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Rational Design of the Microtubule-Targeting Anti–Breast Cancer Drug EM015

Departments of ¹ Cell Biology, ² Digestive Diseases, ³ Chemistry, and ⁴ Pathology, Emory University School of Medicine, Atlanta, Georgia; ⁵ School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India; ⁶ Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China; and ⁷ BR Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

Requests for reprints: Harish C. Joshi, Department of Cell Biology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322. Phone: 404-727-0445; Fax: 404-727-6256; E-mail: joshi{at}cellbio.emory.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, -, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti–breast cancer agent and displays a 20-fold lower IC₅₀ against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic. (Cancer Res 2006; 66(7): 3782-91)

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Microtubules are dynamic intracellular polymers and provide tracks on which bidirectional transport of intracellular organelles and subcellular machines takes place (1, 2). Perhaps the most dramatic physiologic role of microtubule dynamics is in the segregation of duplicated chromosomal DNA during mitosis. Perturbed microtubule dynamics or damaged apparatus halts the cell cycle usually in prometaphase by the activation of cell cycle checkpoints followed by cell death (3). Many microtubule-binding agents that compromise dynamics, such as paclitaxel and Vincas, have now been proposed to work at low concentrations via their subtle effects on microtubule dynamics (4). The first proposed mechanism of cell death following abortive mitosis, the so-called mitotic catastrophe, was suggested by Sir Paul Nurse (5). Subsequently, we and others have observed that drug-treated abnormal mitotic cells exit without cytokinesis, yielding genotoxic amounts of DNA that appears as different-sized blobs of nuclei (micronuclei), causing apoptosis by activating signaling cascades such as the c-jun NH₂-terminal kinase (6–9) and nuclear factor B pathways (10).

The cell cycle mechanisms are built from multicomponent protein machines, genes of which often harbor mutational lesions that attenuate or obliterate checkpoint mechanisms in a variety of tumor types (11–13). Halted cell cycles usually signal to cell death mechanisms, the precise nature of which remains to be known. Nevertheless, small molecules that interfere with microtubule function are clinically used for the management of many cancer types. Although antimicrotubule agents often successfully manage many cancers for varying amounts of time, their success has been somewhat limited because drug-resistant tumors keep recurring (14). Overexpression of drug-efflux pumps, mutations in tubulin itself, overexpression of different tubulin genes, and their posttranslational modifications alter affinities to specific drugs and thus are thought to be important for conferring resistance to treatment (15–18). Therefore, there is a tremendous opportunity to develop novel tubulin/microtubule-binding drugs that preferentially interact with either different binding pockets or different genotypic or posttranslational tubulin isotypes.

We recently discovered the tubulin-binding property (with 1:1 stoichiometry) of a naturally occurring phthalideisoquinoline alkaloid, noscapine (19), known to be a well-tolerated, orally available, nontoxic anti-cough drug in humans (20, 21). Circular dichroism spectra suggest structural changes but the morphology or polymer mass of microtubule arrays is not altered by noscapine treatment because noscapine merely decreases transition frequencies between growth and shortening phases of microtubules over a broad concentration range (19, 22). This represents an improvement over the overpolymerizers/microtubule-binding agents, such as taxanes, or microtubule-depolymerizers, such as Vincas, which cause toxicities in tissues with active mitoses and postmitotic neurons (23). Furthermore, noscapine inhibits proliferation of a wide variety of human cancer cells including lymphoma, thymoma, melanoma, and gliomas, both in vitro and in animal models (6, 19, 22, 24, 25). We now report the rational in silico design of a novel noscapine analogue, EM015, which shows a 20-fold lower IC₅₀ and induces more potent apoptosis, compared with noscapine, in human breast cancer cells but not in primary human cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Materials. Mammalian brain microtubule proteins were isolated by two cycles of polymerization and depolymerization and tubulin was separated from the microtubule binding proteins by phosphocellulose chromatography as described (26). The tubulin solution was stored at –80°C until use. Noscapine was purchased from Sigma (St. Louis, MO).

