Mol Cancer Ther. 2005;4:562-568
© 2005 American Association for Cancer Research
A-432411, a novel indolinone compound that disrupts spindle pole formation and inhibits human cancer cell growth
Zehan Chen1,
Philip J. Merta1,
Nan-Hong Lin1,
Stephen K. Tahir1,
Peter Kovar1,
Hing L. Sham2 and
Haiying Zhang1
1 Cancer Research and 2 Metabolic Disease, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois
Requests for reprints: Zehan Chen, Abbott Laboratories, Global Pharmaceutical Research and Development, Department 47B, AP10/114, 100 Abbott Park Road, Abbott Park, IL 60064. Phone: 847-935-5182; Fax: 847-935-7551. E-mail: zehan.chen{at}abbott.com
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Abstract
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Microtubules are among the most successful targets for anticancer therapies and for the development of new anticancer drugs. A-432411 is a novel small molecule that destabilizes microtubules at high concentration and disrupts normal spindle formation at low concentration. A-432411 is an indolinone that is structurally different from other known synthetic microtubule inhibitors. This compound is efficacious against a variety of human cancer cell lines including drug-resistant HCT-15 that overexpresses Pgp170. Biochemical studies show that A-432411 competes with the colchicine-binding site on tubulin and inhibits microtubule polymerization. Fluorescence-activated cell sorting analysis indicates that A-432411 causes G2-M arrest and induces apoptosis. Cells treated with A-432411 have increased level of phospho-histone H3 at Ser10 and decreased level of phospho-cdc2 at Tyr15. Concurrently, securin and cyclin B1 expression levels remain the same, indicating the activation of the spindle checkpoint. Immunocytochemistry and fluorescence microscopy experiments reveal that 1 µmol/L A-432411 destabilizes microtubules in cells. At 0.1 µmol/L, the compound disrupts normal spindle pole formation possibly through stabilization of microtubule dynamic. Both structural and cellular properties of A-432411 make it an attractive candidate for further development.
Key Words: A-432411 • antimitotics • microtubules • spindle poles
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Introduction
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Microtubules are cytoskeletal protein polymers consisting of 
-tubulin and ß-tubulin dimers (1). They are critical for the maintenance of cell morphology, signal transduction, and cell division. Interference of microtubules prevents dividing cells from proceeding through the spindle checkpoint of the cell cycle and leads to apoptosis (2, 3). Cancer cells acquire unlimited replicative potential and continue to divide without progressing into quiescence and senescence (4). The property of uncontrolled growth and division makes cancer cells extremely dependent upon microtubule dynamics and vulnerable to antimitotic drugs that target microtubules (5). Additionally, proliferating endothelial cells that form neovasculature in the tumor are also sensitive to antimitotic agents (6). These findings suggest that microtubules will continue to be among the most promising cancer targets for the development of new anticancer drugs (7, 8).
Several antimitotic drugs have been successfully used in the clinic for the treatment of cancer. Whereas the mechanism of action for the antimitotic therapies is essentially the same, different chemical structures seem to define their selectivity for different tissue and cancer types. For example, Vinca alkaloids such as vinblastine, vincristine, and vinorelbine are often efficacious against hematologic cancers but less effective against adult solid tumors (8). In contrast, paclitaxel and its semisynthetic analogue docetaxel effectively combat many solid cancers arising from ovarian, breast, and lung tissues but seem ineffective against cancers of the colon, kidney, and hematologic cancers. Colchicine represents another class of antimitotic compounds that inhibit the assembly of microtubules (9). None of the compounds in this class has been successful in the clinic for the treatment of cancers. The success of microtubule as cancer target points to a promising future to explore it further with the development of novel and efficacious inhibitors.
We report here a novel indolinone, A-432411, and its ability to destabilize microtubules in both cancer and proliferating endothelial cells at concentrations above 1 µmol/L. At lower concentrations, this compound disrupts spindle pole development. Cellular and molecular evidence indicates that A-432411 activates the spindle checkpoint and induces apoptosis. The results also show that it inhibits proliferation of cancer cells including Pgp170-positive HCT-15 colon carcinoma cells. Cell biology, biochemistry, and molecular biology data suggest that A-432411 could be a prime candidate for the development of a novel class of antimitotic drugs.
