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[Cancer Research 63, 3211-3220, June 15, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Taccalonolides E and A

Plant-derived Steroids with Microtubule-stabilizing Activity1

Tina L. Tinley, Deborah A. Randall-Hlubek, Rachel M. Leal, Evelyn M. Jackson, James W. Cessac, James C. Quada, Jr.2, Thomas K. Hemscheidt and Susan L. Mooberry3

Departments of Physiology and Medicine [T. L. T., D. A. R-H., R. M. L., E. M. J., J. C. Q., S. L. M.] and Organic Chemistry [J. W. C.], Southwest Foundation for Biomedical Research, San Antonio, Texas 78227, and Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822 [T. K. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the course of a mechanism-based screening program designed to identify new microtubule-disrupting agents from natural products, we identified a crude extract from Tacca chantrieri that initiated Taxol-like microtubule bundling. Bioassay-directed purification of the extract yielded the highly oxygenated steroids taccalonolides E and A. The taccalonolides caused an increased density of cellular microtubules in interphase cells and the formation of thick bundles of microtubules similar to the effects of Taxol. Mitotic cells exhibited abnormal mitotic spindles containing three or more spindle poles. The taccalonolides were evaluated for antiproliferative effects in drug-sensitive and multidrug-resistant cell lines. The data indicate that taccalonolide E is slightly more potent than taccalonolide A in drug-sensitive cell lines and that both taccalonolides are effective inhibitors of cell proliferation. Both taccalonolides are poorer substrates for transport by P-glycoprotein than Taxol. The ability of the taccalonolides to circumvent mutations in the Taxol-binding region of ß-tubulin was examined using the PTX 10, PTX 22, and 1A9/A8 cell lines. The data suggest little cross-resistance of taccalonolide A as compared with Taxol, however, the data from the PTX 22 cell line indicate a 12-fold resistance to taccalonolide E, suggesting a potential overlap of binding sites. Characteristic of agents that disrupt microtubules, the taccalonolides caused G2-M accumulation, Bcl-2 phosphorylation, and initiation of apoptosis. The taccalonolides represent a novel class of plant-derived microtubule-stabilizers that differ structurally and biologically from other classes of microtubule-stabilizers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microtubules are dynamic intracellular structures that play a key role in cellular metabolism, intracellular transport, and mitosis. Microtubule poisons like the Vinca alkaloids and colchicines have been known for decades for their depolymerizing effects on microtubules. In 1980, the mechanism of action of the cytotoxic antitumor agent Taxol was discovered (1) . Taxol, unlike other microtubule-interacting agents previously identified, promotes the polymerization of tubulin into microtubules. In cells, this action of Taxol causes the formation of thick microtubule bundles throughout the cytoplasm. Relatively high concentrations of Taxol are required for these effects and at lower concentrations Taxol suppresses microtubule dynamics without effecting tubulin polymer mass (2) . Disruption of microtubule dynamics interrupts normal microtubule functions, including mitosis, and ultimately leads to the initiation of apoptosis (3 , 4) .

Taxol is a clinically effective anticancer agent and represents perhaps the most significant addition to the pharmacopoeia of cancer chemotherapeutic agents in the last decade (5) . Prompted by the clinical successes of the taxanes, TaxolTM (paclitaxel) and TaxotereTM (docetaxel), significant efforts have been focused on identifying new agents that have a similar mechanism of action yet superior properties such as agents that are poor substrates of Pgp4 -mediated transport and exhibit better solubility in aqueous solutions. These efforts have been successful. Several structurally unrelated classes of microtubule-stabilizing agents have been identified. The epothilones were isolated from the mycobacterium Sporangium cellulosum and are effective against multidrug-resistant cells (6 , 7) . Epothilone B and an analogue, desoxyepothilone B, have antitumor activity in vivo (reviewed in Ref. 8 ). To date, the marine environment has proven to be the richest source for new microtubule-stabilizing agents, yielding discodermolide (9 , 10) , eleutherobin (11) , the sarcodictyins (12) , the laulimalides (13) , and peloruside A (14) . These classes of compounds are structurally unrelated and discodermolide, the epothilones, and eleutherobin competitively displace Taxol, suggesting overlap with the Taxol-binding site (7 , 10 , 12) . In contrast, laulimalide does not displace Taxol, and cell lines with mutations in the Taxol-binding site are not cross-resistant to laulimalide, suggesting that it has a different binding site on tubulin (15) . Whether these new microtubule-stabilizers have clinical efficacy has yet to be determined.

We recently identified a new class of microtubule-stabilizing agents, the taccalonolides. Taccalonolide A was first isolated from Tacca plantaginea by Chen et al. (16) in 1987 and was reported to have cytotoxic activity against P-388 in vitro. Taccalonolide E was isolated in 1991 (17) . We present data showing that these compounds are the first plant-derived microtubule-stabilizing agents to be identified since Taxol and the first natural steroids to exhibit this activity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Collection and Identification.
The roots of Tacca chantrieri were originally collected from the Lyon Arboretum of the University of Hawaii. A voucher specimen was used to identify the sample and was deposited in the University of Hawaii’s herbarium. Other root collections were from potted T. chantrieri plants grown in greenhouses.

