Parthenolide Inhibits Tubulin Carboxypeptidase Activity

Microtubules are centrally involved in cell division, being the principal components of mitotic spindle. Tubulin, the constituent of microtubules, can be cyclically modified on its A-subunit by enzymatic removal of the COOH-terminal tyrosine residue by an ill-defined tubulin carboxypeptidase (TCP) and its readdition by tubulin tyrosine ligase (TTL). We and others have previously shown that suppression of TTL and resulting accumulation of detyrosinated tubulin are frequent in human cancers of poor prognosis. Explanations for the involvement of TTL and detyrosinated tubulin in tumor progression arise from the recent discovery that tubulin detyrosination leads to CAP-Gly protein mislocalization, which correlates with defects in spindle positioning during mitosis. Impaired control of spindle positioning is one factor favoring tumor invasiveness. Thus, TCP could be a target for developing novel therapeutic strategies against advanced stages of cancers. Inhibitors of TCP, by reversing abnormal detyrosinated tubulin accumulation in tumor cells, could impair tumor progression. TCP has never been isolated and this has hampered search of specific inhibitors. In this article, we describe a cell-based assay of TCP activity and its use to screen a library of natural extracts for their inhibitory potency. This led to the isolation of two sesquiterpene lactones. We subsequently found that parthenolide, a structurally related compound, can efficiently inhibit TCP. This inhibitory activity is a new specific property of parthenolide independent of its action on the nuclear factor-KB pathway. Parthenolide is also known for its anticancer properties. Thus, TCP inhibition could be one of the underlying mechanisms of these anticancer properties. [Cancer Res 2007;67(7):3371–8]


Introduction
Microtubules are key components of the cytoskeleton of eukaryotic cells.They are centrally involved in the control and mechanics of cell division, being the principal components of mitotic spindle.The building block of microtubules is the ahtubulin heterodimer.In most eukaryotic cells, the a chain of tubulin can be modified by enzymatic removal of the COOHterminal tyrosine residue by an ill-defined tubulin carboxypeptidase (TCP) and by readdition of this tyrosine by a distinct enzyme, tubulin tyrosine ligase (TTL; refs.1,2).This tyrosination cycle is conserved among eukaryotes (1,3) and generates two tubulin pools: intact tyrosinated a-tubulin (Tyr-tubulin) and detyrosinated a-tubulin (Glu-tubulin), which lacks the COOH-terminal tyrosine.Its physiologic importance in normal cells, tissues, or organisms has been shown with the generation of TTL-null mice.TTL-null mice die within hours after birth due to the disorganization of vital neuronal circuits (4), indicating that TTL is essential for neuronal organization.
TTL is also frequently suppressed during tumor progression (5), with resulting accumulation of Glu-tubulin in tumor cells.TTL suppression and resulting tubulin detyrosination in human cancers are associated with increased tumor aggressiveness (6)(7)(8).Experiments conducted in yeast (9), in TTL nude mice (4), and in TTLnull fibroblasts (10) show that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus tiptracking proteins, such as CLIP-170.In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in spindle positioning during mitosis.Because impaired control of spindle positioning has previously been proposed as one factor favoring tumor invasiveness (11), these results partly shed light on the molecular basis for the apparent stimulatory effect of Glu-tubulin accumulation on tumor progression.
Inhibition of TCP could reverse abnormal Glu-tubulin accumulation in tumor cells and could lead to the restoration of normal Tyr-tubulin levels through tubulin synthesis (4).Such an inhibition could thus impair tumor progression (7,8).Until now, there are no known specific TCP inhibitors (12).Moreover, the TCP gene has never been identified, although biochemical TCP activity has been reported to be present in some subcellular fractions (1,3).Thus, the discovery of specific TCP inhibitors could also be useful for a ligand-based affinity purification of TCP.Finally, TCP inhibition is also necessary for a full understanding of the tyrosination cycle.Detyrosinated tubulin is very abundant in differentiated cells (13).Although tubulin tyrosination is essential for microtubule interaction with some proteins plus tip-tracking proteins, it is not known why eukaryotic cells developed a tyrosination cycle by introducing a detyrosination reaction.
Chemogenomic strategies have proven useful to isolate chemical compounds that could induce a specific phenotype (14,15).In such approaches, also referred as chemical genetics, cells are tested against a library of small molecules using high-throughput screening and then examined for a desired phenotype (16)(17)(18).The subsequent identification of the intracellular protein target of these compounds, although often technically difficult (19), has obtained notable successes (20)(21)(22)(23).We designed a phenotype assay of TCP activity and used this cell-based assay to screen a chemical library to isolate TCP inhibitors.In this report, we describe the assay, its application to a screen of 23,904 natural extracts, and the purification and characterization of two sesquiterpene lactones that were able to specifically inhibit TCP.We tested several structurally related compounds and found that parthenolide, a known nuclear factor-nB (NF-nB) inhibitor (24), can efficiently inhibit TCP activity.Parthenolide has also been shown to present some anticancer properties, but the underlying mechanisms are not fully understood.TCP inhibition could be one of the mechanisms underlying the known anticancer properties of parthenolide.