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Materials and Methods
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Compound Synthesis
A-432411 [6-(4-hydroxy-3-methoxyphenyl)-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one] was prepared by total synthesis starting from the corresponding indolinone material at Abbott Laboratories (Abbott Park, IL).3
Cell Culture
Human lung cancer cell lines NCI-H460 and NCI-H1299, colon cancer cell lines HCT-116 and HCT-15, and the cervical cancer cell line HeLa were obtained from American Type Culture Collection (Manassas, VA). NCI-H460, HCT-116, and HCT-15 were grown in RPMI 1640 supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. NCI-H1299 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, and 0.45% glucose. HeLa cells were grown in MEM supplemented with 10% fetal bovine serum, glutamine, nonessential amino acids, and pyruvate. Human microvascular endothelial (HMVEC) cells were obtained from Clonetics Co. (Walkersville, MD) and maintained in EGM-2MV growth medium as recommended by the vendor (Clonetics).
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, Inner Salt Assay
Exponentially growing cells were seeded in 96-well plates and allowed to attach overnight in the incubator. The cells were then treated with various concentrations of compound for 48 hours. Finally, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt (MTS) reagent (Promega, Madison, WI) was used to assess live cell number. The MTS and cells were incubated from 20 minutes to 2 hours and assayed at 490 nm on Spectra MAX 190 plate reader (Molecular Devices, Sunnyvale, CA). Resulting EC50 values were defined as the compound concentration required to inhibit cell proliferation by 50% in 48 hours. DMSO treatment was used as 100% control.
Cell Cycle Analysis
Flow cytometric analyses were done as described previously (10). Cells were treated with compounds at the indicated concentrations for 24 hours and trypsinized. After spinning at 1,000 x g for 5 minutes, the cells were washed in PBS and fixed in 70% ethanol. The washed cells were treated with RNase A at 37°C for 30 minutes. Finally, the cells were stained with propidium iodide and analyzed by fluorescence-activated cell sorting on a FACSCalibur instrument (Becton Dickinson, San Jose, CA). Cells were counted and analyzed using the CellQuest Pro program provided by the vendor.
Immunocytochemistry and Fluorescence Microscopy
Cells were seeded in 8-well chamber slides, treated with the compounds for 16 to 24 hours, and processed according to a protocol modified from previous reports (11, 12). Briefly, compound-treated cells were washed twice with PBS and fixed in 10% formalin. The slides were then blocked with 2% bovine serum albumin and 0.4% Triton X-100. Microtubule structure was labeled with FITC-conjugated anti--tubulin antibodies (Sigma, St. Louis, MO) and counterstained with Hoechst 33258 (Sigma) for DNA. The slides were air-dried, sealed under a cover glass, and examined under a Zeiss AxioVision 200 microscope (Carl Zeiss, Inc., Thornwood, NY).
Tubulin Competition-Binding Scintillation Proximity Assays
Colchicine and paclitaxel were purchased from Sigma Chemical Co. (St. Louis, MO). [3H]colchicine (specific activity = 61.4 Ci/mmol) and [3H]paclitaxel (specific activity = 2.4 Ci/mmol) were purchased from New England Nuclear (Boston, MA) and Moravek Biochemicals (Brea, CA), respectively. The colchicine and paclitaxel competition-binding scintillation proximity assays were conducted as described previously using biotin-labeled tubulin and streptavidin-coated scintillation proximity assay beads (13–15). Briefly, radiolabeled colchicine or paclitaxel, unlabeled compound, and special long-chain biotin-labeled tubulin were incubated together in the binding buffer containing 80 mmol/L PIPES (pH 6.8), 1 mmol/L EGTA, 10% glycerol, 1 mmol/L MgCl2, and 1 mmol/L GTP for 2 hours at 37°C. Streptavidin-labeled scintillation proximity assay beads were added to each reaction mixture for colchicine and paclitaxel assay, respectively. The inhibition constant (Ki) was calculated using the Cheng-Prussof equation (Biochem Pharmacol 1973; 22:3099).