Mice and cell lines. Eight- to ten-week-old female BALB/c athymic nude mice (nu/nu) were from Harlan-Sprague Laboratories (Indianapolis, IN). Human breast adenocarcinoma cells (MCF-7 and MDA-MB-231) were from American Type Culture Collection (Manassas, VA). Human fibroblast primary cultures (HDF), near-normal human breast epithelial cells (MCF-10A), and other breast cancer cells such as BT-474, SK-Br3, T47D, and ER-MDA-MB-231 were from Emory University (Atlanta, GA). MTR-3 cells were from Dr. Maricarmen Panigone-Silva (Penn State College of Medicine, Hershey, PA) and CEM, CEM/VLB100, and CEM/VM-1-5 cells were from Dr. William Beck (University of Illinois at Chicago, Chicago, IL). MCF-7 and HDF cells were maintained in DMEM with 4.5 g/L glucose and L-glutamine (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). MTR-3 cells were grown in phenol red–free DMEM supplemented with 4 mmol/L L-glutamine, 5% charcoal-cleared FBS (HyClone, Logan, UT) and 1 µmol/L tamoxifen (Sigma). MDA-MB-231, ER-MDA-MB-231, BT-474, SK-Br3, T47D, A2780, A2780/ADR, 1A9, 1A9/PTX10, CEM, CEM/VLB100, and CEM/VM-1-5 were grown in RPMI 1640 supplemented with 10% FBS. All cells were grown at 37°C in 5% CO₂/95% air atmosphere. Cell viability was assessed by counting trypan blue excluding cells using a hemocytometer.

Molecular modeling studies. Nuclear Overhauser and Exchange Spectroscopy was used to measure the distance between spatially close protons in noscapine using the method of Esposito and Pastore (27). This structure was then docked onto the 3.5-Å coordinates obtained from Vinca-induced paracrystals of tubulin (kindly provided by Dr. Kenneth Downing of Lawrence Berkeley Labs, Berkeley, CA). The orientation of noscapine bound tubulin in silico in its putative pocket is clearly marked with respect to significant amino acids and is color coded, with red depicting oxygen, blue depicting nitrogen, yellow showing sulfur, and white representing carbon (Fig. 1A ). As shown in the in silico model (Fig. 1A), there is a gaping hole between the bound noscapine and a nearby positively charged histidine on loop 7, thus providing an opportunity to incorporate an electronegative atom of small electron cloud radius of 1.81 Å, such as chlorine, to minimize the free energy of noscapine-tubulin complex.

View larger version (22K): [in this window] [in a new window]   Figure 1. Rational design and synthesis of a noscapine analogue, EM015. Derived from in silico simulations of in-solution conformer states of noscapine, we docked the most represented conformer onto the 3.5-Å resolution coordinates of tubulin structure. A, noscapine docked onto ß-tubulin near its interaction surface with -tubulin. A flexible loop spans positions 1, 9, and 8 on the isoquinoline ring system of noscapine in an open-palm state [see (B) for numbering scheme]. These positions include an acidic proton at position 9 apposed to a histidine residue in the spanning loop. This gave an opportunity to increase electrostatic interactions by substituting the proton at position 9 with a small electronegative halogen atom such as chlorine (electron cloud = 1.81 Å) as shown in A (right, white arrow, position 9). B, synthetic scheme for chlorination of noscapine to yield EM015. To achieve chlorination without cleavage of vulnerable bonds, particularly the C-C bond between the two ring systems, we used sulfuryl chloride in chloroform at 25°C for 10 hours. Under these mild conditions, the disappearance of one aromatic singlet proton of C-9 at 6.30 ppm in the 1H NMR without disrupting any methoxy groups present on both ring systems of the starting compound is evident from the spectral data provided in Results and Discussion. As expected from our modeling studies, EM015 binds tubulin with higher affinity than noscapine as determined by saturable internal fluorescence quenching of its amino acids, tryptophans and tyrosines, at an excitation wavelength of 295 nm. The fluorescence emission intensity of noscapine and EM015 at this excitation wavelength was negligible. A 0.3-cm path-length cuvette was used to minimize the inner filter effects caused by the absorbance of these agents at higher concentration ranges. In addition, the inner filter effects were corrected using the formula Fcorrected = Fobserved x antilog [(Aex + Aem) / 2], where Aex is the absorbance at the excitation wavelength and Aem is the absorbance at the emission wavelength. The dissociation constant (Kd) was determined by the formula 1/B = Kd / [free ligand] + 1, where B is the fractional occupancy and [free ligand] is the concentration of free noscapine or EM015. The fractional occupancy (B) was determined by the formula B = F / Fmax, where F is the change in fluorescence intensity when tubulin and its ligand are in equilibrium and Fmax is the value of maximum fluorescence change when tubulin is completely bound with its ligand. Fmax was calculated by plotting 1/F versus 1/ligand using total ligand concentration as the first estimate of free ligand concentration. D, left, fluorescence quenching spectrum of tubulin for EM015; right, double reciprocal plot which gives a Kd of 40 ± 8 µmol/L for EM015 binding to tubulin. This is in contrast to data from the parent compound noscapine, as shown in C (left and right). Like noscapine, EM015 does not significantly change the polymerization rate and the steady-state polymer mass of purified tubulin in vitro (E).

Chlorination of noscapine. EM015 was synthesized by dropwise addition (over 1 hour at 5°C) of 36.28 mmol/L sulfuryl chloride in chloroform (100 mL) to a solution of 12.01 mmol/L noscapine in chloroform (200 mL) and stirred for 10 hours at 25°C until subjected to TLC (7% methanol in chloroform; ref. 28). On completion of the reaction, the mixture was poured into 300 mL of water and extracted with chloroform twice. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, evaporated in vacuo, and purified using flash chromatography (7% methanol in chloroform) to yield the desired product, (S)-3-((R)-9-chloro-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxy-isobenzofuran-1(3H)-one (see Results and Discussion for spectral analyses).

Tubulin binding and polymerization assay. Fluorescence titration for determining the tubulin binding variables was done as described (29). In brief, noscapine and EM015 (0-100 µmol/L) were incubated with 2 µmol/L tubulin in 25 mmol/L PIPES (pH 6.8), 3 mmol/L MgSO₄, and 1 mmol/L EGTA for 45 minutes at 37°C. The relative intrinsic fluorescence intensity of tubulin was then monitored in a JASCO FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) using a 0.3-cm path-length cuvette at an excitation wavelength of 295 nm. For the tubulin polymerization assay, mammalian brain tubulin (1.0 mg/mL) was mixed with different concentrations of noscapine (25 or 100 µmol/L) or EM015 (25 or 100 µmol/L) at 0°C in an assembly buffer [100 mmol/L PIPES (pH 6.8), 3 mmol/L MgSO₄, 1 mmol/L EGTA, 1 mmol/L GTP, and 1 mol/L sodium glutamate] and polymerization was initiated by raising the temperature to 37°C in a water bath. The rate and extent of the polymerization reaction were monitored by light scattering at 550 nm using a 0.3-cm path-length cuvette in a JASCO FP-6500 spectrofluorometer for 30 minutes.

In vitro cell proliferation assay. The cell proliferation assay was done in 96-well plates as described (30). In brief, 5 x 10³ cells seeded in each well were incubated with increasing concentrations of noscapine or EM015 for 72 hours. Cells were then fixed with 50% trichloroacetic acid, stained with 0.4% sulforhodamine B, followed by washing with 1% acetic acid to remove the unbound dye. The protein-bound dye was extracted with 10 mmol/L Tris base to determine the absorbance at 564-nm wavelength using a SPECTRAmax PLUS 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

Cell cycle analysis. The flow cytometric evaluation of cell cycle status was done as described (30). Briefly, MCF-7 cells were treated with 50 µmol/L noscapine or 5 µmol/L EM015 for 0, 12, 24, 48, and 72 hours. HDF and MCF-10A cells were treated with a 10-fold higher concentration (50 µmol/L EM015) than the therapeutic 5 µmol/L dose for 0, 24, 48, and 72 hours. After drug incubation, cells were centrifuged, washed twice with ice-cold PBS, and fixed in 70% ethanol for at least 24 hours. Cell pellets were then washed with PBS and stained with 0.5 mL of RNase A (2 mg/mL) and 0.5 mL of propidium iodide (0.1% in 0.6% Triton X-100 in PBS) for 45 minutes in the dark followed by analysis on a FACSCalibur flow cytometer (Beckman Coulter, Inc., Fullerton, CA).