Cell Culture
HeLa cells were purchased from the American Type Culture Collection (Rockville, MD).They were grown in RPMI 1640 with Glutamax supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin.Cells were maintained at 37jC in 5% CO 2 and 95% air.
Dihydroparthenolide was obtained by hydrogenation of parthenolide as described by Kwok et al. (25).

96-Well Format Assay of TCP Activity
Microplate preparation.HeLa cells were cultured as monolayers.The cells were seeded at 30,000 per well in 96-well polystyrene tissue culture plates (reference no.655 083; Greiner Bio-One, Kremsmuenster, Austria) in 90 AL medium.They were allowed to grow for 24 h.
Drug addition.The day following microplate preparation, each well was robotically supplemented with 10 AL RPMI 1640 containing natural extracts and paclitaxel so that the final concentrations were 35 Ag/mL for natural extracts and 5 Amol/L for paclitaxel.Bioinactive controls were obtained with paclitaxel alone (5 Amol/L).The final concentration of DMSO was 0.5%.After drugs dispense, cells were incubated for 2 h at 37jC, 5% CO 2 , in the workstation incubator.
Cell immunostaining.After medium aspiration, cells were washed with TBS/0.1% Tween 20 and fixed for 1 h with 3.7% formaldehyde.Cells were permeabilized with 100 AL methanol for 5 min.
Result analysis.Raw data were exported from the software of the microplate reader to an Excel file and normalized according to the following procedure.Mean values of DMSO and paclitaxel were calculated for each individual microplate using intraplate control data points.Paclitaxel and DMSO mean values were considered as bioinactive control (0% inhibition) and bioactive control (100% inhibition) of TCP activity, respectively.
Assay performance.To evaluate the quality of the assay, the Z ¶ factor was calculated using the following equation according to Zhang et al. (26): where c + is the bioactive controls, c À is the bioinactive controls, l is the mean, and r is the SD.In our experimental conditions, the Z ¶ was comprised between 0.54 and 0.57, indicating that the assay is of good quality for high-throughput screening.

Sample Collection and Extract Preparation
Plant samples were collected worldwide under the general framework of the International Convention on Biological Diversity (Rio de Janeiro, Argentina, 1992).Bases for the collection were the taxonomic identification of the species, botanical diversity, and availability for a second collection.No rare or protected plants belonged to the collection.When possible, the different organs of the plants were separated and treated as different samples (bark, leaves, root, etc.).Each plant sample was dried (55jC) and finely ground.An aliquot of the plant (20 g) was macerated overnight in 200 mL ethyl acetate in an Erlenmeyer flask; after filtration, the solvent was evaporated in vacuo to give the ethyl acetate crude extract.Part of the extract (50 mg) was adsorbed on 500 mg Sigel, which was placed on top of SiO 2 solid-phase extraction cartridge.Successive elutions with CHCl 3 , CHCl 3 /MeOH (17:3), and MeOH provided three ''fractions, '' which were conditioned in 96-well deep-well plates for the screening after evaporation of the solvents and replacement with 1 mL DMSO, along with the ''crude extract'' at a concentration of 50 mg/mL.

