Chantal Etievant

Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid

This study aimed to define the mechanism of action of vinflunine, a novel Vinca alkaloid synthesised from vinorelbine using superacidic chemistry and characterised by superior in vivo activity to vinorelbine in preclinical tumour models. In vitro vinflunine cytotoxicity proved dependent on concentration and exposure duration, with IC50 values (72-hr exposures) generally ranging from 60-300 nM. Vinflunine induced G2 + M arrest, associated with mitotic accumulation and a concentration-dependent reduction of the microtubular network of interphase cells, accompanied by paracrystal formation. These effects, while comparable to those of vincristine, vinblastine or vinorelbine, were achieved with 3- to 17-fold higher vinflunine concentrations. However, vinflunine and the other Vincas all inhibited microtubule assembly at micromolar concentrations. Vinflunine, like vinblastine, vincristine and vinorelbine, appeared to interact at the Vinca binding domain, as judged by proteolytic cleavage patterns, and induced tubulin structural changes favouring an inhibition of GTP hydrolysis. However, vinflunine did not prevent [3H]vincristine binding to unassembled tubulin at concentrations < or = 100 microM, and only weakly inhibited binding of [3H]vinblastine or [3H]vinorelbine. Indeed, specific binding of [3H]vinflunine to tubulin was undetectable by centrifugal gel filtration. Thus, the comparative capacities of these Vincas to bind to or to interfere with their binding to tubulin could be classified as: vincristine > vinblastine > vinorelbine > vinflunine. By monitoring alkylation of sulfhydryl groups, differential effects on tubulin conformation were identified with vinflunine and vinorelbine acting similarly, yet distinctively from vinblastine and vincristine. Overall, vinflunine appears to function as a definite inhibitor of tubulin assembly, while exhibiting quantitatively different tubulin binding properties to the classic Vinca alkaloids.

Vinflunine (20′,20′-difluoro- 3′,4′-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine®), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vincas, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.

In vitro synergistic effects of vinflunine, a novel fluorinated vinca alkaloid, in combination with other anticancer drugs.

Purpose: Vinflunine (20′-20′-difluoro-3′,4′-dihydrovinorelbine), a novel derivative of vinorelbine characterized by marked antitumour activity in vivo in a series of experimental murine and human tumours is currently undergoing phase I evaluation. To investigate its potential for inclusion in combination chemotherapy regimens, this preclinical study was undertaken. The in vitro cytotoxicity of vinflunine incubated simultaneously with one of the following drugs was investigated: camptothecin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, gemcitabine, mitomycin C, paclitaxel or vinorelbine. Methods: The combinations were first evaluated in vitro against the A549 human non-small-cell lung cancer cell line using median-effect analyses. Results: The results revealed synergistic cytotoxicity when vinflunine was combined with cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Synergy was also observed when testing similar combinations against CCRF-CEM human leukaemia cells. Finally, these findings were comparable with those resulting from such combinations involving vinorelbine instead of vinflunine. Conclusion: Vinflunine appears a promising candidate for combining with other anticancer drugs.

DNA damage induce gamma-tubulin-RAD51 nuclear complexes in mammalian cells.

Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, γ-tubulin. This association was confirmed by the reverse immunoprecipitation with γ-tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and γ-tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and γ-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.

Markedly diminished drug resistance-inducing properties of vinflunine (20′,20′-difluoro-3′,4′-dihydrovinorelbine) relative to vinorelbine, identified in murine and human tumour cells in vivo and in vitro

Abstract.Purpose: Vinflunine (VFL) is a novel Vinca alkaloid with markedly superior experimental in vivo antitumour activity to its parent molecule, vinorelbine (Navelbine, NVB), against a panel of murine and human tumours. The aim of this study was to establish whether there are differences in the rate and extent of development of resistance, both in vivo and in vitro, to these two newer Vinca alkaloids under identical selection conditions. Methods: Using P388 leukaemia cells in vivo, it was evident that VFL induced drug resistance far less readily than NVB, as shown by the number of passages required to select for total resistance. Under in vitro conditions, using A549 human lung carcinoma cells, it was also clearly shown by drug sensitivity determinations that VFL was a less-potent inducer of drug resistance than NVB. Resistance resulting from either in vivo or in vitro selection was associated with a classic multidrug resistance profile. Further characterization of the drug-resistance phenotype of the most highly resistant A549 sublines showed that the level of total β-tubulin expression appeared to be modified exclusively in the NVB-resistant cells. Conclusion: The clear demonstration that resistance to VFL developed far less readily than resistance to NVB both in vivo and in vitro may have potential clinical implications.