Tubulin Polymerization Assay
Antimitotic compounds were tested using Tubulin Polymerization kit as specified by the manufacturer (Cytoskeleton, Inc., Denver, CO).
Western Blot Analysis
Western blot analyses were done using a modified version of previously described methods (10, 16). Briefly, cells were seeded in 12-well plates and treated with the reagents for 24 hours. Cells were then harvested by washing once with PBS and then lysing in 1x Laemmli SDS sample buffer containing 0.05% of ß-mercaptoethanol, 1x protease inhibitor mixture (Sigma), 0.5 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L activated sodium orthovanadate, and 200 nmol/L okadaic acid. The samples were heated at 95°C for 5 minutes and separated under denaturing conditions in a mini-gel system (Invitrogen, Carlsbad, CA). Protein samples were transferred to a polyvinylidene difluoride membrane using a semi-dry blotting system according to the vendor's manual (Amersham Biosciences, Buckinghamshire, United Kingdom). The membrane was blocked with 5% nonfat dry milk and probed with specific antibodies against phospho-histone H3 (Upsdate, Charlottesville, VA), phospho-cdc2-Y15 (Cell Signaling, Beverly, MA), securin (Abcam, Inc., Cambridge, MA), and ß-actin (Sigma). Chemiluminescent detection was done using enhanced chemiluminescence reagents according to the vendor's protocol (Amersham Biosciences).
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Results
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A-432411 Is a Novel Antimitotic Compound
A-432411 is an indolinone analogue that has a phenyl group attached to the C6 position of the indolinone ring (Fig. 1). In addition, a pyrrole-methylene group is placed at the C3 position of the ring system. This compound is structurally different from A-289099 (15) and BPR0L075 (17), both of which are colchicine binders that have an indole moiety in their structure. BPR0L075 has a trimethoxyphenylcarbonyl group attached to the C3 position of the indole ring, whereas A-289099 has an oxazoline ring appended to the C6 position of its indole ring.
A-432411 Inhibits the Growth of Pgp170-Positive and Pgp170-Negative Cancer Cells and Endothelial Cells
To test the cytotoxic effect of A-432411, we treated various human cancer cell lines and normal endothelial cells and measured cell survival by MTS assays (Table 1). The cancer cell lines tested included cells with negligible Pgp170 and a cell line, HCT-15, that expresses high levels of Pgp170 (15). A-432411 efficacy was similar against most of the cell lines tested regardless of the expression of Pgp170. Paclitaxel and colchicine were more potent than A-432411 against the sensitive lines including HeLa and HCT-116. However, A-432411 was much more potent against HCT-15 than both paclitaxel and colchicine. These results show that A-432411 is efficacious against a variety of human cancer cell lines including cancer cells expressing high levels of Pgp170. These data also indicate that A-432411 possesses an inhibitory profile that is different from paclitaxel or colchicine.
Normal endothelial cells responded to the three antimitotic agents differently. Paclitaxel and colchicine inhibited HMVEC cell proliferation similar to cancer cells. In contrast, A-432411 inhibited HMVEC proliferation to a lesser extent than paclitaxel and colchicine.
A-432411 Arrests Cells in G2-M and Causes Cell Death
Colchicine-like antimitotic compounds are known to arrest cells in G2-M and induce apoptosis. To determine the effect of A-432411 on the cell cycle, we treated HCT-116, HCT-15, H1299, and HeLa cells with A-432411 and obtained their cell cycle profiles. A representative profile for HeLa cells is shown in Fig. 2. DNA content was indicated as 2N for G0/G1 cells and 4N for G2-M cells. Cells with DNA content between 2N and 4N are in the S phase. The sub-G0/G1 population represents apoptotic cells with less than 2N DNA content. The results showed that untreated cells had a normal distribution of cells in all four phases. Cells treated with A-432411 had an increase of G2-M cells and apoptotic cells. The results were consistent with the activation of a mitotic checkpoint and induction of programmed cell death.
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Figure 2. A-432411 induces cell cycle arrest in G2-M and causes cell death. HeLa cells were treated with A-432411 at the indicated concentrations for 24 h and processed for FACS analysis. DNA content was marked as 2N and 4N. Cells in the G0-G1 phase have 2N DNA content. G2-M phase cells have 4N DNA. Apoptotic cells fall in the sub–G0-G1 region with less than 2N DNA. S-phase cells have DNA content between 2N and 4N.
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A-432411 Destabilizes Microtubules and Disrupts Normal Spindle Pole Formation
A close examination of the cellular mechanism for the G2-M arrest revealed that A-432411 destabilized microtubules at the high concentration (1 µmol/L) and disrupted normal spindle pole formation at the low concentration (0.1 µmol/L). To document the effect on microtubule, we treated HeLa, HCT-116, and HCT-15 cells with 0.1 and 1 µmol/L A-432411 for 18 hours and stained for microtubule and DNA. Figure 3 shows representative views from HeLa and HCT-15 cells. Microtubule and DNA were stained green and red, respectively. The two colors were merged to colocalize microtubule and DNA. Figure 3 (top) presents the views for four HeLa cells treated with high dose A-432411, DMSO vehicle control, and low-dose A-432411. Cell 1 was treated with 1 µmol/L of A-432411. The strong diffused green stain is typical of depolymerized tubulins. Nuclear stain shows condensed DNA with irregular structure indicating that the cell was arrested in the M phase. Cell 2 was treated with DMSO vehicle. This anaphase cell shows normal bipolar spindles and regular DNA separation. Cells 3 and 4 were treated with 0.1 µmol/L of A-432411. The cells maintain their mitotic microtubule mass. However, cell 3 shows multiple spindle poles, whereas cell 4 shows a near normal bipolar spindle. DNA content clearly identifies the two cells in the metaphase. None of the cells treated with low-dose A-432411 went beyond this phase.
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Figure 3. Low concentration of A-432411 disrupts spindle pole formation. HeLa and HCT-15 cells were treated with high (1 µmol/L) and low (0.1 µmol/L) concentrations of A-432411 for 16 h and processed for fluorescence microscopy. Top, HeLa cells stained with anti--tubulin (green) and propidium iodide for DNA (red). Four cells from different treatment conditions were indicated. Cells 1 and 2 were treated with 1 µmol/L A-432411 and DMSO vehicle, respectively. Cells 3 and 4 were treated with 0.1 µmol/L A-432411. Bottom, morphologies of 5 HCT-15 cells treated with 0.1 µmol/L A-432411.
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Figure 3 (bottom) presents the views for HCT-15 cells treated with low-dose A-432411 at 0.1 µmol/L. The five cells in the field exhibit unique DNA and spindle structures that are representative of the results from all cell lines tested. Cells 5 and 6 have multiple spindle poles and disoriented DNA congregation. A normal metaphase cell should have two separate spindle poles and the DNA aligned on the midplane. Cell 7 has only a single spindle pole and disorderly arrayed DNA. Cell 8 seems to have two normal bipolar spindles. However, some of the chromosomes failed to congress to the midplane and comigrated with microtubules to the spindle poles. Cell 9, on the other hand, displays a ring of chromosomes with a monoastrol spindle indicating the failure of spindle pole duplication in mitosis. Previous reports have shown that low concentrations of paclitaxel (18) and vinflunine (19) stabilize microtubule dynamics and interrupt normal chromosome segregation in a similar fashion. Our results show that, at low concentration, A-432411 seems to interrupt spindle pole formation by stabilization of microtubule dynamics.