Bioassay-Guided Fractionation of the Extract from Vernonia perrottetii
Finely ground aerial parts from the plant (97 g) were macerated overnight in 1 L ethyl acetate; the suspension was filtered and the organic filtrate was evaporated in vacuum to give 9.6 g of a gum.Part of this extract (4 g) was dissolved in 8 mL chloroform and adsorbed on a Sigel column (120 g) eluted in 200 mL fractions under 18 p.s.i. at a 30 mL/min flow rate with a chloroform/methanol gradient (10 min: CHCl 3 ; 50 min: CHCl 3 /MeOH (17:3); 10 min: MeOH).The second fraction concentrated the activity and was further purified on a semiprep high-performance liquid chromatography Lichrospher column with a water/acetonitrile gradient.Compound V18 was eluted at 15.6 min and compound V19 was eluted at 16.6 min (gradient system: water/acetonitrile, from 6/4 to 1/19 in 20 min).

Description of the Molecules
V18: crystals from diethyl ether (melting point 136jC), MS, UV, IR, and nuclear magnetic resonance (NMR) spectra identical with those published in the literature (27); V19: amorphous gum with MS, UV, IR, and NMR spectra identical with those published in the literature.

Cytotoxicity Assay
Some of the natural extract fractions were tested for their cytotoxic activity.This cytotoxic activity was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation/cytotoxicity assay.This assay is a colorimetric assay, which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells.Briefly, cells were plated in 96-well flatbottomed microplates at a density of 30,000 per well.Twenty-four hours after plating, the fractions were added and appropriately diluted in DMSO.After 2 h of incubation with the fractions, the medium was replaced with MTT (Sigma-Aldrich) dissolved at a final concentration of 1 mg/mL in serum-free, phenol red-free RPMI 1640 (Life Technologies).The cells were then let to incubate for 3 h to develop purple formazan precipitate.The MTT formazan was then solubilized in DMSO, and the absorbance was measured at a wavelength of 570 nm and a reference wavelength of 630 nm.

Immunofluorescence Procedure
Cells grown for 2 days on glass coverslips were washed with warm PBS and fixed for 6 min in anhydrous methanol at À20jC.After an additional wash in PBS/0.1% (v/v) Tween 20, cells were sequentially incubated with primary antibodies and appropriate secondary antibodies.Antibodies were diluted in PBS/0.1% BSA.YL1/2 and L4 antibodies were used at a 1:4,000 dilution.Cy3-conjugated antibodies and Alexa Fluor 488-conjugated antibodies were used at a 1:1,000 and 1:500 dilution, respectively.Nuclei were stained with Hoechst 33258 (1 Ag/mL).
ID3 antibody used in microinjection experiments was diluted at a concentration of 4 mg/mL in PBS buffer.Before microinjection, the solution was clarified by centrifugation in a Beckman TLA-100 rotor (100,000 Â g, 4jC, 10 min).The supernatant was then loaded into injection needles and injected into HeLa cells.After microinjection, cells were incubated for 4 h.They were then processed for immunofluorescence as described above.

Assay of CPA Activity on Permeabilized Cells
Cells grown for 2 days on glass coverslips were permeabilized with warm OPT buffer [80 mmol/L PIPES, 1 mmol/L EGTA, 1 mmol/L MgCl 2 , 0.5% Triton X-100, 10% glycerol (pH 6.8)].OPT buffer permeabilizes cells while preserving intact microtubules (28).CPA (0.5 Ag/mL) diluted in warm OPT buffer was added with or without inhibitory compounds (1 mmol/L).Cells were incubated for 15 min at 37jC and then fixed and processed for immunofluorescence as described above.Immunofluorescence analysis of the effect on paclitaxel-induced tubulin detyrosination of natural extract fractions selected by the high-throughput screening assay.HeLa cells were incubated for 2 h with DMSO (A ), 5 Amol/L paclitaxel (B), or 5 Amol/L paclitaxel combined with an active fraction (C ).Cells were then fixed, double stained with Tyr-tubulin and Glu-tubulin antibodies as indicated, and processed for immunofluorescence as described in Materials and Methods.

