Antiangiogenic concentrations of vinflunine increase the interphase microtubule dynamics and decrease the motility of endothelial cells.

Angiogenesis is a key event in tumor progression and metastasis. This complex process, which constitutes a potent target for cancer therapy, is inhibited by very low concentrations of microtubule-targeting drugs (MTD). However, the intimate mechanisms of the antiangiogenic activity of MTDs remain unclear. Recently, we have shown that low antiangiogenic and noncytotoxic concentrations of paclitaxel induced an unexpected increase in microtubule dynamics in endothelial cells. In this study, we showed that vinflunine, the newest Vinca alkaloid, increased microtubule dynamic instability in human endothelial cells after 4-hour incubation at low concentrations (29% and 54% at 0.1 and 2 nmol/L). The growth and shortening rates were increased, and the percentage of time spent in pause and the mean duration of pauses were decreased, as previously observed with paclitaxel. As opposed to paclitaxel, the transition frequencies were not significantly disturbed by vinflunine. Moreover, low concentrations of vinflunine did not affect mitotic index and anaphase/metaphase ratio. Interestingly, these low vinflunine concentrations that increased microtubule dynamics exhibited an antiangiogenic effect through the inhibition of both morphogenesis and random motility. Capillary tube formation on Matrigel was decreased up to 44%. The cell speed and the random motility coefficient were decreased (13% and 19% and 13% and 33% at 0.1 and 2 nmol/L, respectively) and the persistent time was statistically increased. Altogether, our results confirm that the increase in microtubule dynamics is involved in MTD antiangiogenic activity and highlight the crucial role of interphase microtubule dynamics in angiogenesis.

Antiangiogenic concentrations of paclitaxel induce an increase in microtubule dynamics in endothelial cells but not in cancer cells.

Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis.

Antiangiogenic activity of paclitaxel is associated with its cytostatic effect, mediated by the initiation but not completion of a mitochondrial apoptotic signaling pathway.

Angiogenesis is a critical event in tumor growth and metastasis, which can be inhibited by conventional anticancer drugs such as the microtubule-damaging agent paclitaxel (Taxol). In this study, we investigate the mechanism of action of paclitaxel on human endothelial cells. We characterize two distinct effects of paclitaxel on human umbilical vein endothelial cell and human microvascular endothelial cell-1 proliferation according to drug concentration: a cytostatic effect at low concentrations and a cytotoxic effect at concentrations > or =10 nmol/L. The cytotoxic effect involves signaling pathways similar to those described in tumor cells (i.e., microtubule network disturbance, G(2)-M arrest, increase in Bax/Bcl-2 ratio, and mitochondria permeabilization) that result in apoptosis. In sharp contrast, the cytostatic effect involves an inhibition of endothelial cell proliferation without apoptosis induction and without any structural modification of the microtubule network. This cytostatic effect is due to a slowing of the cell cycle rather than to an arrest in a specific phase of the cell cycle. In addition, paclitaxel, at cytostatic concentrations, early initiates an apoptotic signaling pathway associated with increases in the mitochondrial reducing potential, mitochondrial membrane potential, p53 expression, and Bax/Bcl-2 ratio. However, this apoptotic pathway is stopped upstream of mitochondria permeabilization and it does not lead to endothelial cell death. Finally, we found that paclitaxel inhibits endothelial cell morphogenesis on Matrigel at all tested concentrations. In conclusion, we describe the mechanism of action of low concentrations of paclitaxel related to the antiangiogenic properties of this drug.

Low concentrations of vinflunine induce apoptosis in human SK-N-SH neuroblastoma cells through a postmitotic G1 arrest and a mitochondrial pathway.

Vinflunine, the newest fluorinated Vinca alkaloid, currently in phase III clinical trials, targets the microtubule network to induce mitotic block and apoptosis by mechanisms that remain unclear. In the current study, we investigated the apoptotic pathways induced by a wide range of vinflunine concentrations in SK-N-SH neuroblastoma cells. The concentrations of vinflunine that inhibited 50 and 70% of cell growth (IC(50) and IC(70)) induced high extents of apoptosis but failed to depolymerize microtubule network and to block cells in G(2)/M. It is interesting that the IC(50) and IC(70) concentrations suppressed microtubule dynamics, slowed down mitotic progression from metaphase to anaphase, and induced a postmitotic G(1) arrest. This G(1) arrest was associated with an increase in p53 and p21 expression and with their nuclear translocation. A high concentration of vinflunine (500 nM) induced both microtubule depolymerization and a canonical G(2)/M block. Mitochondria were involved in apoptotic pathways because all studied concentrations induced cytochrome c release. Bcl-2 family members were differently modulated by the different drug concentrations. Bax was up-regulated and translocated to mitochondria at the IC(50) and IC(70) concentrations, whereas Bcl-2 was phosphorylated only at the highest vinflunine concentration examined (500 nM). Our findings can be extended to other Vinca alkaloids, because similar results were obtained with vinblastine. All together, our results show that low concentrations of vinflunine fail to promote a G(2)/M arrest but are sufficient to induce suppression of microtubule dynamics and subsequent apoptosis. Moreover, mitochondria constitute the point of convergence of apoptotic signals induced by both low and high concentrations of vinflunine.

Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells.

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978-1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC(50)s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G(2)-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

Monolayer versus aggregate balance in survival process for EGF-induced apoptosis in A431 carcinoma cells: Implication of ROS-P38 MAPK-integrin alpha2beta1 pathway.

A431 cells escape EGF-induced apoptosis by forming cell aggregates. We show that these clusters migrate and merge with neighboring ones, resulting in larger structures composed of a multilayer central (3D) population surrounded by a cell monolayer (2D). We found that after 48 hr of 10 nM EGF treatment, 3D structure formation correlates with alpha2beta1 integrin upregulation. Blockade of alpha2 integrin impairs 3D structure formation. We studied the involvement of reactive oxygen species (ROS) in this process. We show that A431 cells express the NADPH oxidase catalytic subunits Nox1. EGF-induced dose-dependent ROS production was inhibited by the NADPH oxidase inhibitor, diphenylene iodonium (DPI), in these cells while rotenone was ineffective. Inhibition of ROS level in A431 cells with DPI or ebselen (glutathione peroxydase mimic) as well as P38 MAP kinase inhibition by SB203580 decreases alpha2 integrin subunit expression and induces a shift to 3D versus 2D populations. Cell cycle analysis of 2D cells shows that DPI, ebselen and SB203580 decrease the number of cells in S/G2 phase without affecting the cell number in mitosis phase. On the contrary, for 3D cells, these treatments increased the proportion of cells in mitosis without modification of the cell number in S/G2 phase. For both populations, apoptosis was increased by DPI and ebselen. Resistance of cell aggregates by paclitaxel to cell death is usually described. We show that DPI abolishes paclitaxel resistance of 3D cell aggregates. We observed a greater than additive effect between paclitaxel and DPI resulting in an increased proportion of cells in S/G2 phase for 3D populations. These results suggested that the ROS-P38 MAP kinase-alpha2beta1 integrin pathway was implicated in the A431 survival process by modulating the balance between 2D/3D cells.

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake.

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.

Specific binding of dehydroepiandrosterone to the N terminus of the microtubule-associated protein MAP2.

The effect of neurosteroids is mediated through their membrane or nuclear receptors. However, no dehydroepiandrosterone (DHEA)-specific receptors have been evidenced so far in the brain. In this paper, we showed by isothermal titration calorimetry that the DHEA specifically binds to the dendritic brain microtubule-associated protein MAP2C with an association constant of 2.7 x 10(7) m-1 and at a molar ratio of 1:1. By partial tryptic digestions and mass spectrometry analysis, we found that the binding involved the N-terminal region of MAP2C. Interestingly, MAP2C displays homologies with 17 beta-hydroxysteroid dehydrogenase 1, an enzyme required for estrogen synthesis. Based on these sequence homologies and on the x-ray structure of the DHEA-binding pocket of 17 beta-hydroxysteroid dehydrogenase 1, we modeled the complex of DHEA with MAP2C. The binding of DHEA to MAP2C involved specific hydrogen bonds that orient the steroid into the pocket. This work suggests that DHEA can directly influence brain plasticity via MAP2C binding. It opens interesting ways for understanding the role of DHEA in the brain.

Insulin-like growth factor-1 downregulates nuclear factor kappa B activation and upregulates interleukin-8 gene expression induced by tumor necrosis factor alpha.

Pretreatment of HT29-D4 epithelial adenocarcinoma colic cells with des-IGF-1 upregulated TNF alpha-mediated activation of IL-8 expression at different levels (protein, mRNA, and hnRNA). RNA transcription but not RNA stabilization was found to be involved. In this cell line, cooperation of NF-kappa B with other factors appeared essential for IL-8 expression. Indeed, TNF alpha-induced NF-kappa B translocation was not sufficient to support enhancement of the transcription and des-IGF-1 did not promote but partly inhibited both the TNF alpha-induced NF-kappa B activation and I kappa B alpha degradation through a PI-3K-dependent pathway. A CCAAT/enhancer binding protein (C/EBP) site located on the IL-8 gene enhancer cooperated with a NF-kappa B binding site and led to the upregulation of IL-8 expression. Binding of C/EBP alpha to this sequence disappeared in IGF-1 treated cells. This event may be important for the cross-talk between IGF-1- and TNF alpha-mediated pathways leading to the control of inflammatory processes and the decision concerning apoptosis or cell survival.

Alpha2beta1-integrin signaling by itself controls G1/S transition in a human adenocarcinoma cell line (Caco-2): implication of NADPH oxidase-dependent production of ROS.

In this work, we report that type IV collagen, mainly via alpha2beta1-integrin ligation, was able to induce cyclin expression and G1/S transition in a colic adenocarcinoma cell line (Caco-2) cultured without soluble growth factors or fetal bovine serum. This process involved Erk 1/2 activation and the production of reactive oxygen species (ROS) by a membrane-bound NADPH oxidase. Data presented here show that NADPH oxidase-dependent production of ROS increased following alpha2beta1-integrin ligation with type IV collagen or with a specific monoclonal antibody (Gi9 mAb). NADPH oxidase activation and, therefore, the production of ROS were shown to be involved in the increase of alpha2beta1-integrin plasma membrane expression, p38 MAPK phosphorylation, cyclin expression, and G1/S transition. We thus identified in this work a new integrin-signaling pathway in colon tumor cells involved in cell cycle regulation by the extracellular matrix.

Suppression of microtubule dynamics by discodermolide by a novel mechanism is associated with mitotic arrest and inhibition of tumor cell proliferation.

Discodermolide is a new microtubule-targeted drug in Phase I clinical trials that inhibits tumor growth and induces G(2)-M cell cycle arrest. It is effective against paclitaxel-resistant cell lines and acts synergistically in combination with paclitaxel. Suppression of microtubule dynamics by microtubule-targeted drugs has been hypothesized to be responsible for their ability to inhibit mitotic progression and cell proliferation. To determine whether discodermolide blocks mitosis by an effect on microtubule dynamics, we analyzed the effects of discodermolide on microtubule dynamics in living A549 human lung cancer cells during interphase at concentrations that block mitosis and inhibit cell proliferation. We found that discodermolide (7-166 nM) significantly suppressed microtubule dynamic instability. At the IC(50) for proliferation (7 nM discodermolide, 72 h), overall dynamicity was reduced by 23%. The principal parameters of dynamic instability suppressed by discodermolide were the microtubule shortening rate and length shortened. In addition, discodermolide markedly increased the frequency of rescued catastrophes. At the discodermolide concentration that resulted in 50% of maximal mitotic block (83 nM, 20 h), most microtubules were completely non-dynamic, no anaphases occurred, and all spindles were abnormal. The dynamicity of the remaining dynamic microtubules was reduced by 62%. The results indicate that a principal mechanism of inhibition of cell proliferation and mitotic block by discodermolide is suppression of microtubule dynamics. Importantly, the results indicate significant additional stabilizing effects of discodermolide on microtubule dynamics as compared with those of paclitaxel that may in turn reflect differences in their binding sites and their effects on tubulin conformation.

Paclitaxel targets mitochondria upstream of caspase activation in intact human neuroblastoma cells.

We previously reported that paclitaxel acted directly on mitochondria isolated from human neuroblastoma SK-N-SH cells. Here, we demonstrate that the direct mitochondrial effect of paclitaxel observed in vitro is relevant in intact SK-N-SH cells. After a 2 h incubation with 1 microM paclitaxel, the mitochondria were less condensed. Paclitaxel (1 microM, 1-4 h) also induced a 20% increase in respiration rate and a caspase-independent production of reactive oxygen species by mitochondria. The paclitaxel-induced release of cytochrome c was detected only after 24 h of incubation, was caspase-independent and permeability transition pore-dependent. Thus, paclitaxel targets mitochondria upstream of caspase activation, early during the apoptotic process in intact human neuroblastoma cells.

Cation binding mode of fully oxidised calmodulin explained by the unfolding of the apostate.

Calmodulin is the most ubiquitous calcium binding protein. The protein is very sensitive to oxidation and this modification has pronounced effects on calmodulin function. In this work, we decided to fully oxidise calmodulin in order to study the consequences on cation binding, domain stability, and alpha helicity. Oxidation of methionines unfolds completely the apostate of the protein, which upon calcium binding recovers the major part of its secondary and tertiary structure. However, the unstructuring of the apostate results in a protein that binds calcium to any site in an independent manner, does not bind magnesium and does not possess auxiliary sites anymore.

First tau repeat domain binding to growing and taxol-stabilized microtubules, and serine 262 residue phosphorylation.