Immunofluorescence microscopy. Cells were grown on poly-L-lysine–coated glass coverslips for immunofluorescence microscopy as described (30). After treatment with 5 µmol/L EM015 for 0, 0.5, 24, and 48 hours, cells were fixed with cold (–20°C) methanol for 5 minutes, washed with PBS for 5 minutes, blocked with 2% bovine serum albumin (BSA)/PBS at 37°C for 15 minutes, and incubated with a mouse monoclonal antibody against -tubulin (DM1A, Sigma; 1:500 in 2% BSA/PBS) for 2 hours at 37°C. Cells were then washed with 2% BSA/PBS for 10 minutes before incubating with a 1:200 dilution of a FITC-labeled goat anti-mouse immunoglobulin G antibody (Jackson ImmunoResearch, Inc., West Grove, PA) at 37°C for 1 hour. Coverslips were rinsed with 2% BSA/PBS for 10 minutes and incubated with propidium iodide (0.5 µg/mL) for 15 minutes before mounting with Aquamount (Lerner Laboratories, Pittsburgh, PA) containing 0.01% 1,4-diazobicyclo(2,2,2)octane (Sigma). Cells were examined using confocal microscopy for microtubule morphology, nuclear morphology, and the number of cells in mitosis (at least 100 cells were examined per condition).

Annexin V staining and terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling assay for apoptosis. For Annexin V staining, MCF-7 cells were exposed to 5 µmol/L EM015 for 48 hours. Adherent cells harvested by mild trypsinization were pooled together with detached cells followed by staining with Alexa Fluor 488–conjugated Annexin V and propidium iodide using the Vybrant Apoptosis Assay Kit from Molecular Probes (Eugene, OR) as per protocol of the manufacturer. The 3' ends of DNA breaks were identified and quantified by using the terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL) assay. Briefly, cells were incubated with 5 µmol/L EM015 for 72 hours, pelleted, washed with ice-cold PBS twice, fixed in 1% paraformaldehyde, and apoptosis was detected using the APO-BrdU TUNEL Assay Kit (Molecular Probes) according to the instructions of the manufacturer. Propidium iodide (total cellular DNA) and Alexa Fluor 488 (apoptotic cells) were the two dyes used. Two-color flow cytometric analyses for both assays were done on a FACSCalibur equipped with an argon ion laser. Confocal micrographs were also obtained for Annexin V– and TUNEL-stained cells using a 63x objective (numerical aperture, 1.4).

In vivo analysis of breast carcinoma progression. Eight- to 10-week-old female athymic nude mice were housed in the Emory University Animal Care Facility. Suspensions of 10⁶ MDA-MB-231 cells in 0.2 mL of PBS were inoculated s.c. into the anterior flank. When tumors were palpable and measurable, usually after 7 to 10 days, mice were randomly divided into three groups of eight animals each. One group of mice received the vehicle solution alone (water, pH 4.0) by daily gavage whereas the other two groups were orally fed 300 mg/kg body weight of EM015 or noscapine in the same vehicle solution. We included the noscapine treatment group for a comparison. Tumor volumes were determined daily by measuring three perpendicular diameters using vernier calipers and the volume was calculated as /6 (length x width x height) (31). The rapid growth of s.c. injected MDA-MB-231 breast cancer cells required that control vehicle-treated animals be euthanized at day 24 after inoculation owing to their exceedingly large tumor volumes and in compliance with the criteria set by the Institutional Animal Care and Use Committee. This served as an "end point" for control animals. The two treatment groups (EM015 and noscapine) with regressed tumors were further followed for long-term survival and were sacrificed on day 80. Thus, in survival studies, the end point of treatment groups was 80 days.

Evaluation of immune cells. At the end point of each experimental group, blood was collected from the retro-orbital sinuses of mice and mononuclear cells were isolated using Histopaque-1077 (Sigma) polysucrose density gradient centrifugation. After centrifugation, they appear as an opaque band at the interface between plasma and Histopaque-1077, which were collected by aspiration with a siliconized pasteur pipet and subjected to fluorescence-activated cell sorting analysis (FACS) analysis after staining. The four-color panels included CD4/NK1.1/CD8/CD3 and immunoglobulin M/CD3/B220/immunoglobulin D in FITC, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin channels, respectively.

Histopathologic and hematologic analyses. At the end points of control and treatment groups (24 and 80 days, respectively), blood was collected by cardiac puncture and complete blood count analysis was done using a complete blood count instrument (CDC Technologies, Oxford, CT). Liver, kidney, spleen, lung, heart, brain, intestines, sciatic nerve, and tumors were stained with H&E as described (22). The sciatic nerve was also stained using Luxol fast blue/periodic acid-Schiff stain. Microscopic evaluation was done blindly by two pathologists.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The rational design of EM015. The first step in our strategy to rationally design more potent noscapine analogues was to model the docking of noscapine onto the target molecule tubulin in silico. The crystal structures of both noscapine and its target molecule tubulin are known (32, 33) and this allowed us to dock various in silico noscapine conformer states onto 3.5-Å tubulin coordinates (kindly provided by Dr. Kenneth Downing) to determine the lowest free-energy bound state. The criteria included considerations of surface charge, -, van der Waals, and hydrogen bonding interactions.