Results
Screen for chemicals that decrease paclitaxel-induced tubulin detyrosination.The rationale of the assay relies on the substrate properties of TCP and TTL.TTL, the enzyme that catalyzes the tubulin tyrosination reaction, adds tyrosine to soluble, detyrosinated tubulin and does not react with tubulin once it is assembled into microtubules (3,(29)(30)(31).TCP is likely to prefer polymerized substrate, and its binding to microtubules may be required for its function (30,32,33).TCP acts slowly on microtubules, whereas TTL recharges Glu-tubulin quickly on release from microtubules (30).As a consequence of the substrate specificity of TTL and of the kinetic differences of TTL and TCP, Tyr-tubulin is the main component of dynamic microtubules, being the dominant tubulin variant in cycling cells in vitro, whereas Glutubulin is a marker of long-lived stable microtubules (3,30,(34)(35)(36).Depending on the cell type, these Glu-microtubules either are not detectable or represent a small subpopulation of the cellular microtubules.In HeLa cells, these Glu-microtubules are almost absent (28).However, they do accumulate on paclitaxel treatment, indicating that TCP is active in these cells.We set up an assay amenable to automation, in which TCP inhibitors were selected according to their ability to inhibit accumulation of Glu-tubulin in HeLa cells with paclitaxel-stabilized microtubules.Briefly, compounds were added to HeLa cells grown in 96-well microplates.Cells were treated with paclitaxel immediately after addition of the compounds.After 2 h of incubation, cells were rinsed, fixed, and permeabilized with methanol.Glu-tubulin was then quantified by immunoluminescence using a specific antibody (6,28).The Glutubulin signal obtained after treatment of the cells with paclitaxel alone was defined as 0% of inhibition, whereas the basal Glutubulin signal obtained after treatment with only DMSO was taken as 100% of inhibition.The different steps of the assay are illustrated in Fig. 1.
Screening of partially purified natural extracts.A total of 23,904 partially purified extracts, corresponding to 3,252 natural species (81.5% plants, 2.5% insects, and 16% marine organisms), was screened for potential TCP-inhibiting activity using the above described assay (Fig. 1).Among these, 268 extracts (1.1% hit rate) were scored as positive, showing >50% of inhibition.However, in such a cell-based assay, a lowered Glu-tubulin signal could result from a global toxic effect of the extracts on the cells.We thus assayed the extract cytotoxicity using a MTT assay.A subset of 127 extracts showed some cytotoxicity after 2 h of incubation on the cells and was discarded.Among the 141 remaining extracts, 49 extracts showed an apparent inhibitory activity superior to 70%.These most active extracts were set aside for further characterization.
Bioguided fractionation and identification of TCP inhibitors.The 49 extracts were submitted to fractionation by combining chromatographies (normal and reverse C18 phases), and each fraction was tested again using several different biological complementary assays (bioguided fractionation).The aim of these other assays was to discriminate between false-and true-positive compounds.All fractions were first screened using tubulin immunofluorescence microscopy to examine their effects on microtubule structure (data not shown).Fractions able to depolymerize the microtubule network were classed as potential false positive.Indeed, such compounds could have competed with paclitaxel during the high-throughput screening assay and thus led to a diminished Glu-tubulin signal that does not result to TCP inhibition.Such depolymerizing fractions were discarded, and only fractions showing no detectable effect on the microtubule network were kept.These last fractions were checked for their inhibitory effect on paclitaxel-induced tubulin detyrosination using tubulin immunofluorescence microscopy (Fig. 2).When cells were only treated with DMSO (Fig. 2A), the Glu-microtubules represented a small subpopulation of cellular microtubules as expected.After paclitaxel treatment (Fig. 2B), Glu-microtubules did accumulate, reflecting TCP action on stabilized microtubules.This accumulation was prevented when cells were treated with the fractions selected by the screening strategy, together with paclitaxel (Fig. 2C), although paclitaxel still exerted its stabilizing effect as indicated by the presence of tyrosinated microtubule bundles.
Finally, the inhibitory effect of the remaining fractions was investigated with another assay of TCP activity using microinjection experiments (Fig. 3).In such an assay, TCP activity is assessed after blocking TTL activity in cells by microinjection of ID3, a monoclonal antibody directed against the active site of TTL (29,37).The activity of TCP is revealed in injected cells by the resulting accumulation of Glu-tubulin (Fig. 3A).In the presence of a TCP-inhibiting fraction, such an accumulation does not occur.In that case, the level of detyrosinated tubulin (Fig. 3B) is as low in microinjected cells as in non-microinjected cells, which can be localized thanks to Hoechst 33258 staining of the nuclei.