A-432411 Inhibits Microtubule Polymerization
To investigate the role of A-432411 on microtubule polymerization, we examined the effects of A-432411 using an in vitro microtubule polymerization assay (Fig. 4). An increase in the absorbance at 340 nm indicates an increase in tubulin polymerization. DMSO was used as control for the experiments. As expected, paclitaxel results in a dose-dependent increase in absorbance above the control. Colchicine has little effect at 1 µmol/L but strongly inhibits microtubule polymerization at 10 µmol/L. Similarly, A-432411 strongly inhibits microtubule polymerization at 10 µmol/L. However, it exhibits a much stronger inhibitory effect on microtubule polymerization than colchicine at 1 µmol/L. The data indicate that A-432411 destabilizes microtubules in a similar but more potent manner to colchicine.
A-432411 Competes with the Colchicine-Binding Site
Microtubule-destabilizing drugs often have specific microtubule binding sites. We assessed the interaction of A-432411 with tubulin by determining whether it competes for binding with either colchicine or paclitaxel. A competition-binding scintillation proximity assay shows that A-432411 competes strongly with [3H]colchicine with an IC50 value of 0.13 µmol/L over a compound concentration range up to 3 µmol/L (Fig. 5). However, the compound does not compete with [3H]paclitaxel. These data show that A-432411 binds the colchicine-binding site on the tubulins.
Molecular Events Show Activation of the Spindle Checkpoint
To better understand the mechanism of A-432411, we analyzed its effect on key molecular events in the cell cycle. We chose H1299 cells to determine the molecular events because of its distinct cell cycle profile (10). In higher eukaryotes, phosphorylation of the TyrY15 inactivates cdc2 kinase and prevents cells from entering mitosis (20). A 4N DNA content in the fluorescence-activated cell sorting analysis and a high level of phosphorylated cdc2 at Y15 suggest that the cells arrest in the G2 phase. The G2 checkpoint is often induced under DNA damage conditions such as treatment of cells with doxorubicin (10, 21). In contrast, activation of the mitotic checkpoint can be assessed by the increase of phosphorylation of histone H3 at Ser10 in the presence of paclitaxel or colchicine. The onset of anaphase in the cell cycle requires destruction of cyclin B1 and securin (22, 23). When cells were treated with doxorubicin, phospho-cdc2-Y15 levels increased, indicating an arrest of the cells in the G2 phase (Fig. 6). A-432411, paclitxel, and colchicine, on the other hand, reduced phospho-cdc2-Y15 and increased the levels of phospho-histone H3, indicating that cells were arrested in the M phase. Concomitantly, cyclin B1 and securin levels remained unchanged, indicating that cell cycle progression stopped before entry into anaphase through activation of the spindle checkpoint. It is noteworthy that the mitotic arrest was in the presence of 0.1 µmol/L A-432411. These data imply that low concentration of A-432411 is sufficient to stabilize the microtubule dynamic and induce the spindle checkpoint, eventually leading to apoptosis.
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Figure 6. A-432411 activates the spindle checkpoint. Cells were treated for 24 h with 0.1 µmol/L A-432411 and other control compounds, including doxorubicin (DOX) for G2 and paclitaxel (TAX) and colchicine (COL) for M phase of the cell cycle. Western blot analysis of protein expression levels for phospho-cdc2 at Tyr15 (cdc2-Y15), phospho-histone H3 at Ser10 (pH 3), securin, and cyclin B1. Level of ß-actin as a loading control.
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Discussion
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Here we present the biological properties of a structurally novel compound that displays strong antimitotic activity against cancer and proliferating endothelial cells. Antimitotic drugs have not only been established as one of the most efficacious anticancer therapies, but they have also provided ample opportunities for new applications and developments. It is predicted that antimitotic drugs will continue to be the therapy of choice to combat cancer for many years to come. In fact, new antimitotic compounds have been reported recently including synthetic small molecules such as A-289099 (15), ABT-751 (24), and BPR0L075 (17).
Antimitotic drugs used in the clinic include paclitaxel, docetaxel, vinblastine, vincristine, and vinorelbine. All of them stabilize microtubule dynamics at their lowest effective concentrations and induce mitotic blocks that lead to apoptosis (8). An unresolved question lies in the fact that despite the common cellular mechanism, these agents show striking differences in their clinical efficacies against tumors at various tissue types. These unique patterns of selectivity are probably related to the nature and availability of a particular antimitotic agent. A-432411 represents a novel structure in the colchicine class and provides an opportunity for potential new indications.
Like other microtubule-destabilizing compounds, A-432411 destabilizes microtubules at high concentrations (>1 µmol/L) and stabilizes microtubule dynamics at low concentration (0.1 µmol/L), which leads to abnormal spindle pole formation. Stabilization of microtubule dynamic and perturbation of DNA segregation at low concentration have been well documented for antimitotic compounds including paclitaxel, Vinca alkaloid, and colchicine (8). A 3.5-Å structure of tubulin in complex with colchicine and the stathmin-like domain may shed some light on the concentration-dependent mechanisms (9). Stathmin is a microtubule regulatory protein that can sequester the -tubulin and ß-tubulin heterodimers and thereby regulate the microtubule dynamic instability (25–27). In this model, colchicine binds and stabilizes the tubulin heterodimers at a curved conformation that prevents lateral contact of the subunits at the newly formed end of the microtubule. High concentration of the compound, however, favors the formation of the ligand-tubulin complexes and causes dynamic instability of microtubules. When the compound concentration is low, small numbers of the curved tubulin heterodimers only hinder the lateral interaction of the tubulin subunits without significantly affecting the longitudinal growth of the microtubules. This condition favors the stabilization of microtubule dynamic and agrees well with previous observations (28). It has been reported that the treadmilling dynamic of microtubules might be more prevalent in mitotic cells than in interphase cells (29). A-432411 may perturb spindle pole formation through modulation of treadmilling microtubules.
Failure in cancer chemotherapy is often related to multidrug resistance. Our results show that sensitivity to paclitaxel differs by >30-fold between high and low Pgp170 expressing cancer cells. Similarly, colchicine shows >10-fold difference. In contrast, A-432411 exhibits a similar potency regardless of the Pgp170 status. The results indicate that A-432411 is not a Pgp substrate, thereby suggesting that it is superior to other antimitotic agents in this regard.
One observation in this report is that A-432411 seems less potent against normal HMVEC cells than paclitaxel and colchicines. As a representative normal cell type, HMVEC's response to A-432411 suggests that it may have a large therapeutic window in which to treat cancer cells. Paclitaxel and colchicine show no such selectivity. On the other hand, if HMVEC represents precursor cells for neovasculature, paclitaxel and colchicine may be more potent inhibitors than A-432411. The potency of A-432411 on the other aspects of HMVEC cells such as migration and tube formation remains unclear and requires additional investigation. At this time, it is clear that A-432411 offers some unique properties and selectivity profiles over other antimitotic agents.
In conclusion, we present a structurally novel antimitotic compound that binds tubulin at the same site as colchicine. A-432411 exhibits biochemical, molecular, and cellular mechanisms that are consistent with other natural and synthetic antimitotic compounds. The unique structural and biological properties of A-432411 make it an attractive candidate for further development toward potential clinical applications.
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Acknowledgments
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We thank Saul H. Rosenburg for critical review of the article, Thomas J. Sowin and Shi-Chung Ng for helpful discussions on the subject, and Jieyi Wang and Ping-Ping Lou for providing the HMVEC cells.
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Footnotes
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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.
3 Lin N-H, Ping X, Kovar P, et al. Synthesis and biological evaluation of 3-Ethylidine-1,3-dihydro-indol-2-one as novel checkpoint kinase 1 inhibitors. Abstracts of Papers, 229th ACS National Meeting, San Diego, CA, March, 2005, MEDI-146. 
Received 9/ 1/04;
revised 1/21/05;
accepted 2/ 7/05.
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Abstract
Introduction
), paclitaxel at 1 µmol/L (
) and 10 µmol/L (
), colchicine at 1 µmol/L (
) and 10 µmol/L (
), and A-432411 at 1 µmol/L (x) and 10 µmol/L (*). A shift of the curve to the left of the control is an increase of polymerized microtubule. A shift to the right is a decrease in the rate of polymerization.
) and [3H]paclitaxel (
). Points, mean; bars, ±SE.