Gaetano Marverti

Synthesis, cytotoxic and combined cDDP activity of new stable curcumin derivatives

New curcumin derivatives are synthesized in order to improve chemical properties of curcumin. The aromatic ring glycosylation of curcumin provides more water-soluble compounds with a greater kinetic stability which is a fundamental feature for drug bioavailability. The glycosylation reaction is quite simple, low cost, with high yield and minimum waste. NMR data show that the ability of curcumin to coordinate metal ion, in particular Ga(III), is maintained in the synthesized products. Although the binding of glucose to curcumin reduces the cytotoxicity of the derivatives towards cisplatin (cDDP)-sensitive and -resistant human ovarian carcinoma cell lines, the compounds display a good selectivity since they are much less toxic against non-tumourigenic Vero cells. The combination of cDDP with the most active glycosyl-curcuminoid drug against both cDDP-sensitive and -resistant as well as against Vero cell lines is tested. The results show an improvement of cDDP efficacy with higher selectivity towards cancer cells than non-cancer cells. These studies indicate the need for developing new valid components of drug treatment protocols to cDDP-resistant cells as well.

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The “LR” peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

Newly synthesized curcumin derivatives: crosstalk between chemico-physical properties and biological activity.

New curcumin analogues (ester and acid series) were synthesized with the aim to improve the chemical stability in physiological conditions and potential anticancer activity. Cytotoxicity against different tumorigenic cell lines (human ovarian carcinoma cells -2008, A2780, C13*, and A2780/CP, and human colon carcinoma cells HCT116 and LoVo) was tested to evaluate cellular specificity and activity. Physico-chemical properties such as acidity, lipophilicity, kinetic stability, and free radical scavenging activity were investigated to shed light on the structure-activity relationship and provide new attractive candidates for drug development. Most of ester derivatives show IC(50) values lower than curcumin and exhibit selectivity against colon carcinoma cells. Especially they are extremely active after 24 h exposure showing enhanced inhibitory effect on cell viability. The best performances of ester curcuminoids could be ascribed to their high lipophilicity that favors a greater and faster cellular uptake overcoming their apparently higher instability in physiological condition.

Modulation of cis-diamminedichloroplatinum (II) accumulation and cytotoxicity by spermine in sensitive and resistant human ovarian carcinoma cells

The effect of spermine (Sp), a natural polycationic amine, on cisplatin (CDDP) sensitivity and accumulation of a human ovarian CDDP-sensitive cell line (2008) and its resistant variant (C13*) was investigated. Survival was also studied. The C13* cells were approximately 20-fold resistant to CDDP, yet were found to be just as sensitive to Sp as 2008 cells. When Sp was concurrently added with CDDP to the colony-forming assay, the IC50 dose was approximately 3-fold lower than that of CDDP alone. This decrease was the result of a synergistic interaction, as assessed by median effect analysis. The incubation of cells with the approximate IC50 dose of Sp for 1-8 h indicated that this synergism could be due to stimulation of CDDP accumulation, showing maximal uptake after 4 h of Sp exposure. This stimulation may be the result of a modulation of cellular membrane permeability by Sp, as assessed by the accumulation of [3H]mannitol. Exposure to Sp concentrations active on CDDP uptake also significantly increased [3H]mannitol accumulation in both cell lines. The triamine spermidine (Spd) did not significantly affect either the sensitivity of the two cell lines or CDDP and [3H]mannitol accumulation. These results suggest that Sp is a positive modulator of CDDP uptake, and thus of its cytotoxicity, even in resistant cells, where the phenotype is partly due to a CDDP accumulation defect.

N1,N12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines

The results presented here demonstrate that a cis‐diamminedichloroplatinum(II) (DDP)‐resistant human ovarian‐carcinoma cell line is also cross‐resistant to the spermine analogue N1,N12‐bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20‐fold resistant to DDP, similarly showed 7‐fold resistance to BESPM by colony‐forming assay with an IC50 value of 24.6 ± 2 μM vs. 3.4 ± 0.8 μM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/spermine N1‐acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross‐resistance, the combination of the 2 drugs resulted in supra‐additive and synergistic effects in both cell lines, depending on concentration, as assessed by median‐effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G2/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP‐sensitive and DDP‐resistant cells involves multiple mechanisms that are differently modulated by the DDP‐resistant phenotype. Int. J. Cancer 78:33–40, 1998.© 1998 Wiley‐Liss, Inc.

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.

Collateral sensitivity to novel thymidylate synthase inhibitors correlates with folate cycle enzymes impairment in cisplatin-resistant human ovarian cancer cells

The cytotoxicity of two novel folate cycle inhibitors with quinoxalinic structure, 3-methyl-7-trifluoromethyl-2(R)-[3,4,5-trimethoxyanilino]-quinoxaline (453R) and 3-piperazinilmethyl-2[4(oxymethyl)-phenoxy]quinoxaline (311S), was tested against a panel of both cisplatin(cDDP)-sensitive and -resistant carcinoma cell lines. Interestingly, the cisplatin-resistant human ovarian line, C13 cells, exhibited collateral sensitivity towards the two compounds when compared to its sensitive parental 2008 cells. In this resistant line, which showed elevated expression of the folate cycle enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), due to cisplatin-resistance phenotype, collateral sensitivity correlated with the greater reduction of enzyme expression. In addition, TS and DHFR expression of the other resistant lines, the human ovarian carcinoma A2780/CP cells and the human breast cancer MDA/CH cells, were decreased in accordance with the similar sensitivity or the low level of cross-resistance to these compounds in comparison to their respective parental lines. Noteworthy, unlike 5-fluorouracil, both drugs reduced the level of TS without inducing ternary complex formation with the co-substrate and the nucleotide analogue. Median effect analysis of the interactive effects of cisplatin with the two quinoxalines mainly showed additive or synergistic cell killing, depending on schedules of drug combinations. In particular, synergistic effects were more often obtained, even on the resistant cells, when cisplatin was added at the beginning of the treatment. These results indicate that, despite the possibility of other mechanisms being involved, inhibition of TS cycle enzymes plays an important role in the pharmacology of these compounds, which might also represent a useful component in drug treatment protocols against cDDP-resistant cells.

Inhibitor of ovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase.

Human thymidylate synthase (hTS) was targeted through a virtual screening approach. The most optimal inhibitor identified, 2-{4-hydroxy-2-[(2-hydroxybenzylidene)hydrazono]-2,5-dihydrothiazol-5-yl}-N-(3-trifluoromethylphenyl)acetamide (5), showed a mixed-type inhibition pattern, with a K(i) of 1.3 μM and activity against ovarian cancer cell lines with the same potency as cisplatin. X-ray studies revealed that it binds the inactive enzyme conformation. This study is the first example of a nonpeptidic inhibitor that binds the inactive hTS and exhibits anticancer activity against ovarian cancer 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.