The overall process allowed the selection of an extract from Vernonia perrottetii, and this led to the isolation of two compounds (V18 and V19).Their structures were established by analysis of their NMR data and comparison with published values.These compounds were structurally related sesquiterpene lactones (V18 and V19; Fig. 4).They showed a potent TCP inhibitory activity, as assessed by ID3 microinjection experiment, for a concentration of 10 Amol/L.
Preliminary structure-activity relationships.We tested the effect of 17 available structural analogues of the active compounds (Fig. 4), at a final concentration of 20 Amol/L, on TCP activity evaluated by ID3 microinjection experiments.Among these compounds, we found that only the known anti-inflammatory sesquiterpene lactone parthenolide, originally extracted from feverfew Tanacetum parthenium, and, to a much lesser extent, cnicin displayed a TCP inhibitory activity (Fig. 5D).
Parthenolide showed a similar TCP inhibitory activity than the two compounds isolated from V. perrottetii (Fig. 5A).This indicates that the C8 lateral chain, present on V18 and V19, is not necessary for TCP inhibitory activity.
There are strong indications that alkylation of cysteine(s) is a general mechanism for sesquiterpene lactones, which possess a,hunsaturated carbonyl structures, such as a-methylene lactones (24).These structural elements preferably react with nucleophiles, especially sulfhydryl groups, by a Michael-type addition.To test the importance of such an unsaturated a, h-lactone, we synthesized dihydroparthenolide and assayed it for TCP inhibition.Dihydroparthenolide did not show any activity (Fig. 5B), indicating that the unsaturated a, h-lactone is in fact necessary for TCP inhibitory activity.However, we found that the presence of the unsaturated a, h-lactone, even if it is necessary, is not sufficient for TCP inhibition.For instance, costunolide showed no TCP inhibitory activity (Fig. 5C).Costunolide possesses an unsaturated a, h-lactone and differs from parthenolide by the lost of an epoxide function.This underlies the importance of the epoxide function in TCP inhibitory activity.The replacement of this epoxide function by an alcohol (cnicin) leads to a partial loss of activity (Fig. 5D).
We tested the cytotoxicity of parthenolide and of the compounds V18 and V19.HeLa cells were exposed to increasing concentrations of these molecules, and cytotoxicity was measured after 48 h using MTT assay.We found that parthenolide, V18, and V19 had similar cytotoxic properties with an IC 50 of 8, 3.5, and 7.1 Amol/L, respectively.
Does the observed parthenolide inhibition of TCP activity result from NF-KB inhibition?One well-described activity of parthenolide is its potent anti-inflammatory effect, which is mainly achieved through its inhibitory effects on NF-nB activation.Thus, the observed TCP inhibition by parthenolide could actually be an indirect result of NF-nB inhibition.Costunolide, an also known NF-nB inhibitor, has no effect on TCP activity, indicating that TCP inhibition does not result from NF-nB inhibition.However, this conclusion remains uncertain due to the different NF-nB inhibitory potencies of parthenolide and costunolide (24).To get insight into that question, we tested the effect of sulindac, an inhibitor of NF-nB activation (38,39) that is not structurally related to parthenolide or costunolide, on TCP activity.We found that 1 mmol/L sulindac has no inhibitory effect on the generation of Glu-tubulin induced by ID3 microinjection (data not shown).This result strongly shows that parthenolide inhibition of TCP activity is independent of NF-nB inhibition.
Specific action of parthenolide on TCP.In vitro, CPA is able to excise the COOH-terminal tyrosine of tubulin as well as does TCP in cells (40).We tested if parthenolide could also inhibit CPA using a cell-based assay of CPA activity.In such an assay, the effect of the application of exogenous CPA on the cellular microtubule network is observed by the generation of Glu-tubulin in permeabilized cells.The buffer used for the permeabilization preserves microtubule network (28).In the absence of CPA, the microtubule network is predominantly composed of Tyr-tubulin, whereas Glu-tubulin is almost absent (Fig. 6A).
After an exposition of 15 min to CPA, the Glu-tubulin staining is largely enhanced, extending to the entire microtubule network (Fig. 6B).This augmentation is prevented when orthophenanthroline is added to cells in combination with CPA (Fig. 6C).Orthophenanthroline is a known CPA inhibitor with no effect on TCP (12).When cells are exposed with CPA in combination with parthenolide, the Glu-tubulin staining is the same as the Glutubulin staining observed with CPA alone (Fig. 6D).Although parthenolide is a reactive compound because of its unsaturated a, h-lactone, this result shows that it has no effect on CPA even at the highest concentration (1 mmol/L) tested.