F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103

Purpose: F 11782 (2′′,3′′-bis pentafluorophenoxyacetyl-4′′,6′′-ethylidene-β-d-glucoside of 4′-phosphate-4′-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin, intoplicin and TAS-103, and established its novel mechanism of action. Methods: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. Results: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. Conclusion: These results emphasize the novel mechanism of action of F 11782 vis-à-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development.

The centrosome protein NEDD1 as a potential pharmacological target to induce cell cycle arrest

BackgroundNEDD1 is a protein that binds to the gamma-tubulin ring complex, a multiprotein complex at the centrosome and at the mitotic spindle that mediates the nucleation of microtubules.ResultsWe show that NEDD1 is expressed at comparable levels in a variety of tumor-derived cell lines and untransformed cells. We demonstrate that silencing of NEDD1 expression by treatment with siRNA has differential effects on cells, depending on their status of p53 expression: p53-positive cells arrest in G1, whereas p53-negative cells arrest in mitosis with predominantly aberrant monopolar spindles. However, both p53-positive and -negative cells arrest in mitosis if treated with low doses of siRNA against NEDD1 combined with low doses of the inhibitor BI2536 against the mitotic kinase Plk1. Simultaneous reduction of NEDD1 levels and inhibition of Plk1 act in a synergistic manner, by potentiating the anti-mitotic activity of each treatment.ConclusionWe propose that NEDD1 may be a promising target for controlling cell proliferation, in particular if targeted in combination with Plk1 inhibitors.

Low-dose twice-daily fractionated X-irradiation of ovarian tumor cells in vitro generates drug-resistant cells overexpressing two multidrug resistance-associated proteins, P-glycoprotein and MRP1.

Failure of chemotherapy is frequently observed in patients previously treated with radiotherapy. To establish a cellular model for examining this resistance phenotype a series of mammalian tumor cell lines were exposed in vitro to fractionated X-irradiation and were then shown to express resistance to multiple antitumor drugs, including vincristine, etoposide and cisplatin. In these experiments the radiation was delivered as 10 fractions of 5 Gy (dose resulting in 1 log cell kill) given intermittently over several months. We now report that a comparable multidrug-resistance profile is expressed by human SK-OV-3 human ovarian tumor cells exposed in vitro to low dose (2 Gy) twice-daily fractions of X-rays given for 5 days on two consecutive weeks, essentially mimicking clinical practice, involving an overexpression of two MDR-associated proteins, P-glycoprotein and the multidrug resistance protein 1 (MRP1), with the latter being readily detectable by immunocytochemistry.

Novel artificial endonucleases inhibit base excision repair and potentiate the cytotoxicity of DNA-damaging agents on L1210 cells.

A series of molecules with apurinic/apyrimidic (AP) endonuclease activity targeted to abasic sites in DNA, which incorporate an intercalating moiety linked to a purine base by a polyamino chain and recognize and cleave abasic sites in DNA with high efficiency, has been studied. The aim was first to establish whether these compounds were inhibitors of base excision DNA repair, since abasic sites are generated during this process. Using an extension of a recently established methodology, two members of this series have been identified as definite repair inhibitors. Secondly, the potential of using such compounds as sensitizers of alkylating agents has been investigated by determining the cytotoxic activity of these compounds on L1210 cells in culture. A concentration-dependent potentiation of nitrosoureas has been demonstrated, but interpretation is complicated by the inherent cytotoxic properties of the test compounds themselves. Such molecules, however, provide interesting lead compounds for new strategies aimed at enhancing the cytotoxic potential of clinically useful DNA-damaging agents.

Pharmacological screening of bryophyte extracts that inhibit growth and induce abnormal phenotypes in human HeLa cancer cells.

Antitumor activities of substances from natural sources apart from vascular plants and micro‐organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell‐based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes.