3-Iodoacetamido benzoyl ethyl ester (3-IAABE) is a new compound synthesized in our laboratory. The primary action of 3-IAABE is to inhibit microtubule assembly by interacting with −SH groups on tubulin. In contrast to other known microtubule disrupters, 3-IAABE caused a double blockade in the cell cycle at G1-S transition and in M phase. The blockade was determined by cell cycle analysis and chromosome distribution. Kinase activities of cyclin E and cyclin-dependent kinase 2 responsible for the G1-S transition were increased, as were the activities of mitotic cyclin B and cdc2. 3-IAABE treatment also increased p53 expression and dephosphorylated (or activated) retinoblastoma protein. Investigation of the signal transduction pathway showed that 3-IAABE induced bcl-2 phosphorylation, followed by activation of caspase-9, -3, and -6, but not caspase-8. DNA fragmentation factor and poly(ADP-ribose) polymerase, the downstream substrates of caspase-3 and -6, were cleaved after 3 h of exposure to 3-IAABE, followed by DNA fragmentation. Pretreatment of the cells with inhibitors of caspase-9, -3, or -6, respectively, inhibited the cleavage of DNA fragmentation factor and poly(ADP-ribose) polymerase and thus inhibited the onset of apoptosis. 3-IAABE showed antitumor activities in the panel of 60 National Cancer Institute human tumor cell lines with total growth inhibition in the range of 0.22–4.3 μm for solid tumor lines and 0.025–0.22 μm for leukemia/lymphoma cell lines. The 3-IAABU total growth inhibition of phytohemagglutinin-stimulated healthy human lymphocytes was 450-fold greater than that of leukemic cells. 3-IAABE significantly inhibited the growth of human hepatocarcinoma (BEL-7402) in nude mice by 72% in tumor volume, more strongly than did vincristine (43% inhibition). Besides being a novel lead for the design of new anticancer tubulin ligands, the activity of 3-IAABE in the cell cycle may also help us to understand the molecular pharmacology of microtubule-active drugs.

Microtubules are highly dynamic assemblies of α/β-tubulin and play important roles in a variety of biological functions, especially in governing the movement of chromosomes during mitosis. Microtubule dynamics speed up by 100-fold in mitosis as compared with interphase. The half-time of exchanging spindle tubulin with tubulin in the free tubulin pool is about 10 s (1, 2). Tubulin ligands that target the dynamic process induce mitotic arrest in the cell cycle (3, 4, 5, 6). Such compounds have been used in the clinic for cancer chemotherapy (Vinca alkaloids, podophyllotoxin, and the taxanes), karyotype analysis (vinblastine and Colcemid), the treatment of gout (colchicine), and as antiparasitic drugs (mebendazole).

Tubulin ligands also have other important biological effects, however, which cannot be explained simply by their primary action of mitotic arrest. Experiments in vitro showed that tubulin disrupters increased chemical-induced cell differentiation (7). Arsenic trioxide (As2O3), which serves in the clinic as a differentiation agent, interrupts the microtubule-tubulin equilibrium (8, 9).

In a recent study of drugs active on microtubules, a unique tubulin ligand, 3-IAABE3 (Table 1a), was found in our laboratory. 3-IAABE was originally designed and synthesized to improve the solubility of the parent ligand 3-IAABU (Table 1b; Refs. 10 and 11). As expected, the solubility was remarkably improved without abrogating the inhibitory effect on microtubule assembly. However, the rational modification rendered the daughter ligand a new function of inducing significant cell cycle blocks at the G1-S transition in addition to its activity in M phase. This property differentiates 3-IAABE from the known drugs active on microtubules. The primary action, molecular mechanism, and anticancer activity of this new tubulin ligand are presented below.

Reagents.

3-IAABE was synthesized in our laboratory (Ref. 11; United States patent). The molecular weight of this compound is 289. The compound was dissolved in a mixture of N,N-dimethyl acetamide, propylene glycol, and Tween 80 (1:2:1, v/v/v) at 10 mg/ml as a stock solution. Dilution was made in medium before use. Vincristine, vinblastine, and paclitaxel were from Sigma-Aldrich (St. Louis, MO).

Inhibition Assay of Microtubule Assembly and Disassembly.

The CytoDYNAMIX Microtubule Polymerization Screens-2 (CDS-02) kits were from Cytoskeleton, Inc. (Denver, CO). Tubulin protein was resuspended (120 μg/well) in the wells of a 96-well plate with 125 μl of G-PEM [80 mm PIPES (pH 6.9), 1.0 mm MgCl2, 0.5 mm EGTA, and 1.0 mm GTP] containing the test compound. The absorbance (A) was read (Spectromax 250; Molecular Devices, Inc.) at 340 nm once per min for 60 min. Temperature was set at 37°C. A is proportional to the concentration of polymerized tubulin. Reducing DTT and β-ME were from Sigma (St. Louis, MO). The methods used for disassembly assays have been reported previously (12). The solvents did not affect polymerization or depolymerization at concentrations <1% (v/v) as used in the experiments.

Morphology.

Slides were prepared by a Cytospin centrifuge (Shandon Southern Products Ltd.) at 700 × g for 5 min. The slides were air dried, fixed in methanol, stained with Giemsa (Harleco) at room temperature for 15 min, and read by a light microscope. Cells in mitotic phase were recognized by the appearance of chromosomes dispersed in the cytoplasm and by the disappearance of nuclear membranes.

Detection of Kinase Activity.

Cells were exposed to 0.35 μm (0.1 μg/ml) 3-IAABE for the indicated time and lysed with 0.5 ml of lysis buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 10 mm EDTA, 10 mm EGTA, 1 mm NaF, 1 mm DTT, 1 mm PMSF, 1% NP40, and 10 μg/ml aprotinin and leupeptin. The lysate was incubated with anti-cyclin A, B, D1, and E, anti-cdk2, and anti-cdc2 antibodies (PharMingen, San Diego, CA), respectively, and with protein A/G-conjugated agarose beads at 4°C overnight. After centrifugation at 4°C at 15,000 × g for 5 min, the immunoprecipitates were washed three times with lysis buffer and once with reaction buffer. The kinase reaction was started by the addition of 30 μl of kinase reaction buffer containing 100 μm Tris-HCl (pH 7.4), 10 mm Mg2Cl, 5 mg of histone H1 (Boehringer Mannheim), 1 μm ATP, and 1 μCi of γ-[32P]ATP (Amersham, Arlington Heights, IL) at 30°C for 15 min. The reaction was terminated by addition of 4× Laemmli sample buffer containing 62.5 mm Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% β-ME, and 0.05% bromphenol blue and resolved by 10% SDS-PAGE. The kinase activity was determined by either autoradiography or counting radioactivity in histone H1 protein.

Immunoblot for bcl-2, p53, and Rb Proteins.

The immunoblot method was described previously (10). Briefly, cell aliquots were taken and lysed in lysis buffer [50 mm Tris-HCl (pH 7.4), 0.1% Triton X-100, 1% SDS, 250 mm NaCl, 15 mm MgCl2, 1 mm DTT, 2 mm EDTA, 2 mm EGTA, 25 mm NaF, 1 mm PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. The DC protein assay kit (Bio-Rad, Hercules, CA) was used to determine the protein concentration. Samples with equal protein concentration (30 μg/test) were subjected to electrophoresis in a 10% polyacrylamide gel containing 0.1% SDS. The blots were transferred onto nitrocellulose filters and then blocked with 5% nonfat milk in TBST buffer (100 mM Tris-Cl pH 7.5, 0.9% NaCl, 0.1% Tween-20) at room temperature for 1 h. Bcl-2, p53, and Rb proteins were detected with the anti-bcl-2 mAb, anti-p53 mAb (PharMingen), and anti-ppRb mAb (New England BioLabs, Beverly, MA), respectively, followed by goat antimouse HRP. The protein signals were detected by ECL.

Detection of Caspase Activation.

Activities of the studied caspases were measured by ApoAlert CPP32/caspase-3 assay kit from Clontech (Palo Alto, CA) or by caspase-9/Mch6, caspase-8/FLICE, and caspase-6/Mch2 colorimetric assay kits from BioVision (Palo Alto, CA). CEM cells were treated with 3-IAABE at 0.35 μm for 0, 1, 3, 6, 12, and 24 h, respectively. Cells were washed twice in PBS and then aliquoted at about 4 × 106 cells/test. The experiments were performed following the steps recommended by vendors. Raw data obtained by measuring the absorbance at 405 nm were calibrated according to the protein concentrations.

Western Blot Analysis for DFF and PARP.

Cells were treated for different times with 0.35 μm 3-IAABE and washed once in PBS, and the pellets were frozen at −80°C before use. After resuspension of the pellets in lysis buffer [1% Triton X-100; 150 mm NaCl; 25 mm Tris (pH 7.4); 1 μg/ml leupeptin, antipain, aprotinin, benzamidine HCl, chymostatin, and pepstatin; and 0.5 mm PMSF] and centrifugation, the supernatant fluid was used for analysis for DFF. The pellets were extracted, mixed with loading buffer B [62.5 mm Tris (pH 6.8), 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5% β-ME] at a ratio of 4:1 (v/v), sonicated for 20 s, and incubated for 15 min at 65°C. For DFF, 50 μl of the lysis buffer supernatant (containing 200 μg of protein) were resolved by 15% SDS-PAGE, transferred to nitrocellulose, probed with anti-DFF antibody (Upstate Biotechnology, Lake Placid, NY) followed by goat antirabbit HRP, and visualized by ECL.

For the immunodetection of PARP, 100,000 cells (treated with 3-IAABE or untreated) were spun down in a microfuge at 12,000 rpm for 5 min. The cell pellet was washed once with 1 ml of PBS, resuspended in 40 μl of SDS-PAGE loading buffer A [62.5 mm Tris (pH 6.8), 6 m urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5% β-ME], sonicated for 20 s, incubated for 15 min at 65°C, and applied to a 10% acrylamide gel containing 0.1% SDS. After electrophoresis and transfer to nitrocellulose (Hybond ECL; Amersham), filters were incubated overnight at 4°C with 1:2500 diluted anti-PARP mAb (BioMol, Plymouth Meeting, PA), followed by a 1-h incubation at room temperature with 1:3000 diluted antimouse HRP (Amersham). Immunoreactive bands were visualized using the ECL detection system as suggested by the manufacturer.

Determination of Apoptosis.

Quantitative measurement of apoptosis by flow cytometry has been described previously (11, 13). Cell samples (106 cells/50 μl) were fixed with 1% paraformaldehyde and treated with methanol to make them permeable for the metachromatic fluorochrome acridine orange staining. Intact DNA was measured from the intensity of luminescence with a flow cytometer (FACScan; Becton Dickinson, San Jose, CA).

Human Tumor Cells and Determination of IC50 and TGI.

Sixty human tumor cell lines (NCI screening panel) representing nine major tumor categories (14) were screened for sensitivity to 3-IAABE. These cells were cultured, as reported previously (15), in RPMI 1640 plus 5% FBS, 2 mm glutamine, and 1% antibiotics. Daudi/w and Daudi/MDR cells (provided by Dr. T. Ohnuma, Mount Sinai School of Medicine-New York University, New York, NY), human hepatocarcinoma BEL-7402 cells (provided by Dr. Y. S. Zhen, Chinese Academy of Medical Sciences, Beijing, China), and normal human prostate fibroblast PC-41 cells (provided by Dr. L. G. Wang, Mount Sinai School of Medicine) were cultured in the same medium. Normal human endothelial cell line HU-VEC was from American Type Culture Collection (Manassas, VA) and was cultured in endothelial basal medium with 10% FBS. Normal human lymphocytes were isolated from the blood of healthy individuals using Ficoll-Hypaque gradients and cultured in RPMI 1640 plus 10% FBS. Anticancer activity of 3-IAABE was determined in vitro in our laboratory and by the NCI Developmental Therapeutics Program using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method (16). Growth inhibition was expressed as a drug concentration inhibiting 50% (IC50) or 100% (TGI) growth as compared with untreated cells.

Anticancer Effect in Vivo.

Nude mice (male, 4–8 weeks of age, 15–20 grams in weight) were obtained from Center of Experimental Animals, Chinese Academy of Medical Sciences (Beijing, China) and used for human hepatocarcinoma xenografts (BEL-7402). Tumor tissue grafts were processed carefully to remove the connective tissues and blood clots before inoculation. Nude mice received s.c. trocar grafts of BEL-7402 (6–8 mm3/mouse) on their backs. Five days later, tumor-bearing mice were randomly divided into solvent control and treatment cages with 6–8 mice/group. 3-IAABE or vincristine was first administered i.p. on day 6 postimplantation. 3-IAABE was given at 10 or 35 mg/kg, and vincristine was given at 0.3 mg/kg (17). The regimens were continued with four other injections at 7-day intervals. Therapeutic response was monitored by measuring tumor volume every 7 days until the tumor volume in controls was >2000 mm3, the criterion for euthanasia (18). Two perpendicular tumor diameters, width and length, were obtained with calipers and used to calculate tumor volume with the following formula: tumor volume = length × width2 × 0.52 (19).

Effect of 3-IAABE on Microtubules.

A wide range of concentrations of 3-IAABE were measured for activity against microtubules (Fig. 1,A). 3-IAABE inhibited the microtubule assembly process with an ID50 of 3.1 μm but had no effect on disassembly. The inhibition of microtubule assembly by 3-IAABE was significantly reduced in the presence of reducing agent DTT (with 2 −SH/molecule) or β-ME (1−SH/molecule), suggesting a competitive binding to 3-IAABE between the reducing agents and the thiol (−SH) groups of tubulin. DTT showed a stronger inhibitory effect on 3-IAABE compared with β-ME (Fig. 1 B). The inhibitory effect on 3-IAABE appeared to be positively correlated with the numbers of thiol groups on the reducing agents. The −SH group of cysteine residues on tubulin seemed to be the binding domain for 3-IAABE (20).

Blockade at G1-S Transition and M Phase.

Flow cytometric analysis (Fig. 2) showed that blockade at the G1-S transition by 3-IAABE occurred at 4 h, became prominent at 12 h, and was followed by the appearance of G2-M peak at 24 h. About 25% of the cells at the 24 h time point had dispersed chromosomes characteristic of mitotic arrest (Fig. 3) when the concentrations ranged between 0.05 and 0.15 μg/ml. CEM cells treated with <0.01 μg/ml 3-IAABE had a cell cycle profile identical to that of untreated cells, with only 5% of cells at M phase by morphological characteristics. CEM, Daudi, and U937 cell lines exhibited a similar cell cycle profile and similar morphological features.

To further prove these observations, kinase activities of cell cycle regulators responsible for each stage of a cell cycle were measured in CEM cells. As shown in Fig. 4, kinase activities of cyclin E and cdk2, which are responsible for the G1-S transition, were increased within the first hour of 3-IAABE treatment (Fig. 4, A, a and B, b); kinase activities of mitotic cyclin B and cdc2 were also elevated parallel to that of cdk2 (Fig. 4, C, c and D, d). Using Western blot, we found that the expression of cyclin E, cdk2, and cyclin B was increased concordant with the time course of kinase activities (data not shown). Kinase activities and expression of cyclin A and D1, which are relevant to S and G1 phases, remained unchanged after exposure to 3-IAABE (Fig. 4, E, e and F, f).

Expression of wild-type p53 and ppRb proteins was also examined in cells treated with 3-IAABE (Fig. 5). Increased expression of p53 was detected in CEM cells treated with 3-IAABE (0.35 μm, 4 and 12 h). Using a mAb specific for ppRb (inactivated protein, phosphorylated at Ser807/Ser811), we found that the ppRb signal was significantly decreased in cells exposed to 3-IAABE for 4 and 12 h, indicating a shift from ppRb to Rb/Rb protein (activated form).

Activation of Apoptotic Pathway.

3-IAABE at a concentration of ≥0.16 μm caused phosphorylation of bcl-2 (Fig. 6), indicated by the appearance of slower-migrating bands in the PAGE electrophoresis (pbcl-2). The earliest bcl-2 phosphorylation was seen 1 h after treatment. Known antimicrotubule agents (i.e., paclitaxel and vinblastine), used as positive controls under identical conditions, demonstrated the same bcl-2 phosphorylation-inducing activity in a similar concentration range.

To identify caspases involved in the mechanism, we measured the catalytic activity of several caspases. Caspase-9 was rapidly elevated within the first hour of 3-IAABE treatment and peaked at 6 h (Fig. 7,A). This was followed by induction of caspase-3 activity at 3 h and induction of caspase-6 at 6 h (Fig. 7,B). Caspase-8, which is critical for the Fas- or tumor necrosis factor receptor-mediated apoptotic pathway, was unaffected by 3-IAABE (Fig. 7 A).

Because DFF and PARP are downstream substrates of caspase-3 and -6 in this cascade, changes in DFF and/or PARP in 3-IAABE-treated tumor cells should be recognizable events after caspase-3 and caspase-6 activation. Indeed, the Mr 45,000 DFF was cleaved into clear Mr 32,000 and Mr 12,000 fragments 3 h after 3-IAABE treatment, indicating activation of the inert DFF (Fig. 8,A, Lanes 1, 2, 4, 6, 8, and 10). Similarly, intact PARP (Mr 116,000) was cleaved into the Mr 85,000 signature fragment (the activated form) starting at 3 h and progressing throughout the 24-h treatment course (Fig. 8 B, Lanes 1, 2, 4, 6, 8, and 10). Apoptotic DNA became visible 6 h after 3-IAABE treatment, and the signal increased with time (data not shown).

Inhibitors of caspase-3, -6, -8, and -9 were used, respectively, to see whether pretreatment of CEM cells could inhibit apoptosis caused by 3-IAABE. As shown in Table 2, the strongest inhibitory effect was observed in cells pretreated with inhibitors of caspase-3 or -9 in which apoptosis caused by 3-IAABE (at 0.1 μg/ml, or 0.35 μm) was reduced to <13–19%. Pretreatment of CEM cells with a caspase-6 inhibitor resulted in partial protection from apoptosis. Inhibition of caspase-8 had no protective effect, even when 3-IAABE was as low as 0.05 μg/ml. The results were further supported by the DFF and PARP analyses using DEVD-FMK (a specific inhibitor of caspase-3), because pretreatment of the cells with DEVD-FMK (10 μm for 24 h) almost completely inhibited the cleavage of DFF and PARP by 3-IAABE (Fig. 8, A and B, Lanes 3, 5, 7, 9, and 11).

Anticancer Activity of 3-IAABE in Human Tumor Cell Lines.

Anticancer activity of 3-IAABE was examined in a wide spectrum of tumor cell lines representing 10 different human tumor types. The experiments were performed in our laboratory (21 selected cell lines) and by the NCI (60 cell lines). The responses of 34 3-IAABE (sensitive to 3-IAABE) sensitive cell lines are presented in Table 3. 3-IAABE had TGI in the range of 0.22–4.3 μm for solid tumor lines and 0.025–0.22 μm for leukemia/lymphoma cell lines. The most sensitive cell line was Daudi (TGI = 0.025 μm), and the least sensitive one was non-small cell lung cancer (TGI = 4.3 μm).

Three nonmalignant normal human cell lines representing three different types of normal tissue (lymphocytes, endothelial cells, and fibroblast tissues) were also tested (Table 3). Concentrations of 3-IAABE required for TGI in these normal cells were much higher than those in tumor cells. TGI of 3-IAABE in PHA-stimulated normal human lymphocytes was 11.3 μm, about 450 times that in Daudi cells and 50 times that in colon cancer and melanoma. Cell cycle analysis showed a mild elevation in G1-S transition in PHA-stimulated lymphocytes treated with 3-IAABE (1.0 μg/ml, 12 h), and the profile returned to normal when the compound was removed (Fig. 9).

Anticancer Effect of 3-IAABE in Human Hepatocarcinoma (BEL-7402) Cells in Nude Mice.

Because BEL-7402 appeared to be one of the most sensitive solid tumor cell lines to 3-IAABE, we chose this hepatocarcinoma cell line to investigate the in vivo activity of the compound. As shown in Fig. 10, when the average tumor volume of the solvent-treated BEL-7402 group was >2000 mm3 on day 42 postimplantation, the 3-IAABE (35 mg/kg, i.p.) group had a tumor size about one-fourth of the solvent controls (72% inhibition). The group treated with low-dose 3-IAABE (10 mg/kg, i.p.) showed no difference in tumor size compared with controls. Apparent toxicity and loss of body weight in 3-IAABE-treated mice (35 mg/kg, i.p.) were not observed. We should also mention here that the parent compound, 3-IAABU, which induced mitotic arrest only (10), showed about 45% inhibition of the tumor volume. The data are not presented because it was hard for us to tell whether the increased inhibition was due to the additional blocking at G1-S transition or the improved solubility.

Comparison of 3-IAABE with Vincristine.

Vincristine, a commonly used anticancer agent in the clinic, was used in this study as a control of known microtubule-active agent. Vincristine showed higher activities in vitro than did 3-IAABE in both malignant (BEL-7402) and nonmalignant cells (HU-VEC); however, the ratio of IC50 (HU-VEC):IC50 (BEL-7402) of 3-IAABE was similar to that of vincristine (Table 4). Furthermore, vincristine (i.p.) at a dose close to that causing severe neurotoxicity (17) showed only 43% growth inhibition of human hepatocarcinoma (BEL-7402) in nude mice, lower than the 72% inhibition by 3-IAABE (Fig. 10).

It is well known that tubulin drugs induce mitotic block. Among these agents, arsenic trioxide (As2O3) is of interest. Arsenic is a differentiation agent and has attracted attention because of its therapeutic effect in the clinic for acute promyelocytic leukemia (21, 22, 23). Experiments showed that arsenic targeted tubulin and caused G1- and M-phase blocks (8, 24, 25). Although the significance of this observation needs to be explored, tubulin-binding compounds may have unknown biological activities in addition to mitotic arrest. As a new tubulin ligand, 3-IAABE provoked our curiosity, mainly because of its in vivo activity against cancer and the double blockade in the cell cycle.

The mechanism by which tubulin ligands cause cell cycle arrest at the G1-S transition needs clarification. Theoretically, there could be at least two explanations for the mechanism by which 3-IAABE induced blockade at G1-S transition. First, 3-IAABE may have molecular target(s) other than β-tubulin; second, 3-IAABE may target the mitochondria-bound tubulin (26, 27), thus interrupting ATP/ADP operation and resulting in blockage of the G1-S transition. [3H]3-IAABE was synthesized to identify the primary targets in tumor cells. Treated cells had all of the radioactivity (cpm) in their protein portion (not in the DNA or RNA fraction); the radioactivity was largely removed (>95%) from the protein pool by immunoprecipitation of tubulin, indicating a binding at tubulin; we failed to identify target protein(s) other than tubulin in the cytosol. For the second possibility, we have detected a considerable amount of [3H]3-IAABE associated with the mitochondrial fraction in the treated cells (data not shown). The primary target of 3-IAABE other than tubulin and the mechanism(s) for the block at G1-S transition are under investigation in our laboratory.

In exploring the interaction between tubulin and 3-IAABE, reducing agents were used. Cysteine-SH is most likely the domain that forms a covalent bond with the iodoacetamido group of 3-IAABE (19, 20, 28), and the interaction might cause a structural modification of tubulin. Comparing the structures of 3-IAABE with 3-IAABU in Table 1, a and b, we assume that the ethyl ester group of 3-IAABE appears to be the key factor responsible for the cell cycle blockade at G1-S transition.

Because it is known that an arrest of cell cycle at G1-S transition can prevent the subsequent accumulation of M phase (29), it is not unexpected to encounter a minor G2-M block compared with the major one at the G1-S transition. To prove that the new peak at G1-S transition was not an artificial one caused by G2-M cells undergoing DNA degradation, p53 and Rb proteins that provide a cell cycle checkpoint at G1-S (30, 31) were analyzed. The increased expression of wild-type p53 and dephosphorylation of ppRb (from the inactivated form to the activated one; Ref. 32) found in 3-IAABE-treated tumor cells provided supporting data for the G1-S arrest found in cell cycle analysis. M-phase block was further examined using morphological criteria, with which we found about 20–25% of 3-IAABE-treated cells (12–24 h) full of scattered metaphase chromosomes in the cytoplasm. This indicates that the G2-M peak was due to blockage of M-phase cells but not G2 cells. The elevated S fraction could be a reflection of the overlap of cells arrested at G1-S transition and M phase or DNA degradation straight out of mitotic arrest.

The cell cycle is an operating biochemical mechanism constructed from a set of regulatory enzymes (cyclins and cdks). The activities of the enzymes alternate cyclically, concordant with the cell cycle downstream processes (33). Cell cycle arrest caused by drugs should therefore be reflected in the kinase activities of cyclins and cdks (34). 3-IAABE caused increases of the kinase activities of cyclin E and cdk2, consistent with the accumulation of cells at the G1-S transition. The enhanced kinase activity of mitotic cyclin B was consistent with the M-phase arrest (29, 34, 35). Because cdc2 is activated by phosphorylation in both the G1-S transition and M phase (33), the elevated activity of cdc2 abetted the accumulation of cells at these two checkpoints. To test the specificity, kinase activities of cyclin A and D1 (relevant to S and G1 phases) were also examined and were without change. The effect of 3-IAABE on the cell cycle (G1-S and M blockade) may make this compound function as a double secure lock to halt tumor cells from proliferation.

3-IAABE-induced phosphorylation of bcl-2 protein was similar to that of other tubulin ligands (36, 37). The posttranslational modification made bcl-2 incapable of forming heterodimers with bax (37, 38) and thus incapable of controlling the mitochondrial permeability transition pores (39, 40). The results of bcl-2 phosphorylation, elevation of caspase activity, DFF cleavage, PARP cleavage, and DNA fragmentation were temporally consistent. The bcl-2 phosphorylation and increased activity of caspase-9 occurred rapidly, within 1 h of treatment. Caspase-3 and -6 followed soon after, leading to cleavage of DFF and PARP at 3–6 h and DNA laddering at 6–9 h. Because the pathway of Fas and tumor necrosis factor receptors (associated with apoptosis caused by inflammation and/or infection) leading to activation of caspase-8 (41) was not involved, the bcl-2-caspase-9 pathway is clearly and solely engaged by 3-IAABE (42, 43). The pretreatment experiments with various caspase inhibitors provided solid support for this conclusion. For the sake of consistency, most of the results of mechanism studies presented in this paper are in CEM, one of the most sensitive tumor cell lines to 3-IAABE. Experiments with other cell lines such as Daudi, U937, BEL-7402, and DND-1A (human melanoma) showed an action similar to that of CEM.

Because of the above-mentioned characteristics, 3-IAABE is considered to be a new class of cancericidal tubulin ligand. Diversity of susceptibility to 3-IAABE was noted in human tumor cell lines. Hematological tumors were, generally speaking, sensitive to 3-IAABE. Non-small cell lung cancer was the least susceptible. An impressive selectivity was seen between malignant and nonmalignant cells. MDR+ lymphoma cells (Daudi/MDR) showed a similar sensitivity to 3-IAABE as MDR− lymphoma cells (Daudi/w), indicating that this compound was not a substrate of P-glycoprotein. Compared with 3-IAABE, vincristine was more potent to human hepatocarcinoma cells in culture but was also more toxic to human nonmalignant HU-VEC cells. Their safety ratios [IC50 (HU-VEC):IC50 (BEL-7402)] were close. The high inhibitory efficiency of Vinca alkaloids in microtubule assembly (ID50 in the 0.3–0.4 μm range) is largely responsible for the discrepancy (44, 45). In in vivo experiments, 3-IAABE demonstrated a stronger inhibitory effect than vincristine on the growth of human hepatocarcinoma in nude mice. These results indicate that 3-IAABE might have other cellular target(s) in addition to tubulin molecules through its interaction with −SH groups and/or a favorable bioavailability in vivo.

Because 3-IAABE has a specific target, induced a double blockade in the cell cycle, used a direct pathway in tumor cells leading to apoptosis, and showed a promising activity against human solid tumor in vivo, we believe that 3-IAABE is a lead compound for designing new cancericidal tubulin ligands and merits consideration for clinical trial.

Fig. 1.

Inhibition of microtubule assembly by 3-IAABE. A, microtubule assembly in the absence or presence of 3-IAABE was measured by absorbance at 340 nm; 3-IAABE showed a significant inhibition on the assembly at concentrations over 1.5 μm. B, inhibitory effect of 3-IAABE (7.5 μm) on microtubule polymerization was reduced in the presence of either DTT (10 μm) or β-ME (10 μm).

Fig. 1.

Inhibition of microtubule assembly by 3-IAABE. A, microtubule assembly in the absence or presence of 3-IAABE was measured by absorbance at 340 nm; 3-IAABE showed a significant inhibition on the assembly at concentrations over 1.5 μm. B, inhibitory effect of 3-IAABE (7.5 μm) on microtubule polymerization was reduced in the presence of either DTT (10 μm) or β-ME (10 μm).

Close modal
Fig. 2.

Blockade of cell cycle at the G1-S transition and M phase in CEM cells treated with 3-IAABE. Cell cycle analysis of cells treated with 0.35 μm 3-IAABE was done by flow cytometry at 0, 4, 12, and 24 h (a−d) after treatment. The major block was at the G1-S transition at 12 h (arrow), and the minor one was at G2-M phase at 24 h (arrow).

Fig. 2.

Blockade of cell cycle at the G1-S transition and M phase in CEM cells treated with 3-IAABE. Cell cycle analysis of cells treated with 0.35 μm 3-IAABE was done by flow cytometry at 0, 4, 12, and 24 h (a−d) after treatment. The major block was at the G1-S transition at 12 h (arrow), and the minor one was at G2-M phase at 24 h (arrow).

Close modal
Fig. 3.

Metaphase block by 3-IAABE in human leukemia/lymphoma cell lines. A, untreated CEM cells; B, treated CEM cells; C, treated Daudi cells; D, treated U937 cells. The cells shown in B−D were treated with 0.35 μm 3-IAABE for 24 h. Untreated Daudi and U937 cells had a morphology similar to A. Giemsa staining (×400).

Fig. 3.

Metaphase block by 3-IAABE in human leukemia/lymphoma cell lines. A, untreated CEM cells; B, treated CEM cells; C, treated Daudi cells; D, treated U937 cells. The cells shown in B−D were treated with 0.35 μm 3-IAABE for 24 h. Untreated Daudi and U937 cells had a morphology similar to A. Giemsa staining (×400).

Close modal
Fig. 4.

Effects of 3-IAABE on kinase activities of cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1. CEM cells were exposed to 0.35 μm 3-IAABE. At the indicated time points, cells were sampled for kinase activity determination (see “Materials and Methods”). A−F, autoradiography of 32P incorporation into histone H1 by cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1, respectively; a−f, relative kinase activities of cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1, as determined by counting 32P incorporation into histone H1. The experiment was repeated twice.

Fig. 4.

Effects of 3-IAABE on kinase activities of cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1. CEM cells were exposed to 0.35 μm 3-IAABE. At the indicated time points, cells were sampled for kinase activity determination (see “Materials and Methods”). A−F, autoradiography of 32P incorporation into histone H1 by cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1, respectively; a−f, relative kinase activities of cyclin E, ckd2, cyclin B, cdc2, cyclin A, and cyclin D1, as determined by counting 32P incorporation into histone H1. The experiment was repeated twice.

Close modal
Fig. 5.

Increased expression of wild-type p53 and dephosphorylation (activation) of ppRb in CEM cells treated with 3-IAABE. CEM cells were treated with 3-IAABE (0.35 μm) for 0 (control), 4, and 12 h, followed by protein extraction. Immunoblot was done with the method described in “Materials and Methods.” Increased p53 expression and reduced signal of ppRb (dephosphorylation and activation of ppRb) became visible 4 h after treatment. ppRb, Rb protein phosphorylated at Ser807/Ser811.

Fig. 5.

Increased expression of wild-type p53 and dephosphorylation (activation) of ppRb in CEM cells treated with 3-IAABE. CEM cells were treated with 3-IAABE (0.35 μm) for 0 (control), 4, and 12 h, followed by protein extraction. Immunoblot was done with the method described in “Materials and Methods.” Increased p53 expression and reduced signal of ppRb (dephosphorylation and activation of ppRb) became visible 4 h after treatment. ppRb, Rb protein phosphorylated at Ser807/Ser811.

Close modal
Fig. 6.

3-IAABE induced bcl-2 phosphorylation in leukemic cells. CEM cells were treated with 3-IAABE for 24 h at concentrations of 0, 0.08, 0.16, 0.32, 0.8, and 1.6 μm, respectively. Paclitaxel and vinblastine (0.12 μm, 24 h for both) were used in the experiment as positive controls. pbcl-2, phosphorylated bcl-2.

Fig. 6.

3-IAABE induced bcl-2 phosphorylation in leukemic cells. CEM cells were treated with 3-IAABE for 24 h at concentrations of 0, 0.08, 0.16, 0.32, 0.8, and 1.6 μm, respectively. Paclitaxel and vinblastine (0.12 μm, 24 h for both) were used in the experiment as positive controls. pbcl-2, phosphorylated bcl-2.

Close modal
Fig. 7.

Increased caspase activities in CEM cells treated with 3-IAABE (0.35 μm). A, activator caspases: activity of caspase 9 (right-hatched bars), but not caspase 8 (cross-hatched bars), was increased beginning the first hour after treatment. B, executioner caspases: activity of caspase 3 (stippled bars) increased within 3 h, before the elevation of caspase 6 (horizontal-lined bars).

Fig. 7.

Increased caspase activities in CEM cells treated with 3-IAABE (0.35 μm). A, activator caspases: activity of caspase 9 (right-hatched bars), but not caspase 8 (cross-hatched bars), was increased beginning the first hour after treatment. B, executioner caspases: activity of caspase 3 (stippled bars) increased within 3 h, before the elevation of caspase 6 (horizontal-lined bars).

Close modal
Fig. 8.

Cleavage of DFF and PARP by 3-IAABE in CEM leukemic cells. A, the intact and cleaved DFF are at Mr 45,000, 32,000, and 12,000, respectively. B, the intact and cleaved PARP are at Mr 116,000 and 85,000, respectively. Lane 1, untreated CEM cells; Lanes 2, 4, 6, 8, and 10, 1, 3, 6, 12, and 24 h after treatment with 3-IAABE (0.35 μm), respectively; Lanes 3, 5, 7, 9, and 11, pretreatment with DEVD-FMK (caspase-3 inhibitor; 10 μm for 24 h) followed by 1, 3, 6, 12, and 24 h treatment of 3-IAABE (0.35 μm), respectively.

Fig. 8.

Cleavage of DFF and PARP by 3-IAABE in CEM leukemic cells. A, the intact and cleaved DFF are at Mr 45,000, 32,000, and 12,000, respectively. B, the intact and cleaved PARP are at Mr 116,000 and 85,000, respectively. Lane 1, untreated CEM cells; Lanes 2, 4, 6, 8, and 10, 1, 3, 6, 12, and 24 h after treatment with 3-IAABE (0.35 μm), respectively; Lanes 3, 5, 7, 9, and 11, pretreatment with DEVD-FMK (caspase-3 inhibitor; 10 μm for 24 h) followed by 1, 3, 6, 12, and 24 h treatment of 3-IAABE (0.35 μm), respectively.

Close modal
Fig. 9.

Cell cycle analysis of human PBLs treated with 3-IAABE. PBLs were pretreated with PHA for 24 h, producing PHA-activated PBLs (PHA-PBLs). PHA-PBLs were then treated with 3-IAABE (1.0 μm) for 12 h. Cell cycle was analyzed in the PHA-PBL samples [a, cells with no 3-IAABE treatment; b, cells treated with 3-IAABE for 12 h; c, cells after 12 h of treatment with 3-IAABE], the compound was removed by centrifugation, and the cells were reincubated in the fresh medium for 24 h.

Fig. 9.

Cell cycle analysis of human PBLs treated with 3-IAABE. PBLs were pretreated with PHA for 24 h, producing PHA-activated PBLs (PHA-PBLs). PHA-PBLs were then treated with 3-IAABE (1.0 μm) for 12 h. Cell cycle was analyzed in the PHA-PBL samples [a, cells with no 3-IAABE treatment; b, cells treated with 3-IAABE for 12 h; c, cells after 12 h of treatment with 3-IAABE], the compound was removed by centrifugation, and the cells were reincubated in the fresh medium for 24 h.

Close modal
Fig. 10.

Inhibition of human hepatocarcinoma (BEL-7402) by 3-IAABE in nude mice. BEL-7402 tumor tissue (6–8 mm3) was s.c. transplanted into the mice. Six days later, the mice with visible tumor mass were randomly distributed into cages (6–8 mice/group). Treatment (10 or 35 mg/kg 3-IAABE, i.p.; 0.3 mg/kg vincristine, i.p.) was started on day 6, when the tumor size was about 90 mm3 (“Materials and Methods”). 3-IAABE at 10 mg/kg (i.p.) was without significant effect, and the curve is not shown to preserve clarity. Compounds were given at the time tumor volume was measured. Each point represents the mean ± SE of the tumor volume.

Fig. 10.

Inhibition of human hepatocarcinoma (BEL-7402) by 3-IAABE in nude mice. BEL-7402 tumor tissue (6–8 mm3) was s.c. transplanted into the mice. Six days later, the mice with visible tumor mass were randomly distributed into cages (6–8 mice/group). Treatment (10 or 35 mg/kg 3-IAABE, i.p.; 0.3 mg/kg vincristine, i.p.) was started on day 6, when the tumor size was about 90 mm3 (“Materials and Methods”). 3-IAABE at 10 mg/kg (i.p.) was without significant effect, and the curve is not shown to preserve clarity. Compounds were given at the time tumor volume was measured. Each point represents the mean ± SE of the tumor volume.

Close modal

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

Supported by the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research and by National Natural Science Foundation of the People’s Republic of China (Grant 398708889 and 39930190).

3

The abbreviations used are: 3-IAABE, 3-iodoacetamido benzoyl ethyl ester; 3-IAABU, 3-iodoacetamido benzoylurea; cdk, cyclin-dependent kinase; Rb, retinoblastoma; ppRb, phosphorylated Rb protein; DFF, DNA fragmentation factor; PARP, poly(ADP-ribose) polymerase; NCI, National Cancer Institute; TGI, total growth inhibition; PHA, phytohemagglutinin; PMSF, phenylmethylsulfonyl fluoride; mAb, monoclonal antibody; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; β-ME, β-mercaptoethanol; MDR, multidrug-resistant; PBL, peripheral blood lymphocyte.

Table 1

Chemical structures of 3-IAABE (a) and 3-IAABU (b)

Chemical structures of 3-IAABE (a) and 3-IAABU (b)
Chemical structures of 3-IAABE (a) and 3-IAABU (b)
Table 2

Effects of caspase inhibitors on apoptosis caused by 3-IAABE

TreatmentPretreatment with inhibitor (10 μm)aCytotoxicity
Survival (%)Apoptosis (%)b
Solvent control None 96 ± 7 4 ± 0.6 
3-IAABE None 9 ± 1 91 ± 5 
 Caspase-8 8 ± 1 92 ± 9 
 Caspase-9 87 ± 10 13 ± 3 
 Caspase-3 81 ± 9 19 ± 2 
 Caspase-6 54 ± 8 46 ± 4 
TreatmentPretreatment with inhibitor (10 μm)aCytotoxicity
Survival (%)Apoptosis (%)b
Solvent control None 96 ± 7 4 ± 0.6 
3-IAABE None 9 ± 1 91 ± 5 
 Caspase-8 8 ± 1 92 ± 9 
 Caspase-9 87 ± 10 13 ± 3 
 Caspase-3 81 ± 9 19 ± 2 
 Caspase-6 54 ± 8 46 ± 4 
a

CEM cells were pretreated with 10 μm caspase inhibitor for 24 h, and 0.1 μg/ml (0.35 μm) 3-IAABE was added to induce apoptosis. The inhibitors of caspase-8, -9, -3, and -6 were Z-IETD-FMK, LEHD-FMK, Z-DEVD-FMK, and VEID-FMK, respectively.

b

Apoptosis was measured by flow cytometry (see “Materials and Methods”).

Table 3

Cancericidal activity of 3-IAABE in vitro

Human tumor typeCell lineCytotoxic activity (μm)a
IC50TGI
Leukemia CCRF-CEM 0.018 0.048 
 Molt-4 0.031 0.22 
 SR 0.035 0.15 
Burkit lymphoma Daudi/w 0.013 0.025 
 Daudi/MDR 0.012 0.031 
Hepatocarcinoma BEL-7402 0.12 0.46 
Non-small cell lung cancer NCI-H23 0.17 0.73 
 NCI-H226 2.05 4.3 
 Hop-62 0.38 0.84 
 Hop-92 0.62 1.24 
Colon cancer HCT-15 0.13 0.46 
 HT-29 0.088 0.22 
 COLO 205 0.17 0.61 
CNSb cancer SF-539 0.218 0.518 
 SF-295 0.88 1.96 
 U251 0.13 0.49 
Melanoma LOX IMVI 0.126 0.332 
 M14 0.098 0.35 
 SK-Mel-2 0.2 0.69 
 SK-Mel-5 0.1 0.23 
Ovarian cancer IGROV1 0.244 0.557 
 OVCAR-3 0.27 0.76 
 OVCAR-4 0.53 3.02 
 SK-OV-3 1.66 3.12 
Renal cancer 786-0 0.155 0.289 
 CAKI-1 0.2 0.39 
 TK-10 0.83 2.07 
 UO-31 0.24 0.63 
Prostate cancer PC-3 1.14 2.43 
 DU-145 1.85 3.35 
Breast cancer MDA-MB-435 0.208 0.484 
 BT-549 0.39 1.56 
 MCF7 0.36 1.33 
 NCI/ADR-RES 0.4 
Normal human cells    
Lymphocytes PHA stimulated 5.3 11.4 
Endothelial cells HU-VEC 2.6 7.3 
Prostate fibroblasts PC-41 12.3 
Human tumor typeCell lineCytotoxic activity (μm)a
IC50TGI
Leukemia CCRF-CEM 0.018 0.048 
 Molt-4 0.031 0.22 
 SR 0.035 0.15 
Burkit lymphoma Daudi/w 0.013 0.025 
 Daudi/MDR 0.012 0.031 
Hepatocarcinoma BEL-7402 0.12 0.46 
Non-small cell lung cancer NCI-H23 0.17 0.73 
 NCI-H226 2.05 4.3 
 Hop-62 0.38 0.84 
 Hop-92 0.62 1.24 
Colon cancer HCT-15 0.13 0.46 
 HT-29 0.088 0.22 
 COLO 205 0.17 0.61 
CNSb cancer SF-539 0.218 0.518 
 SF-295 0.88 1.96 
 U251 0.13 0.49 
Melanoma LOX IMVI 0.126 0.332 
 M14 0.098 0.35 
 SK-Mel-2 0.2 0.69 
 SK-Mel-5 0.1 0.23 
Ovarian cancer IGROV1 0.244 0.557 
 OVCAR-3 0.27 0.76 
 OVCAR-4 0.53 3.02 
 SK-OV-3 1.66 3.12 
Renal cancer 786-0 0.155 0.289 
 CAKI-1 0.2 0.39 
 TK-10 0.83 2.07 
 UO-31 0.24 0.63 
Prostate cancer PC-3 1.14 2.43 
 DU-145 1.85 3.35 
Breast cancer MDA-MB-435 0.208 0.484 
 BT-549 0.39 1.56 
 MCF7 0.36 1.33 
 NCI/ADR-RES 0.4 
Normal human cells    
Lymphocytes PHA stimulated 5.3 11.4 
Endothelial cells HU-VEC 2.6 7.3 
Prostate fibroblasts PC-41 12.3 
a

Cell growth inhibition was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method of NCI screening program.

b

CNS, central nervous system.

Table 4

Comparison of 3-IAABE with vincristine in vitro

CompoundIC50m)Ratioa
HU-VECBEL-7402
3-IAABE 2.6 ± 0.3 0.12 ± 0.2 22 
Vincristine 0.1 ± 0.007 0.004 ± 0.001 25 
CompoundIC50m)Ratioa
HU-VECBEL-7402
3-IAABE 2.6 ± 0.3 0.12 ± 0.2 22 
Vincristine 0.1 ± 0.007 0.004 ± 0.001 25 
a

Ratio, IC50(HU-VEC):IC50(BEL-7402).

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