Noscapine docks onto ß-tubulin near the interface with its dimerization partner, -tubulin (34). This is consistent with our previously found 1:1 stoichiometry of tubulin binding (19). A closer look at the binding site revealed interactions with interesting side chains surrounding the putative binding pocket and the presence of an empty space around position 9 of noscapine (Fig. 1A). This position is bounded by a flexible loop between the sixth -helix and the seventh ß-sheet and has positively charged amino acids (e.g., histidine). This space can possibly accommodate small chemical moieties (Fig. 1A). The orientation of noscapine-bound tubulin in its putative pocket is clearly marked with respect to significant amino acids and is color coded, with red depicting oxygen, blue depicting nitrogen, yellow showing sulfur atom, and white representing carbon (Fig. 1A). In our lead compound, noscapine, the hydrogen at position 9 occupies a 1.2-Å radius electron cloud, as determined by Gaussian ab initio calculations. We envisioned that an electronegative halogen atom, such as a chlorine, which has an electron cloud radius of 1.81 Å, can fit in the vicinity of this flexible loop. This allowed us to hypothesize that it might confer additional electrostatic interactions and may lead to enhanced biological activity.

Selective chlorination of noscapine. To test this hypothesis, we synthesized a chlorinated analogue of noscapine, EM015 (Fig. 1B). EM015 was prepared under mild conditions by aromatic halogenation of noscapine using sulfuryl chloride in chloroform at 25°C yielding the desired compound (ref. 28; Fig. 1B). The chlorination took place regioselectively on the isoquinoline ring nucleus at position 9 and was confirmed by NMR spectra that show the disappearance of C-9 proton at 6.30 ppm. Furthermore, the structure was confirmed using high-resolution mass spectroscopy. The detailed spectral data are shown below (see Box 1).

EM015 significantly inhibits proliferation of cancer cells resistant to tamoxifen, vinblastine, teniposide, adriamycin, and paclitaxel. The growth and aggressiveness of breast cancer cells are often dependent on the hormones, estrogen and progesterone, which are bound by receptors such as Her-2. We used a panel of six breast cancer cells with diverse hormone receptor status and tested the efficacy of EM015 in inhibiting cellular proliferation using the standard sulforhodamine assay (cell types are listed in Table 1 ). Interestingly, EM015 inhibited cellular proliferation in all cell lines with an IC₅₀ that ranged between 2 and 10 µmol/L, which is 15- to 20-fold lower than that of noscapine. We also investigated the activity of EM015 against drug-resistant tumor types (refs. 35–38; Table 2 ). A wide variety of parental cancer cells as well as their drug-resistant variants show similar IC₅₀ values, suggesting equal sensitivity to EM015 (Table 2). The higher potency of EM015 over noscapine is best exemplified by MCF-7 cells, which display a 20-fold lower IC₅₀ as compared with noscapine (Fig. 2A ). The question that now remains is the mechanism by which EM015 inhibited cell proliferation.

View larger version (57K): [in this window] [in a new window]   Figure 2. EM015 is more active than noscapine in inhibiting proliferation of human breast cancer cells. MCF-7 cells were treated with increasing concentrations of noscapine or EM015 for 72 hours. A, 20-fold lower IC50 value of EM015 than that of noscapine. This in vitro assay was done using the standard sulforhodamine B. Points, average of three independent experiments with five replicates each. B, apoptotic index and mitotic index as a function of time of treatment with 50 µmol/L noscapine or 5 µmol/L EM015 (10-fold less concentration). At 24 hours, the percentage of mitotic cells was 60.5% and 65.7% for noscapine and EM015 treatments, respectively. The higher activity of EM015 than noscapine was also evident by the higher percentage of cells with degraded DNA, in that, 28.6% and 40% of cells with <2N DNA content were observed on treatment with 50 µmol/L noscapine and 5 µmol/L EM015 for 72 hours, respectively. C, increase in population of cells with degraded DNA (sub-G1 amount in the far left) as a function of time by the three-dimensional FACS analysis of DNA amounts. A 10-fold lower concentration of EM015 shows an effective DNA degradation as noscapine. D, effect of 50 µmol/L EM015 on the cell cycle profiles of near-normal human breast epithelial cells (MCF-10A) and normal human primary fibroblast cells (HDF). Representative of three experiments done in triplicate. E, confocal micrographs of MCF-7 cells treated for 0, 0.5, 24, and 48 hours. As expected, mitotic figures are abundant at 24 hours whereas apoptotic figures appear at 48 hours. Bar, 30 µm.