Tau phosphorylation plays a crucial role in microtubule stabilization and in Alzheimer's disease. To characterize the molecular mechanisms of tau binding on microtubules, we synthesized the peptide R1 (QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI), reproducing the first tau microtubule binding motif. We thermodynamically characterized the molecular mechanism of tubulin assembly with R1 in vitro, and measured, for the first time, the binding parameters of R1 on both growing and taxol-stabilized microtubules. In addition, we obtained similar binding parameters with R1 phosphorylated on Ser262. These data suggest that the consequences of Ser262 phosphorylation on tau binding to microtubules and on tubulin assembly are due to large intramolecular rearrangements of the tau protein.

DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.

DNA gyrase is a major bacterial protein that is involved in replication and transcription and catalyzes the negative supercoiling of bacterial circular DNA. DNA gyrase is a known target for antibacterial agents since its blocking induces bacterial death. Quinolones, coumarins, and cyclothialidines have been designed to inhibit gyrase. Significant improvements can still be envisioned for a better coumarin-gyrase interaction. In this work, we obtained the crystal costructures of the natural coumarin clorobiocin and a synthetic analogue with the 24 kDa gyrase fragment. We used isothermal titration microcalorimetry and differential scanning calorimetry to obtain the thermodynamic parameters representative of the molecular interactions occurring during the binding process between coumarins and the 24 kDa gyrase fragment. We provide the first experimental evidence that clorobiocin binds gyrase with a stronger affinity than novobiocin. We also demonstrate the crucial role of both the hydroxybenzoate isopentenyl moiety and the 5'-alkyl group on the noviose of the coumarins in the binding affinity for gyrase.

Tubulin is an inherent component of mitochondrial membranes that interacts with the voltage-dependent anion channel.

We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated alpha-tubulin and is also enriched in the class III beta-tubulin isotype but contains very little of the class IV beta-tubulin isotype. The mitochondrial tubulin is likely to be organized in alpha/beta dimers and represents 2.2 +/- 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore.

Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis.

Arsenic trioxide (As(2)O(3)) at low concentrations (1-10 microM) is effective in the treatment of acute promyelocytic leukemia (APL) and lymphoma and is in clinical trials for treatment of solid tumors. Paclitaxel, an antimicrotubule agent, is highly efficacious in the treatment of adult tumors and is in clinical evaluation in childhood tumors. This study is the first to investigate the combination of arsenic and paclitaxel in the range of clinically achievable concentrations. We found that the simultaneous combination was antagonistic on proliferation of the neuroblastoma SK-N-SH cell line by using the combination index (CI) method. Moreover, a 40+/-5% decrease in paclitaxel-induced apoptosis in cells co-treated with As(2)O(3) confirmed the antagonism. The mechanism of antagonism was studied at the cellular level with 200 nM paclitaxel, twice the IC(50) value, and with 1 microM As(2)O(3) which administered singly did not affect cell survival or the microtubule network. As(2)O(3) antagonized the effects of paclitaxel on tubulin and microtubules. Paclitaxel-induced mitotic block was decreased by 20+/-2% and bundles induced by 200 nM paclitaxel were less condensed in the presence of 1 microM As(2)O(3). As(2)O(3) (10-200 microM) induced a concentration-dependent inhibition of tubulin polymerization in vitro which was maintained in presence of paclitaxel. Spectrophotometric and spectrofluorometric measurements indicated an interaction of As(2)O(3) with tubulin SH groups, without modification of the stoichiometry of paclitaxel binding to tubulin. Moreover, 4 microM As(2)O(3) inhibited the release of cytochrome c from isolated mitochondria by 78+/-10%. Our results show that As(2)O(3) and paclitaxel act antagonistically on mitochondria and microtubules and illustrate the need for careful evaluation of drug combinations.

Binding of ATP to heat shock protein 90: evidence for an ATP-binding site in the C-terminal domain.

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181-37186). In this paper, the interactions of ATP with native hsp90 and its recombinant N-terminal (positions 1-221) and C-terminal (positions 446-728) domains were studied by isothermal titration calorimetry, scanning differential calorimetry, and fluorescence spectroscopy. Results clearly demonstrate that hsp90 possesses a second ATP-binding site located on the C-terminal part of the protein. The association constant between this domain of hsp90 and ATP-Mg and a comparison with the binding constant on the full-length protein are reported for the first time. Secondary structure prediction revealed motifs compatible with a Rossmann fold in the C-terminal part of hsp90. It is proposed that this potential Rossmann fold may constitute the C-terminal ATP-binding site. This work also suggests allosteric interaction between N- and C-terminal domains of hsp90.