for protein kinase C-membrane binding. This study indicates that spermine may regulate the activation of protein kinase C and affects the calcium requirement for the association of this enzyme with the phospholipid bilayer. The results suggest a possible role for polyamines in signal transduction when protein kinase C is involved.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.