Next, we examined the microtubule arrays and nuclear morphologies of cells at various stages of EM015 treatment using confocal microscopy (Fig. 2E). Treatment at 0 to 0.5 hours did not have any affect (Fig. 2E) whereas at 24 hours, many abnormal multipolar mitotic figures appeared. In clear contrast, at 48 hours, the population of mitotic cells decreased whereas cells with micronucleated and multilobed nuclei were abundant (Fig. 2E).

EM015 causes apoptosis. A consequence of the mitotic abnormalities induced by EM015 in tumor cells could be the activation of apoptotic pathways. Biochemically, the apoptotic process is first recognized by alterations of surface lipid composition, in that, phosphatidylserine, which is normally on the inner leaflet of plasma membrane, translocates to the outer leaflet (see green ring pointed by the solid arrowhead in Fig. 3A, left ), which can be measured fluorescently by Annexin V binding. A cell-impermeant DNA-binding dye, propidium iodide, cannot enter the cells unless they are under stages of late apoptosis when membrane permeability is compromised (Fig. 3A, left, open arrowhead). This can be quantitated in large populations of cells by FACS analysis (Fig. 3A, middle and right). The control untreated cell cultures contained very few apoptotic cells (6.3%), which were assigned as the background cell death due to regular trauma during cell culture (Fig. 3A, middle). We found that 5 µmol/L EM015 treatment of MCF-7 cells resulted in 46.7% early apoptotic cells within 48 hours (Fig. 3A, right). This is consistent with the observations with confocal microscopy that revealed cells in the beginning, intermediate, and terminal stages of apoptosis at 48 hours, suggesting ongoing apoptosis and an active proapoptotic stimulus. The terminal stages of apoptosis display cleaved 3' ends of DNA that can be visualized by specific labeling by TUNEL assay as abundant green staining (Fig. 3B, left, red fluorescent staining denotes propidium iodide). The quantitative FACS analysis showed 89% TUNEL-positive cells on treatment with 5 µmol/L EM015 for 72 hours (Fig. 3B, right). In contrast, control untreated cells showed rare TUNEL-positive cells (Fig. 3C, left, solid arrowhead). This is consistent with the quantitative FACS data that show 93% control cells containing intact 2N genomic DNA in the lower region (below the dividing black line) of the cytogram (Fig. 3C, right).

View larger version (40K): [in this window] [in a new window]   Figure 3. EM015 at 5 µmol/L kills cancer cells by inducing potent apoptosis. This is first revealed by the lipid asymmetry of the outer leaflet of their plasma membranes, detected by staining for the newly displayed phosphatidylserine, a signal thought to be recognized by professional phagocytes. A, left, fluorescent staining of the outer leaflet of these early apoptotic cells (solid arrowheads) when the membranes are intact and impermeable to other dyes such as the DNA-binding red dye propidium iodide. In later stages of apoptosis, when the plasma membranes gain limited permeability to such small dyes, one can also see the staining of DNA within living cells (open arrowheads). These early and late apoptotic cells can be analyzed quantitatively by a two-color flow cytometric analysis. Middle and right, control untreated cells and cells treated with 5 µmol/L EM015 for 48 hours, respectively. The density plots illustrate four cell populations (live, apoptotic, necrotic, and late apoptotic/dead) defined by their fluorescence characteristics. Live cells are Annexin V negative and propidium iodide negative. In these cells, phosphatidylserine translocation has not occurred and the plasma membrane is still intact. Early apoptotic cells are Annexin V positive and propidium iodide negative; their membranes are not permeable. Necrotic cells are Annexin V negative and propidium iodide positive because of damaged cell membranes. The late apoptotic and dead cells are both Annexin V positive and propidium iodide positive. B, the fragmented DNA in the terminal stages of apoptosis was visualized using terminal deoxynucleotidyl transferase–mediated bromo-dUTP reaction (TUNEL assay) in EM015-treated cells. This can be seen by confocal microscopy as an abundance of TUNEL-positive cells (left). This is also evident by a quantitative FACS analysis of these data, which shows that a majority of cells treated with 5 µmol/L EM015 for 72 hours are TUNEL positive (right, cells above the dividing black line in the cytogram). C, parallel TUNEL data for control untreated cells which are in clear contrast with treated cells, in that these cells show minimal apoptosis both by microscopy and by FACS analysis. Bar, 30 µm. Representative of three independent experiments.

