Alessio Ligabue

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.

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.

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.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels

Background: NucS, new bipolar nuclease acting on branched DNA repair substrates, interacts with proliferating cell nuclear antigen (PCNA). Results: PCNA and NucS form a stable 1:1 complex and PCNA directs the activity of NucS toward single-stranded/double-stranded DNA junctions in branched DNA substrates. Conclusion: PCNA regulates NucS activity, preventing the nonspecific cleavage of NucS on the chromatin. Significance: This study will help understand how PCNA regulates its client enzymes. Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression

Background 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70–80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G1 phase and hTS is localized in the nuclei during S and G2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies.

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.

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.

Translational repression of thymidylate synthase by targeting its mRNA

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.