Discussion
By screening a natural extract library on a cell-based assay of TCP activity, we have isolated two structurally related sesquiterpene lactones that produce a strong inhibition on cellular TCP activity.
When searching for therapeutic agents, cell-based assays are particularly valuable because they select not only for activity against a particular molecular target but also for other desirable properties, such as the ability to permeate cells and to retain activity in tissue culture medium and in cells (23).Moreover, by screening for inhibitors on the entire cell, multiple potential targets, direct or indirect, are tested, allowing the cell to dictate the best target.
Although the screen that we set up could lead to the selection of some false positives, the overall strategy was discriminative and led to the isolation of only two compounds.This is noteworthy as regards the high number of compounds tested.We have screened extracts, which could be considered as mixtures of natural substances.Thus, the true number of different compounds tested is much higher than 23,904 (i.e., the number of extracts tested).
We tested several commercially available analogues of the compounds that were isolated by the screening procedure and found that parthenolide, but not the closely related compound costunolide, has also a potent inhibitory effect on TCP.Parthenolide, as well as other sesquiterpene lactones, contains an a-methylene-h-lactone moiety, which is highly reactive with cellular thiols, resulting in alkylation of sulfydryl residues through Michael-type addition.We found that this a-methylene-h-lactone moiety was indispensable for the inhibitory effect of parthenolide on TCP.If the observed inhibitory effect of parthenolide is the consequence of a direct reaction of parthenolide with TCP, this result indicates that TCP could belong to the class of cysteine proteases.Moreover, as parthenolide has no effect on CPA and as known metalloproteinases inhibitors have no effect on TCP (12), it seems that TCP is not a metalloproteinase.
Numerous bioactivities, probably as a consequence of its high reactivity, have been described for parthenolide and other related sesquiterpene lactones.These bioactivities include, for example, anti-inflammatory effect through inhibition of NF-nB activation (41) and signal transducers and activators of transcription signaling pathway (42) or induction of apoptosis through several different mechanisms, such as caspase activation and mitochondrial dysfunction (43,44).Contrary to the panel of actions described for sesquiterpene lactones, TCP inhibition looks to be a specific action of parthenolide and of the two Vernonia compounds selected.
We have shown that the inhibitory effect of parthenolide on TCP does not seem to result from the parthenolide action on NF-nB pathway.However, we cannot exclude that TCP inhibition is an indirect consequence of an action of parthenolide on another known or unknown target.For example, it cannot be excluded that parthenolide inhibits regulating enzymes, such as kinases or phosphatases.It has been shown that inhibitors of serine/ threonine phosphatases seem to dissociate TCP from microtubules (45).Such an inhibition could thus represent an indirect form of TCP inhibition.
Our initial aim was to use the inhibitor selected by the screening procedure as a hook, in an attempt to isolate TCP, as it has been successfully done in some chemical genetic approaches.However, because of its high reactivity and its numerous targets, parthenolide is no suitable for such a strategy.We are currently testing other chemical libraries in an attempt to isolate a compound more convenient for TCP purification.
The anticancer potential of parthenolide has been described both in vitro (43,(46)(47)(48)(49) and on animal models (44,50).Moreover, it has been shown that parthenolide could reduce metastasis (50).We believe that TCP inhibition, which could antagonize the abnormal accumulation of detyrosinated tubulin in tumor cells, could be one of the mechanisms underlying the anticancer and antimetastatic properties of parthenolide.Because parthenolide has shown safety in phase I clinical trials, our results have direct clinical relevance.

Figure 1 .
Figure 1.Schematic illustration of the different steps of the cell-based assay of TCP activity used for high-throughput screening of TCP inhibitors.TCP activity on paclitaxel-stabilized microtubules is measured by quantifying the production of detyrosinated tubulin using a specific antibody and an immunoluminescence procedure as described in Materials and Methods.The inhibitory effect of active natural extracts is detected by a decrease of the luminescent signal.

Figure 2 .
Figure 2. Immunofluorescence analysis of the effect on paclitaxel-induced tubulin detyrosination of natural extract fractions selected by the high-throughput screening assay.HeLa cells were incubated for 2 h with DMSO (A ), 5 Amol/L paclitaxel (B), or 5 Amol/L paclitaxel combined with an active fraction (C ).Cells were then fixed, double stained with Tyr-tubulin and Glu-tubulin antibodies as indicated, and processed for immunofluorescence as described in Materials and Methods.

Figure 4 .
Figure 4.Chemical structures of the two compounds (V18 and V19), isolated from V. perrottetii , and of the 17 structural analogues tested.

Figure 3 .
Figure3.Analysis of the effect of natural extract fractions on TCP activity revealed by accumulation of Glu-tubulin after ID3 microinjection.HeLa cells were microinjected with the TTL inhibitory antibody ID3, incubated for 4 h with (A) or without (B ) a TCP-inhibiting fraction, fixed, and processed for immunofluorescence as described in Materials and Methods.Cells were triple stained using an antimouse Cy3-conjugated antibody to mark ID3 microinjected cells, L4 antibody and Alexa Fluor 488-conjugated secondary antibody to visualize Glu-tubulin, and Hoechst 33258 to visualize nuclei.In control cells (A), Glu-tubulin is generated in the microinjected cells and not in a nonmicroinjected cell (arrow ), which can be localized thanks to Hoechst 33258 staining.B, example of a fraction showing an inhibitory effect on TCP activity.In the presence of such a fraction, the level of Glu-tubulin is as low in microinjected cells as in nonmicroinjected cells (arrows ).

Figure 5 .
Figure 5.Comparison of the effect of several structural analogues of V18 and V19 on TCP activity evaluated after ID3 microinjection.HeLa cells were microinjected with the TTL inhibitory antibody ID3 and incubated for 4 h with the different compounds as indicated.They were then fixed and processed for immunofluorescence as described in Materials and Methods.Cells were triple stained using an antimouse Cy3-conjugated antibody to mark ID3 microinjected cells, L4 antibody and Alexa Fluor 488-conjugated secondary antibody to visualize Glu-tubulin, and Hoechst 33258 to visualize nuclei as indicated.

Figure 6 .
Figure 6.Test of the effect of parthenolide on CPA.Permeabilized cells were exposed to buffer alone (A) or to CPA (B-D ) alone (B) or in combination with orthophenanthroline (C ) or with parthenolide (D ) as described in Materials and Methods.After 15 min of exposition, cells were fixed and processed for immunofluorescence.Cells were double stained with Tyr-tubulin antibody and Glu-tubulin antibody as indicated.