View larger version (31K): [in this window] [in a new window]   Figure 4. Orally administered 300 mg/kg EM015 efficiently reduces the tumor volume significantly within 17 days of treatment after 7 days of tumor establishment. Preestablished palpable tumor-bearing mice were randomly chosen and divided into three groups as described in Materials and Methods. A, representative example of vehicle-treated control and EM015-treated animal with such xenografted tumors. B, kinetics of tumor growth in vehicle-treated animals compared with noscapine and EM015 treatments. C, tumor volume measurement on day 24, the day of euthanasia of control vehicle-treated mice because of overgrown tumors, in compliance with the experimental protocol approved by Institutional Animal Care and Use Committee of the Emory University School of Medicine. After 17 days of EM015 treatment, preestablished tumors shrunk by 84% in volume compared with control vehicle-treated animals (1.21 ± 0.051 and 0.2 ± 0.025 cm3, average tumor volume ± SE for vehicle-treated and EM015-treated groups, respectively; P < 0.01). Animals treated with noscapine showed a reduction in tumor volume by 66% (0.41 ± 0.043 cm3; P < 0.01). As shown in (D), not only EM015 and noscapine treatments were well tolerated but mice in any group also did not suffer from general body weight loss. E, Kaplan-Meier analysis of EM015 therapy that shows a significantly increased life span of mice by 3-fold compared with vehicle-treated tumor bearing mice (tumor volume shown as cm3 ± SE; *, P < 0.01, B-D; log-rank test, E).

EM015 therapy causes no detectable toxicity to tissues and does not affect the hematopoietic system and organ functions. Toxicity in many tissues following chemotherapy is a major clinical concern. Therefore, the search for a safe, well-tolerated regimen has been a major goal of clinical research. Antimitotic drugs that bind free or polymerized tubulin, such as Vinca alkaloids and taxanes, although effective treatment agents, are known to be cytotoxic to dividing cells (23). These agents halt the mitotic growth of dividing cells but also might block microtubule-track dependent intracellular transport causing peripheral neuropathies (23). This is because neurons, which rely on intact cellular microtubules for the maintenance of presynaptic termini, are adversely affected by these agents. These drugs thus exhibit toxicities as revealed by histopathologic analysis of long nerves associated with peripheral neuropathies (23). In addition, they cause toxicities associated with cell division in normally dividing somatic cells such as myelosuppression, colitis, and alopecia.

To determine whether 300 mg/kg EM015 treatment for 80 days results in toxicities to normal tissues, we examined the liver, kidney, spleen, lung, heart, brain, gut, and the sciatic nerve of tumor-bearing mice (Fig. 5 ). Evaluation of numerous histologic sections of these tissues from animals bearing human breast tumor xenografts did not indicate any detectable pathologic abnormalities as examined by H&E staining. There was a complete absence of any metastatic lesions in these organs in the EM015-treated group. The long sciatic nerve from EM015-treated and vehicle-treated animals showed no morphologic abnormalities by H&E and Luxol fast blue/periodic acid-Schiff stainings (Fig. 5C). The tumor xenograft microsections from EM015-treated animals showed large areas of tumor cell death and these areas were replaced by normal looking healthy cells. Consistent with the therapeutic effect of EM015, there was a loss of tumorigenic cells in EM015-treated animals (Fig. 5D, right, solid arrowhead). Some viable tumor cells were observed at the periphery of cell death zones. Microsections from vehicle-treated tumor tissues revealed sheets of tumor cells with high-grade pleomorphic nuclei and angiolymphatic invasion (Fig. 5D, left, solid arrowhead).