Chemical Purification and Identification of Taccalonolides E and A.
Roots were collected from the rhizomes of live plant specimens of T. chantrieri, cut into 2-cm pieces, and frozen at -80°C until processed. The root material was freeze-dried, then quickly ground to a fine powder with mortar and pestle. The powder was extracted three times with dichloromethane/isopropanol [7/3 (v/v)] for 24 h with gentle agitation at room temperature. The solids were filtered off, and the combined organic extract was evaporated to dryness in vacuo. The residue was dissolved in methanol/water [9/1 (v/v)] and extracted three times with equal volumes of hexanes. The methanol phase was diluted with water to yield an 80% aqueous methanol solution and then extracted three times with equal volumes of toluene. The toluene extracts were combined and evaporated to dryness in vacuo. The residue was dissolved in a small amount of dichloromethane and applied to a silica gel column equilibrated in diethyl ether, followed by elution with diethyl ether. Active fractions were identified by bioassay, combined, and evaporated in vacuo. The residue was additionally purified on a silica gel column equilibrated in 10% ethyl acetate in diethyl ether followed by elution with 10% ethyl acetate in diethyl ether. The material was additionally purified by high-performance liquid chromatography using a C18 radial compression module with detection by UV absorbance at 205 nm, yielding a peak containing taccalonolides E and A, as defined by analysis of proton NMR data. Taccalonolides E and A were separated by preparative thin layer chromatography using diethyl ether or by additional high-performance liquid chromatography. The compound present in each band or peak was identified by comparison of the proton NMR spectra with published data (16 , 18) .

Taccalonolides E and A were solubilized in 100% ethanol, and the stock solutions were stored at -20°C with no evidence of chemical degradation over time.

Cell Culture.
A-10 cells (an embryonic rat aortic smooth muscle cell line), SK-OV-3 cells (a human ovarian carcinoma cell line), and HeLa cells (a human cervical carcinoma cell line) were purchased from American Type Culture Collection (Manassas, VA). The A-10, HeLa, and SK-OV-3 cells were grown in Basal Medium Eagle containing Earle’s salts, 50 µg/ml gentamicin, and 10% fetal bovine serum (Hyclone, Logan, UT). The human breast adenocarcinoma line MDA-MB-435 was obtained from the Lombardi Cancer Center (Georgetown University, Washington, DC). MDA-MB-435 cells were maintained in IMEM (Richter’s Medium; Biosource, Camarillo, CA) with 10% fetal bovine serum and 25 µg/ml gentamicin. The NCI/ADR cell line, which was previously called MCF7/ADR (19) , was obtained from the NCI (Bethesda, MD). The drug-sensitive parental cell line 1A9 and the Taxol- and epothilone A-resistant PTX 10, PTX 22, and 1A9/A8 cell lines were obtained from Dr. Evi Giannakakou, Winship Cancer Institute (Emory University School of Medicine, Atlanta, GA). The NCI/ADR, 1A9, and 1A9/A8 cell lines were maintained in RPMI 1640 (Biosource, Camarillo, CA) containing 50 µg/ml gentamicin and 10% fetal bovine serum. The PTX 10 and PTX 22 cell lines were grown in RPMI 1640 containing 50 µg/ml gentamicin, 10% fetal bovine serum, 15 ng/ml Taxol, and 5 µg/ml verapamil to retain selection pressure. The cells used in experiments were grown without drugs for 1 week.

Indirect Immunofluorescence.
A-10 and HeLa cells were grown on glass coverslips and then treated with drugs as described previously (13) . After incubation with drugs, the cells were fixed with methanol (4°C) for 5 min, blocked for 20 min with 10% calf serum in calcium- and magnesium-free PBS, and incubated for 60–90 min with monoclonal ß-tubulin antibody (T-4026; Sigma, St. Louis, MO) and a polyclonal antibody to {gamma}-tubulin (T-3559; Sigma). After a series of washes, the cells were incubated with FITC-conjugated sheep antimouse IgG (F-3008; Sigma) and Texas Red-conjugated goat antirabbit IgG (T-6391; Molecular Probes, Eugene, OR) for 1 h. The coverslips were washed, stained with 0.1 µg/ml DAPI, and examined and photographed using a Nikon ES800 fluorescence microscope with a digital camera. The images were colorized and compiled using MetaMorph software.

Inhibition of Cell Proliferation.
The SRB assay was used to evaluate the ability of the taccalonolides and Taxol to inhibit cellular proliferation of drug-sensitive and drug-resistant cells (20 , 21) . Absorbance of the SRB solution was read at 560 nm for maximum assay sensitivity. The IC50s were determined by least squares analysis and regression of the linear portion of the log-dose response curves. An IC50 was determined for each experiment, and the data are expressed as the means of three experiments ± SD.

Cell Cycle Analysis.
MDA-MB-435 cells were treated with the IC85 concentration of taccalonolide E or A or vehicle for 6, 12, 18, or 24 h. The cells were fixed in 70% ethanol, treated with RNase A, and stained with propidium iodide as previously described (22) or analyzed fresh and stained with Krishan’s reagent (23) . The DNA content was analyzed using a Becton Dickinson FACScan flow cytometer, and the data were plotted as number of events versus propidium iodide fluorescence intensity. Quality control standards were run before each experiment to assess the instrument’s linearity and resolution.

Immunoblot Analysis.
MDA-MB-435 cells were treated with taccalonolide E or A at the IC85 concentration for inhibition of proliferation for 0–30 h. After drug exposure, the cells were harvested and cellular proteins extracted in modified radioimmunoprecipitation buffer in the presence of protease inhibitors as described previously (22) . The protein concentrations of the samples were determined with Coomassie Plus (Pierce, Rockford, IL) and equal protein loading for each set of lysates confirmed by Simply Blue protein stain. Cell lysate aliquots containing equal amounts of protein were separated by SDS-PAGE, transferred, probed with specific antibodies, and detected as described previously (13) . The p85 PARP fragment antibody, which is specific for the caspase cleaved Mr 85,000 fragment of PARP, was obtained from Promega (Madison WI), the Bcl-2 antibody was purchased from PharMingen (San Diego, CA), and the phospho-ERK1/2 (p44/42) MAPK, phospho-p38 MAPK, and p116 PARP antibodies were from Cell Signaling (Beverly, MA).

