Maria Giuseppina Monti

POLYAMINE DEPLETION PROTECTS HL-60 CELLS FROM 2-DEOXY-D-RIBOSE-INDUCED APOPTOSIS

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.

Characterization of the cell growth inhibitory effects of a novel DNA-intercalating bipyridyl-thiourea-Pt(II) complex in cisplatin-sensitive and—resistant human ovarian cancer cells

SummaryThe cellular effects of a novel DNA-intercalating agent, the bipyridyl complex of platinum(II) with diphenyl thiourea, [Pt(bipy)(Ph2-tu)2]Cl2, has been analyzed in the cisplatin (cDDP)—sensitive human ovarian carcinoma cell line, 2008, and its—resistant variant, C13* cells, in which the highest accumulation and cytotoxicity was found among six related bipyridyl thiourea complexes. We also show here that this complex causes reactive oxygen species to form and inhibits topoisomerase II activity to a greater extent in the sensitive than in the resistant line. The impairment of this enzyme led to DNA damage, as shown by the comet assay. As a consequence, cell cycle distribution has also been greatly perturbed in both lines. Morphological analysis revealed deep cellular derangement with the presence of cellular masses, together with increased membrane permeability and depolarization of the mitochondrial membrane. Some of these effects, sometimes differentially evident between the two cell lines, might also be related to the decrease of total cell magnesium content caused by this thiourea complex both in sensitive and resistant cells, though the basal content of this ion was higher in the cDDP-resistant line. Altogether these results suggest that this compound exerts its cytotoxicity by mechanisms partly mediated by the resistance phenotype. In particular, cDDP-sensitive cells were affected mostly by impairing topoisomerase II activity and by increasing membrane permeability and the formation of reactive oxygen species; conversely, mitochondrial impairment appeared to play the most important role in the action of complex F in resistant cells.

Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression.

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.

Modulation of the expression of folate cycle enzymes and polyamine metabolism by berberine in cisplatin-sensitive and -resistant human ovarian cancer cells

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.

Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis(ethyl)spermine involves transcriptional and post-transcriptional regulation

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analogue, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study, we investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin-sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 microM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 microg/ml actinomycin-D (Act-D), suggesting that transcription plays a major role in the analogue-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilised in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels.

Effect of spermine on membrane-associated and membrane-inserted forms of protein kinase C

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form).In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry.Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on Nmax; spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes.Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected.

Spermidine/spermine N1-acetyltranferase modulation by novel folate cycle inhibitors in cisplatin-sensitive and -resistant human ovarian cancer cell lines

OBJECTIVE Polyamines have been shown to play a role in the growth and survival of several solid tumors, including ovarian cancer. Intracellular polyamine depletion by the inhibition of biosynthesis enzymes or by the induction of the catabolic pathway leads to antiproliferative effects in many different tumor cell lines. Recent studies showed that the thymidylate synthase inhibitor 5-fluorouracil (5-FU) affects polyamine metabolism in colon carcinoma cells through the induction of the key catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). METHODS We therefore examined whether combinations of novel folate cycle inhibitors with quinoxaline structure and drugs that specifically target polyamine metabolism, such as diethylderivatives of norspermine (DENSPM) or spermine (BESpm), have synergistic effect in killing cisplatin-sensitive and drug-resistant daughter human ovarian cell lines. RESULTS Our results showed that simultaneous drug combination or quinoxaline pre-treatment synergistically increased SSAT expression, depleted polyamines, increased reactive oxygen species production, and produced synergistic tumor cell killing in both cell lines. Of note, this combined therapy increased the chemosensitivity of cisplatin-resistant cells and cross-resistant to the polyamine analogues. On the contrary, some pre-treatment regimens of Spm analogues were antagonistic. CONCLUSIONS These results show that SSAT plays an important role in novel folate cycle inhibitors effects and suggest that their combination with analogues has potential for development as therapy for ovarian carcinoma based on SSAT modulation.

Effect of spermine on association of protein kinase C with phospholipid visicles

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.

Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells

Acquired resistance to cisplatin (cDDP) is a multifactorial process that represents one of the main problems in ovarian cancer therapy. Distamycin A is a minor groove DNA binder whose toxicity has limited its use and prompted the synthesis of derivatives such as NAX001 and NAX002, which have a carbamoyl moiety and different numbers of pyrrolamidine groups. Their interaction with a B-DNA model and with an extended-TATA box model, [Polyd(AT)], was investigated using isothermal titration calorimetry (ITC) to better understand their mechanism of interaction with DNA and therefore better explain their cellular effects. Distamycin A interactions with Dickerson and Poly[d(AT)6] oligonucleotides show a different thermodynamic with respect to NAX002. The bulkier distamycin A analogue shows a non optimal binding to DNA due to its additional pyrrolamidine group. Cellular assays performed on cDDP-sensitive and -resistant cells showed that these compounds, distamycin A in particular, affect the expression of folate cycle enzymes even at cellular level. The optimal interaction of distamycin A with DNA may account for the down-regulation of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and the up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) caused by this compound. These effects seem differently modulated by the cDDP-resistance phenotype. NAX002 which presents a lower affinity to DNA and slightly affected these enzymes, showed a synergic inhibition profile in combination with cDDP. In addition, their combination with cDDP or polyamine analogues increased cell sensitivity to the drugs suggesting that these interactions may have potential for development in the treatment of ovarian carcinoma.

Polyamines and the catalytic domain of protein kinase C

The effect of polyamines on the catalytic domain of protein kinase C from rat brain was investigated. It was found that the addition of spermine strongly inhibited phosphorylation activity toward histone H1 as substrate. This tetramine, at millimolar concentrations, was most potently effective while triamines and diamines were almost uneffective, therefore the inhibitory action appeared to be structural specific. Data shown here suggest that polyamine by interacting with the catalytic domain of the enzyme may contribute to its regulation.