View larger version (142K): [in this window] [in a new window]   Figure 5. Daily 300 mg/kg EM015 treatment fails to reveal any detectable pathologic abnormalities in normal tissues that are active in normal cell proliferation. A, H&E staining of paraffin-embedded, 5-µm-thick sections of the liver, kidney, spleen, lung, heart, and brain from untreated and EM015-treated groups of mice seen under 200x magnification. There were no observable histopathologic differences in these tissues. The liver showed normal hepatic lobular architecture (solid arrowhead, intact central vein with trapped red cells in a liver section from EM015-treated animal). The kidneys revealed normal glomeruli, proximal and distal tubules, interstitium, and blood vessels. The splenic follicles and vascular sinusoids were indistinguishable between the EM015-treated and vehicle-treated control groups (solid arrowheads). The lung tissue showed normal alveoli (solid arrowhead) and the heart muscle showed normal morphology among the two groups. Microsections of brain did not reveal any infarcted areas. The cerebral cortex and gray and white matters appeared normal. B, parallel H&E staining of duodenum, colon, and pancreas. These tissues were also indistinguishable among EM015-treated and vehicle-treated control groups. The gut showed normal mucosa, submucosa, and muscularis mucosa. In addition, the crypts that are crucial sites for cell division were intact. The pancreatic acini and islets of Langerhans were normal (solid arrowhead). Sciatic nerve was also observed for any signs of toxicity. C, no apparent distinctions between EM015-treated and vehicle-treated control groups as revealed by H&E staining (top) and Luxol fast blue/periodic acid-Schiff staining (bottom). Consistent with the observed tumor reduction in EM015-treated mice compared with the vehicle-treated control mice, the histologic analysis of the tumor xenograft areas (which stain darker) in vehicle-treated mice show abundance of tumor cells (left) compared with EM015-treated tumor microsections that reveal large tumor cell death areas (right), consistent with the therapeutic effects of EM015.

View larger version (40K): [in this window] [in a new window]   Figure 6. EM015 and noscapine treated animals show no deviation in the toxicity profile when compared with vehicle-treated controls. Figure shows no toxicities related to hematologic variables (A), organ functions (B and C), electrolyte balances (D), and systemic homeostasis (E). All show indistinguishable profiles among all variables examined for vehicle-treated, noscapine-treated, and EM015-treated groups. F to I, flow cytometry–based immunophenotyping assays showing no alterations in the CD4, CD8, natural killer (NK), and B-cell counts from vehicle-treated, noscapine-treated, and EM015-treated groups of mice.

EM015 does not compromise the immune surveillance. The risk for recurring malignancies after prolonged cancer chemotherapy has been shown in numerous case reports and epidemiologic studies. This can be partly ascribed to the immunotoxic and immunosuppressive effects of currently used chemotherapeutic drugs. Immunotoxic effects might result in tumor recurrence by reduced immune surveillance through B- and T-cell response against tumors. Both B- and T-lymphocyte lineage cells showed no appreciable difference among noscapine-treated, EM015-treated, and vehicle-treated groups as evidenced by absence of alterations in the B- and T-cell subsets [CD3+, CD4+ T-helper cells (Fig. 6F); CD3+, CD8+ cytotoxic T cells (Fig. 6G); NK1.1+ cells (Fig. 6H); and B220+ cells (Fig. 6I)]. Thus, our treatment strategy does not cause any immunosuppression.

Our rationally in silico designed EM015 can significantly cause tumor regression and increase the survival of human tumors grown in xenograft mice models. EM015 is orally available and thus bypasses parenteral and i.v. administrations that cause complications such as hypersensitivity reactions and thrombosis of blood vessels or embolisms. The novelty of our drug lies in its oral bioavailability and absence of toxicities that are associated with currently used antimicrotubule drugs. Because EM015 treatment does not deplete immune cells and is well tolerated, it might also offer the possibility of combination chemotherapy with immunotherapeutic regimens for the management of breast carcinomas. Our results compel us to continue to examine the effects of EM015 on other human and murine neoplasms with the final goal of taking it to the clinic.

	Acknowledgments

 
Grant support: NIH (H.C. Joshi).

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.

We thank Drs. Liotta and Snyder group and Drs. Wu and Wang group of the Chemistry Department, Emory University, for guidance and help; Drs. Lily Yang and Ruth O'Regan from the Winship Cancer Institute, Emory University, for providing various breast cancer cells; Drs. William Beck and Maricarmen Silvia for providing various cell lines used in this study and advice; the Dermatology Department of Emory University for primary cultures of human fibroblasts; and members of the Joshi laboratory for discussions.

Received 8/19/05. Revised 1/24/06. Accepted 2/ 2/06.

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