In Vivo Tubulin Assembly.
A quantitative measure of the ability of taccalonolide E and Taxol to stimulate the formation of tubulin polymers in vivo was used (24 , 25) . In this assay, MDA-MB-435 cells in log-phase growth were treated with taccalonolide E, Taxol, or vehicle (ethanol) for 1 h, and after incubation, the cells were lysed in hypotonic buffer containing protease inhibitors and the cell constituents separated by centrifugation at 14,000 rpm for 10 min at room temperature in a table-top microcentrifuge. After centrifugation, the supernatants containing soluble, cytosolic tubulin were removed and the pellets, containing particulate fractions, including polymerized, cytoskeletal tubulin, were resuspended in buffer. The cytosolic, soluble (S) and particulate (P) fractions were diluted into 2x sample buffer. The proteins were resolved by SDS-PAGE, transferred to Immobilon, and immunoblotted with a ß-tubulin antibody.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Taccalonolides on Interphase Microtubules.
The crude lipophilic extract of the herbaceous tropical plant T. chantrieri caused dramatic reorganization of cellular microtubules reminiscent of the effects of Taxol. Bioassay-directed purification of the sample yielded the hexacyclic steroids taccalonolides E and A (Fig. 1)Citation . These two taccalonolides differ only in the acetate group at the C-11 position. Studies with the purified compounds showed that they both caused Taxol-like reorganization of microtubules in interphase and mitotic cells.



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  		Fig. 1. Chemical structures of taccalonolides E and A.

 
The effects of the taccalonolides on cellular microtubules were examined in detail in A-10 cells. The A-10 cell line is an embryonic smooth muscle cell line that is used because they are relatively large, flat, and have easily visualized interphase and mitotic microtubules. Cells were treated with either taccalonolide A, E, or Taxol for 18 h, and microtubules and centrosomes were visualized by indirect immunofluorescence techniques. An antibody for ß-tubulin was used to visualize interphase and mitotic microtubule structures and an antibody for {gamma}-tubulin to visualize the centrosomes. Vehicle-treated cells exhibited a normal filamentous microtubule array, with microtubules radiating from the centrosomes in the central regions of the cell to the cell periphery (Fig. 2A)Citation . Although the microtubule network is extensive, it does not occupy the entire cytosol. Taccalonolide E, at a concentration of 1 µM, caused an increase in the density of cellular microtubules, and higher concentrations, beginning at 5 µM, caused the cytoplasm to be filled with microtubules and in the majority of cells, the formation of thick bundles of microtubules (Fig. 2B)Citation . These bundles of microtubules vary in length, some are quite short, and others are longer (Fig. 2B)Citation . Cells treated with 10 µM taccalonolide E exhibited total rearrangement of the microtubule network into short bundles or tufts of microtubules, many of which nucleate independent of the centrosomes (Fig. 2C)Citation . The majority of cells contained two centrosomes, however, a few cells with four centrosomes were present in treated interphase cells. The microtubule bundles in taccalonolide-treated cells were much shorter than the bundles found in Taxol-treated cells (Fig. 2D)Citation . The effects of taccalonolide A on interphase microtubules were identical to the effects of taccalonolide E (data not shown). Although the effects of the taccalonolides on microtubules are similar to the effects of Taxol (Fig. 2D)Citation , the microtubules in Taxol-treated cells consistently appear to fill more of the cytoplasm and form longer bundles of microtubules in contrast to the short tufts of microtubules that are found in taccalonolide-treated cells.



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  		Fig. 2. Effects of taccalonolide E and Taxol on cellular microtubule organization. Cellular microtubules were visualized in A-10 cells by indirect immunofluorescence techniques using a monoclonal ß-tubulin antibody after an 18-h incubation with vehicle control, ethanol (A), 5 µM taccalonolide E (B), 10 µM taccalonolide E (C), or 10 µM Taxol (D). The images are representative of three experiments.

 
Effects of the Taccalonolides on Mitotic Spindles and Mitotic Progression.
A common characteristic of antimicrotubule agents is their ability to disrupt normal mitosis because of inhibition of the highly dynamic mitotic spindles. The effects of the taccalonolides on mitotic spindles were examined in A-10 and HeLa cells. In both cell lines, taccalonolide E and A caused the appearance of abnormal multipolar mitotic spindles. In untreated or vehicle-treated A-10 cells, the mitotic spindles are bipolar (Fig. 3A)Citation , and normal metaphase alignment of the DNA (Fig. 3BCitation , blue color) and the presence of a centrosome (Fig. 3BCitation , yellow color) in each of the bipolar spindle poles can be observed. After treatment with 1 µM taccalonolide E, the formation of abnormal spindle poles was observed (Fig. 3C–F)Citation . Some cells had three or four mitotic spindle pole structures (Fig. 3C)Citation , and others had five or more spindle poles (Fig. 3D)Citation . It is interesting to note that although there are numerous mitotic spindle poles, only two centrosomes, as detected by {gamma}-tubulin (Fig. 3, D and FCitation , yellow color), were present in most cells. Abnormal alignment of the DNA was apparent, and normal metaphase alignment of the DNA in the equator of the cell was not observed in any of the cells with multipolar spindles. The presence of three or more mitotic spindle poles was observed with a 1 µM concentration of taccalonolide E and with increasing concentrations, a higher percentage of mitotic cells had more than two spindle poles. Taxol- and taccalonolide A-treated A-10 cells exhibited similar effects. The number of mitotic spindle poles in mitotic cells were counted and plotted against taccalonolide E concentration, and the data are presented in Fig. 4Citation . All vehicle-treated cells and cells treated with 0–500 nM taccalonolide E exhibited normal bipolar spindle poles. At a concentration of 1 µM taccalonolide E, the majority of mitotic cells had three or more spindle poles (Fig. 4)Citation , and at a 5 µM concentration, <12% of the mitotic cells contained a bipolar spindle and 70% had five or more spindle poles. These abnormal mitotic spindles appeared ineffective in aligning the DNA in metaphase. The effects of taccalonolide E on mitotic spindles were also evaluated in HeLa cells with similar results (data not shown).



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  		Fig. 3. Effects of taccalonolide E on mitotic spindles. Mitotic spindles were visualized in A-10 cells by indirect immunofluorescence after an 18-h treatment with drug vehicle (A and B) or 1 µM taccalonolide E (C–F). The images were captured with a digital camera and colorized (A, C, E) and images from three different emission filters overlaid (B, D, F). The green structures are microtubules detected by FITC-ß-tubulin, the yellow structures by dual staining of structures with green (FITC-ß-tubulin) and red (Texas Red {lambda}-tubulin), and the blue represents DAPI staining. The images are representative of three experiments.

 


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  		Fig. 4. The concentration-dependent effects of taccalonolide E on mitotic spindle poles. The numbers of mitotic spindle poles were counted in A-10 cells treated with taccalonolide E. The entire coverslip was examined and all mitotic figures included. The data expressed are the means from two experiments.

 
To determine whether the taccalonolide-initiated formation of abnormal mitotic spindles caused cells to accumulate in the G2-M phase of the cell cycle, the effects of the taccalonolides on cell cycle progression were examined by flow cytometry. Asynchronous populations of MDA-MB-435 cells in log-phase growth were treated with 5 µM taccalonolide E, the IC85 for inhibition of proliferation or vehicle. Normal cell cycle distribution was observed in the vehicle-treated MDA-MB-435 cells (Fig. 5)Citation , and mitotic accumulation was evident within 6 h of taccalonolide E treatment. Additional mitotic accumulation occurred at 12 h with complete loss of the G1 peak (Fig. 5)Citation . Mitotic accumulation was complete by 24 h, and there was no evidence of release from mitotic blockade. After 12 h of treatment, a population of cells, <5%, with 8n DNA was observed and maintained at 24 h. This suggests that a small population of cells had successful mitosis, as indicated by DNA content, but did not undergo cytokinesis. The appearance of the sub-G1 peak, beginning at 12 h and amplifying by 24 h, suggests that the cells were undergoing apoptotic cell death (26) . Mitotic accumulation was also observed in taccalonolide A-treated cells (data not shown).



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  		Fig. 5. Cell cycle distribution of cells treated with taccalonolide E. MDA-MB-435 cells in log-phase growth were treated with vehicle or 5 µM taccalonolide E for 6, 12, or 24 h and then stained with Krishan’s reagent and analyzed by flow cytometry.

 
Taccalonolides Initiate the Formation of Micronuclei.
Taxol and laulimalide cause the breakdown of the nucleus into multiple micronuclei (13) . Untreated or vehicle-treated A-10 cells have an intact nucleus as visualized with the DNA binding agent DAPI (Fig. 6A)Citation . Cells treated with taccalonolide E exhibited abnormal nuclear organization, beginning with convolution of the nucleus and leading to the breakdown of the nucleus into multiple micronuclei (Fig. 6B)Citation . The breakdown of the nucleus appears to occur from the inside out to the periphery of the nucleus, as nuclei with holes in the middle are sometimes observed (Fig. 6BCitation , lower left side). This nuclear configuration, the lack of condensed chromatin, and interphase microtubule arrays suggests that micronucleation is occurring in interphase cells. These effects were seen after treatment with taccalonolide A and micronucleation also occurred with both taccalonolides in HeLa cells (data not shown). These nuclear changes appear to be independent of mitotic arrest in both cell types. The percentage of cells containing micronuclei were calculated and the results shown in Fig. 6CCitation . Very few cells with micronuclei were observed in vehicle-treated cells or cells treated with 0–500 nM taccalonolide E. Concentrations of taccalonolide E of 1–5 µM caused ~15% of the cells to contain micronuclei, and higher concentrations of taccalonolide E did not cause a greater percentage of micronucleation but did cause noticeable cell loss. The ability of taccalonolide E to initiate micronucleation in interphase cells is similar to the effects of both Taxol and laulimalide (13) . Whether micronucleation induced by the taccalonolides in A-10 cells involves apoptotic or nonapoptotic cell death has not yet been determined.



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  		Fig. 6. Effects of taccalonolide E on nuclear structure. A-10 cells were treated for 18 h with vehicle control (A) or 10 µM taccalonolide E (B) and DNA visualized by DAPI staining. The percentages of cells containing micronuclei were counted in 10 microscope fields/slide (C). The data are representative of the effects observed in three experiments.

 
Effects of Taccalonolides on the Proliferation of Drug-sensitive and Multidrug-resistant Cell Lines.
The SRB assay was used to determine efficacy, maximal effect, and potency of taccalonolides E and A against the drug-sensitive cell lines SK-OV-3 and MDA-MB-435 and the drug-resistant cell line NCI/ADR, which has high levels of expression of Pgp (27) . The results presented in Table 1Citation show that taccalonolide E has an IC50 of 0.78 and 0.99 µM against the two drug-sensitive cell lines, and taccalonolide A is slightly less potent, with IC50s of 2.6 µM for the two drug-sensitive cell lines. Both taccalonolides are significantly less potent than Taxol. Although not as potent as Taxol, both taccalonolides are equally effective inhibitors of proliferation, i.e., both agents were able to completely inhibit cell proliferation and initiate cell death in the two drug-sensitive cell lines. One limitation of Taxol is that it is a good substrate for transport by the drug efflux protein Pgp. The ability of the taccalonolides and Taxol to inhibit proliferation in the multidrug-resistant NCI/ADR was evaluated, and relative resistance factors for each of the drugs was calculated by dividing the IC50 for inhibition of proliferation in the NCI/ADR cell line by the IC50s obtained in the MDA-MB-435 drug-sensitive cell line. In the presence of verapamil, the IC50s for inhibition of proliferation of the NCI/ADR cells are equal to the IC50s of this drug-sensitive cell line (28) . The calculated resistance factors for Taxol are 827, for taccalonolide E they are 27, and for taccalonolide A they are 10.7. The dose-response curves show that although the taccalonolides differ in potency, both are effective inhibitors of proliferation and initiate cytotoxicity in this multidrug-resistant cell line. These data suggest that the taccalonolides are poorer substrates for transport by Pgp than Taxol.


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  		Table 1 Effects of taccalonolides E and A and Taxol on cellular proliferation of drug-sensitive and a multidrug-resistant cell line

Cells were treated with a range of concentrations of the various drugs for 48 h, and the inhibition of proliferation was determined using the SRB assay. The values represent the means of three experiments ± the SD.

 
The effects of the taccalonolides against Taxol- and epothilone-resistant cell lines were evaluated to provide information about potential overlap of the cellular-binding sites of the taccalonolides with the Taxol- and epothilone-binding site on ß-tubulin. The Taxol-resistant cell lines, PTX 10 and PTX 22, and the epothilone-resistant cell line, 1A9/A8, were used for these studies. These cell lines are resistant because of single point mutations in the human M40 ß-tubulin isotype in the Taxol-binding region (24 , 25) . The IC50s for inhibition of proliferation were calculated for each of the drugs in the resistant cell lines and compared with values obtained in the parental 1A9 cell line. Resistance factors were calculated by dividing the IC50s measured in the resistant cell lines by the IC50s obtained in the parental cell line. The data, presented in Table 2Citation , indicate that the Taxol-resistant cell lines are substantially less resistant to the taccalonolides as compared with Taxol, but interesting differences were noted. The resistance factors for taccalonolide E were 4.8 and 4.2 for the PTX 10 and 1A9/A8 cell lines, respectively, yet a resistance factor of 12 was calculated for this drug in the PTX 22 cell line. The PTX 10, PTX 22, and 1A9/A8 have resistance factors of ~2–3 for taccalonolide A. Differences in the resistance factors for the PTX 22 cell line were noted between the taccalonolides, suggesting the possibility of subtle differences in binding of the two drugs in the region of Ala364, which is mutated to threonine in the PTX 22 cell line (24) .


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  		Table 2 The IC50s and relative resistance for taccalonolides and Taxol in Taxol and epothilone-resistant cell lines

 
Effects of Taccalonolide E on in Vivo Microtubule Polymerization.
The ability of taccalonolide E to initiate the polymerization of tubulin was evaluated using an in vivo tubulin polymerization assay. Cells were treated for 1 h with taccalonolide E or Taxol, lysed in hypotonic buffer, and polymerized and soluble tubulin separated by centrifugation. The results of these experiments are presented in Fig. 7Citation . In vehicle-treated cells, the immunoblot shows that the amount of soluble tubulin was slightly greater than the amount of polymerized tubulin. The amount of soluble tubulin decreased, and the amount of polymerized tubulin increased in response to exposure of the cells to either taccalonolide E or Taxol (Fig. 7)Citation . Treatment with a 10 µM concentration of taccalonolide E caused the band containing soluble or cytosolic tubulin to be undetectable, whereas a large increase in polymerized, cytosolic tubulin was observed. Similar effects were observed with Taxol. These effects are consistent with the observations obtained using immunofluorescence techniques, where an increased density of cellular microtubules was apparent in both taccalonolide- and Taxol-treated cells. These results suggest that taccalonolide E, like Taxol, causes the in vivo polymerization of tubulin, an effect common to microtubule stabilizers.



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  		Fig. 7. Drug-induced tubulin polymerization in vivo. MDA-MB-435 cells were treated with taccalonolide E or Taxol for 1 h, and soluble (S) and particulate (P) cellular constituents were separated by centrifugation. Samples were separated by PAGE, transferred, and immunoblotted with a ß-tubulin antibody.

 
Taccalonolide Initiates Bcl-2 Phosphorylation, MAPK Activation, and Apoptosis.
Bcl-2 phosphorylation is a common characteristic of antimitotics and is considered to be a marker of mitotic arrest (29) . The ability of taccalonolide E to initiate the cascade of events leading to Bcl-2 phosphorylation was evaluated by immunoblotting techniques using cell lysates of MDA-MB-435 cells treated for 4–30 h with 5 µM taccalonolide E, the IC85 for inhibition of proliferation. The appearance of the characteristically slower migrating form of Bcl-2, consistent with its phosphorylation, was detected by immunoblotting techniques after 4 h of treatment and was sustained through 30 h of treatment (Fig. 8)Citation . Interestingly, although many microtubule-disrupting agents cause the appearance of two slower migrating bands, suggesting multiple phosphorylation states, this was not observed with taccalonolide E, indicating the possibility of subtle differences in Bcl-2 phosphorylation among the microtubule-interacting drugs. The phosphorylation of Bcl-2 also occurred after treatment of MDA-MB-435 cells with taccalonolide A. These data suggest that the taccalonolides initiate a sequence of cellular events leading to the phosphorylation of Bcl-2, consistent with the effects of other microtubule-interacting agents.



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  		Fig. 8. Effect of taccalonolide E on Bcl-2, ERK1/2, and p38 activation and PARP cleavage. MDA-MB-435 cells were treated with vehicle control or 5 µM taccalonolide E for 4, 6, 12, 24, or 30 h, and cell lysates were prepared in the presence of protease inhibitors. Samples containing equal amounts of protein were separated using SDS-PAGE, transferred to blots, and probed with Bcl-2 antibody, phospho-ERK1/2, phospho-p38 MAPK, or p85-cleaved PARP antibodies.

 
The MAPK pathways are activated in cells treated with Taxol (30, 31, 32) . The effect of taccalonolide E on activation of MAPK-signaling pathways was evaluated using phospho (activation)-specific antibodies for ERK1/2 (p44/42) and p38 MAPK. Cell lysates were made from cells treated for various times with 5 µM taccalonolide E, and the activation of specific proteins was evaluated using immunoblotting techniques. Early, modest activation of ERK1/2 was observed (Fig. 8)Citation , with greater activation of ERK2 (p42) as compared with the ERK1 (p44). This effect was previously detected with Taxol in A549 and CHO cells (30 , 33) . There was no change in the activation of p38 MAPK at the early time points, yet at 30 h an increase in the phospho-p38 was detected, indicating that it may be involved in signaling events late in apoptosis but not in the initiation of apoptosis. These results suggest that taccalonolide E causes activation of the MAPK-signaling pathways consistent with the effects of Taxol.

Taccalonolide E appeared to initiate apoptosis as indicated by the detection of a sub-G1 peak by flow cytometry after 12, 24 (Fig. 5)Citation , and 30 h treatments (data not shown). Additional studies were conducted to determine whether taccalonolide E initiated activation of the caspase cascade of proteinases that are responsible for the execution phase of apoptosis. Caspase 3 activation is used as a marker for late events in apoptosis (34) . One of the downstream targets of activated caspase 3 is the Mr 116,000 enzyme PARP (35) , which is cleaved into two fragments of Mr 85,000 and Mr 24,000. Cell lysates were prepared from MDA-MB-435 cells treated with 5 µM taccalonolide E (the IC85 concentration) and evaluated for activation of the enzymatic cascade over 4–30 h by detection of the p85 fragment of PARP. PARP cleavage was detected at 12 h and increased at 24 and 30 h (Fig. 8)Citation . The appearance of the cleaved p85 PARP occurred simultaneously with the loss of p116 PARP, which was diminished at 12 h and not detectable at 24 and 30 h (data not shown). These data are consistent with apoptotic cell death as defined by activation of the caspase cascade. Additionally, taccalonolide A initiated caspase 3 activation in MDA-MB-435 cells (data not shown). Taccalonolides E and A, at the IC85 concentration for inhibition of proliferation, appear to initiate apoptotic cell death in the MDA-MB-435 cell line within 24 h of treatment.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The microtubule-stabilizing activity of the crude plant extract derived from T. chantrieri was detected in a sensitive mechanism-based screening assay. Bioassay-directed purification led to the identification of the active constituents, taccalonolides E and A, which are complex, highly oxygenated steroids. The identification of the taccalonolides as natural product microtubule-stabilizing agents adds to the growing list of agents with this mechanism of action. Taccalonolides E and A share no obvious structural homology with Taxol, discodermolide, the epothilones, eleutherobin, the sarcodictyins, or the laulimalides. Two synthetic steroid derivatives, 3,17ß-diacetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10) -triene and 17ß-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10) -trien-3-ol, were recently reported to have tubulin-stabilizing effects in vitro (36) . Both steroid derivatives enhance the polymerization of tubulin and microtubule protein in vitro, but in contrast to the taccalonolides, these two steroid derivatives are not cytotoxic, and no effects on cellular microtubules were described (36) . There is little obvious structural homology between the taccalonolides and the two synthetic steroids. The taccalonolides display the usual arrangement of three six-membered rings and a five-membered ring characteristic of steroids, however, the common skeleton is modified by the cyclization of the usual side chain to form two additional rings. In contrast, the synthetic steroid derivatives share the common steroid four-ring motif and an expansion of the B-ring to seven carbon atoms (36) . All three structures share a ketone moiety on the B ring that is critically important to taxane-like effects of the synthetic steroid derivatives. With our current data, it is not possible to determine whether the taccalonolides and the steroid derivatives share a common pharmacophore. It is interesting to note although that in binding studies the synthetic steroid derivatives displaced only 20% of the [3H]Taxol (36) . The Taxol-resistant PTX 10 and PTX 22 cell lines and the epothilone-resistant 1A9/A8 cell line provide useful tools to probe the potential overlap of drug-binding sites on ß-tubulin because each of these cell lines has a point mutation in the Taxol-binding region of ß-tubulin (15 , 24 , 25) . We used these cell lines to provide clues to potential interactions between the taccalonolides and the Taxol-binding site on ß-tubulin. The data indicate that these cell lines exhibit little cross-resistance to taccalonolide A, yet some cross-resistance to taccalonolide E, suggesting the possibility of overlapping-binding sites. The Taxol- and epothilone-resistant cell lines are more resistant to taccalonolide E than they are to laulimalide, with relative resistance factors ranging from 4.2 to 12 for taccalonolide E and 1.5 to 2.4 for laulimalide (15) . A comparison of the relative-resistance factors obtained in these cell lines with taccalonolide E and epothilone B suggests that although the PTX 10 cell line exhibited similar resistance toward these two stabilizers with values of 4.8 and 4.1, respectively, the PTX 22 cell line is much more resistant to taccalonolide E with a resistance factor of 12 as compared with the value of 1.9 obtained with epothilone B (15) . Both the PTX 10 and PTX 22 cell lines are much less resistant to taccalonolide E and epothilone B than they are to Taxol. In contrast, the 1A9/A8 cell line is significantly less resistant to both taccalonolide E and Taxol, with resistance factors of 8.0 and 4.2 as compared with a 38-fold resistant to epothilone B (15) . The data indicate that any potential overlap of these drug-binding sites is complex and that additional studies on the nature of the taccalonolide-binding site are warranted.

The taccalonolides have cellular effects similar to other microtubule-stabilizing agents in that they disrupt both interphase and mitotic microtubules, leading to mitotic arrest and apoptosis. There are subtle differences between the effects of the taccalonolides and Taxol in the A-10 cells. In some cases, equal concentrations of the taccalonolides and Taxol caused identical effects, yet more commonly, the microtubules in taccalonolide-treated cells were organized into short tufts of microtubules that appeared to nucleate independent of the microtubule-organizing center. In this regard, the short bundles of microtubules in taccalonolide-treated cells resemble laulimalide-induced effects (13) . Taxol-induced microtubule bundles are generally longer and do not form the short tufts of microtubules initiated by taccalonolides and laulimalide. The in vivo tubulin polymerization experiments confirm the increased density of cellular microtubules observed by indirect immunofluorescence techniques. Taccalonolide E, like Taxol, caused an increase in tubulin polymer and a decrease in cytosolic, soluble tubulin. Consistent with the effects of other microtubule stabilizers, taccalonolide E rapidly caused a shift in the intracellular tubulin equilibrium toward the formation of microtubule polymers. Whether taccalonolide E interacts directly with tubulin to cause these effects has not yet been tested.

Evaluation of centrosomes in taccalonolide-treated cells indicates that a small population of cells had more than two centrosomes as indicated by {gamma}-tubulin reactivity. A few cells with four centrosomes were identified in treated A-10 and HeLa cells, and this is consistent with the small population of cells with 8n DNA content detected by DNA-ploidy analysis. The data suggest that a small population of treated cells progress through normal mitosis but are unable to complete cytokinesis.

Microtubules are dynamic structures, and the mitotic microtubules are significantly more dynamic than interphase microtubules, exhibiting up to 100-fold increases in rates of growing and shortening (37 , 38) . Because of these rapid dynamics, mitotic spindles are very sensitive to disruption by tubulin-interacting drugs. Antimicrotubule agents are also known as antimitotics because they disrupt normal microtubule dynamics, causing the formation of abnormal mitotic spindles and interruption of mitosis. Mitotic spindles formed in the presence of taccalonolide are multipolar, often with four or more spindles, and the abnormal spindles are associated with abnormal chromatin alignment. The aberrant mitotic spindles are indistinguishable from the spindles formed in Taxol-treated cells and are unlike the monoastral-shaped spindles found in laulimalide-treated cells (13) . The mitotic effects of taccalonolide occurred at lower concentrations than the concentrations that caused dramatic interphase microtubule reorganization. These data suggest that the highly dynamic mitotic spindles are more sensitive to taccalonolide, consistent with the effects of other microtubule stabilizing drugs (37) . No evidence of more than two centrosomes was found in the abnormal mitotic cells in either of the two cells lines treated with a range of concentrations of the taccalonolides. In taccalonolide-treated A-10 and HeLa cells, the formation of multiple spindle poles appears to be independent of centrosome amplification and thus is different from the effects of epothilone B in A549 cells (39) . Whether the centrosome effects are cell line- or drug-specific is not currently known but may provide interesting clues as to the differences and similarities among microtubule stabilizers.

As expected from drugs that cause the formation of abnormal mitotic spindles, the taccalonolides inhibit mitotic progression and cause G2-M arrest in cancer cells. The taccalonolides initiates apoptosis and the phosphorylation of Bcl-2, consistent with the effects of other microtubule agents. The phosphorylation of Bcl-2 after taccalonolide treatment may involve a signaling pathway of apoptosis common to all tubulin-binding agents (29) . In the MDA-MB-435 cell line, early Bcl-2 phosphorylation is a common effect of every microtubule stabilizer and depolymerizer we have evaluated; yet, we have never detected any phosphorylated Bcl-2 in our control samples in which ~15% of the cells are in the G2-M phase of the cell cycle. If Bcl-2 phosphorylation is a normal mitotic event, we would expect to detect some phosphorylation of our control samples. In this cell line, our data suggest that the Bcl-2 phosphorylation that occurs in response to these drugs is not only because of accumulation of cells in mitosis but may indicate abnormal mitotic events. In addition to Bcl-2 phosphorylation, the ERK1/2-signaling pathways are activated early in the time course of taccalonolide E treatment, with a low IC85 concentration. The demonstration that these signaling pathways are altered early in the time course of taccalonolide E treatment and at the lowest cytotoxic concentration may help identify pathways of potential clinical relevance (40) .

The cellular effects of the taccalonolides and Taxol are similar, however, the taccalonolides differ from Taxol and all other microtubule stabilizers, with regards to potency. Taxol is effective in the low nanomolar range, with IC50s of ~2 nM in multiple drug-sensitive cell lines; the IC50s for taccalonolide E are 340–990 nM and 2–3 µM for taccalonolide A in these cell lines. The taccalonolides are the least potent stabilizers identified to date. Whether this difference represents a lower affinity for tubulin, an alternate binding site, or differences in cellular uptake are not yet known but will be investigated. Although the taccalonolides differ in potency as compared with Taxol, they are equally effective as inhibitors of proliferation and cytotoxins. Studies on the Vinca alkaloids suggest that the compounds with highest potency exhibit the highest toxicity (41) and that a larger therapeutic index is observed with the less potent alkaloid vinflunine (42) . Whether this relationship will translate to other classes of microtubule-disrupting drugs is not known.

A long-term goal of our work is the discovery of new antimicrotubule agents. A variety of microtubule-interrupting agents are currently in clinical use. There is a clear need for new agents that can circumvent mechanisms of resistance, and this may provide significant advantages for the treatment of refractory tumors. Newer agents such as the epothilones are able to circumvent drug resistance mechanisms both in vitro and in in vivo murine preclinical models (6 , 43) . Ongoing clinical trials will determine whether they will be useful in the treatment of human tumors. The taccalonolides appear to be poorer substrates for transport by Pgp than Taxol. Another limitation of Taxol is its poor aqueous solubility. Efforts have been undertaken to identify taxanes and other compounds with similar mechanisms of action with better aqueous solubility. Taccalonolide E is a polar molecule and whether it will have advantages over Taxol with respect to solubility in aqueous solutions has yet to be determined.

In contrast to the other major types of cytotoxic chemotherapeutic agents, the efficacy of antimicrotubule agents is independent of p53 status (44) . The tumor suppressor gene encoding p53 is the most frequently mutated gene in human cancers, and it is estimated that half of all cancers in the United States exhibit altered p53. This provides a significant advantage for agents that target microtubules. The search for new antimicrotubule agents is also worthwhile because new compounds are expected to have different clinical efficacies. Structure cannot predict efficacy or limiting toxicity. Clinical experience with the Vinca alkaloids and the taxanes has proven this (5 , 42) . Recent studies demonstrate that the epothilones and eleutherobin can substitute for Taxol in a Taxol-dependent cell line but discodermolide cannot support the growth of the cell line. Paradoxically, as with cytotoxins, Taxol and discodermolide had synergistic effects against four human tumor cell lines (45) . These data suggest that all microtubule-stabilizing agents are not created equally (46) , and the possibility that drugs of this class may act synergistically is truly exciting and may lead to breakthroughs in the treatment of cancer.

The taccalonolides represent a new class of microtubule-stabilizers, a group of compounds that have proved to be very useful clinically, and the first class identified from a plant since Taxol. The taccalonolides differ structurally and biologically from the other microtubule-stabilizers. Whether the differences will translate into a unique spectrum of biological activities and superior antitumor effects with lower toxicity are important questions that will be addressed in the near future.


   ACKNOWLEDGMENTS
 
We thank Jennifer Hewett for identifying medicinal plant families of the Pacific, for plant collections, and constructing the voucher specimens and Anne Hernandez, Georgia Tien, Claudia Lupp, and Serafin Colmenares, III, for the generation of preliminary data. We also thank Robert Hirano and Ray Baker of the University of Hawaii’s Lyon Arboretum for their help in collecting the plant samples, Dr. Will McClatchey for taxonomy, and Dr. Richard Criley for the greenhouse propagations. We thank Dr. Pemmaraju N. Rao, Chair of the Department of Chemistry (Southwest Foundation for Biomedical Research) for the use of the NMR and Dr. Evi Giannakakou for creating, characterizing, and providing us the Taxol-and epothilone-resistant cell lines. We also thank April Hopstetter for all her help in the preparation of the figures and manuscript. We also thank our dedicated Nikon team of Vincent Haile and Vijian Dhevan for their excellent support.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Department of Defense Breast Cancer Initiative Grants DAMD17-97-1-7212 and DAMD17-00-1-0284 (to S. L. M.). Back

2 Present address: TransTech Pharma, Inc., 4170 Mendenhall Oaks Parkway, Suite 110, Highpoint, NC 27265. Back

3 To whom requests for reprints should be addressed, at Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, P. O. Box 760549, San Antonio, TX 78245-0549. Back

4 The abbreviations used are: Pgp, P-glycoprotein; NMR, nuclear magnetic resonance; NCI, National Cancer Institute; DAPI, 4',6-diamidino-2-phenylindole; SRB, sulforhodamine B; PARP, poly(ADP-ribose) polymerase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. Back

Received 1/30/03. Accepted 4/ 8/03.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. L. Mooberry, D. A. Randall-Hlubek, R. M. Leal, S. G. Hegde, R. D. Hubbard, L. Zhang, and P. A. Wender
From the Cover: Microtubule-stabilizing agents based on designed laulimalide analogues
PNAS, June 8, 2004; 101(23): 8803 - 8808.
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