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Experimental Therapeutics |
Arkansas Cancer Research Center, Departments of Pathology [C. Y. P., A. M. T., R. D. S.], Anatomy [R. D. S.], Otolaryngology [S. L.], and Biometry [R. S. P.], University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most GAG chains are attached to core proteins to form proteoglycans which are found on cell surfaces and in the extracellular matrix. The syndecans and the glypicans are the major families of cell surface heparan sulfate proteoglycans and there is substantial evidence that syndecan-1 acts as a powerful tumor suppressor. Expression of syndecan-1 on myeloma cells reduces disease-related morbidity in a mouse model (15) . Syndecan-1 shed from the cell surface by sheddases or secretases (16 , 17) retains biological activity and can reduce cell growth and induce apoptosis of myeloma cells in vitro (15) . The syndecan-1 ectodomain also suppresses the growth of CarB, S115 and MCF-7 mouse and human mammary tumor cells in vitro but not NIH3T3, NMuMG or HaCaT normal cell lines (18) . There is also an emerging correlation between loss of cell surface heparan sulfate proteoglycan expression and cancer progression. Simpson-Golabi-Behmel syndrome is the result of mutation of the heparan sulfate proteoglycan glypican-3 gene and is characterized by prenatal and postnatal overgrowth and an increased incidence of tumor development (19 , 20) . Mutations that inhibit heparan sulfate polymerase enzymes cause hereditary multiple exostoses which is characterized by the formation of benign bone tumors (21 , 22) . Also, loss of syndecan-1 expression on tumor cells correlates with a poor prognosis in patients with head and neck cancer, mesothelioma and gastric carcinoma and is thought to contribute to increased metastatic potential of non-small-cell lung and hepatocellular carcinomas (23, 24, 25, 26, 27, 28) . In contrast to these cancers, pancreatic tumors and their metastatic lesions express high amounts of syndecan-1 demonstrating that syndecan-1 expression is not always lost with cancer progression (29) .
To study the tumor suppressor functions of syndecan-1 and to develop its potential as a therapeutic agent requires the isolation of large quantities of the molecule, a process that is severely hampered by the lack of a suitable system for the production of the protein bearing attached and appropriately modified heparan sulfate chains. To overcome this obstacle, we produced synthetic proteoglycans (neoproteoglycans) by linking GAG chains to human serum albumin by carbodiimide (EDAC) conjugation. Although these preparations significantly reduced cell viability, surprisingly, EDAC modified GAG chains prepared in the absence of protein also reduce cell viability in vitro and in vivo. This is in sharp contrast to unmodified heparin and CS which do not reduce cell viability. These novel compounds, called neoglycans, constitute a new class of anticancer agents.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neoglycan Concentration Determination by Carbazole Assay.
Carbazole assays were performed according to the protocols of Blumenkrantz and Asboe-Hansen (30)
and Filisetti-Cozzi and Carpita (31)
. Known concentrations of heparin and CS ranging from 0 to 60 µg were aliquoted into glass test tubes. One µl of each neoglycan, approximately 50 µg, was aliquoted into glass test tubes in duplicate. The volume of each tube was brought to 200 µl with H2O. One ml of 0°C 125 mM sodium tetraborate (Sigma) in sulfuric acid was added to each tube followed by the addition of 35 µl of a carbazole reagent composed of 0.125% carbazole (Sigma) w/v in ETOH. The tubes were mixed carefully and heated to 100°C for 20 min. Cooled aliquots were transferred to a 96-well plate and the absorbance at OD540 was measured on a microplate reader. Known GAG chain concentrations served as a standard curve for the extrapolation of the unknown neoglycan concentrations.
Cells and Cell Culture.
ARK, ARP-1 and CAG cells were established at the Arkansas Cancer Research Center from bone marrow aspirates of multiple myeloma patients (32
, 33)
. U266 myeloma cells and ARH-77 cells [an EBV-transformed cell line established from a patient with plasma cell leukemia, (34)
] were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors by Ficol-Histopaque separation of whole blood and were mitogen activated for 3 days with 2.5 µg/ml PHA (Sigma) before neoglycan treatment. The cell lines and PBMCs were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin sulfate. HBL-100, Hs578t, MCF-7, MDA-MB-231, MDA-MB-435 and MDA-MB-436 breast cancer cell lines were obtained from the ATCC and were maintained in culture media as suggested by the ATCC. Normal cell lines including CHO-K1 chinese hamster ovary cells, NIH3T3 Swiss murine embryo fibroblast cells, NMuNG murine mammary cells, MDCK canine kidney cells and MCF-10A and MCF-10F human mammary cell lines were all purchased from the ATCC and grown in media specified by the ATCC. All of the cells were cultured at 37°C in an atmosphere of 5% CO2 and adherent cell lines were harvested using standard trypsinization procedures.
Determination of Cell Viability by MTT Assay.
Cells were plated in the appropriate media on 96-well plates in a 100 µl total volume. Nonadherent cells were plated at a density of 2 x 104 cells/well. Adherent cell density varied based on the growth characteristics of each cell line and these cells were plated one day before neoglycan addition to allow the cells to attach. Triplicate wells were treated with media, 10 µM dexamethasone, 50 µg heparin or CS, and 3.2 µg, 1.6 µg, and 0.32 µg of neoheparin or neoCS. The plates were incubated at 37°C in 5% CO2 for 72 h. Cell viability was determined based on mitochondrial conversion of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) to formazan. The amount of MTT converted to formazan is indicative of the number of viable cells (35)
. Each well was supplemented with 50 µl of a 2 mg/ml solution of MTT in complete media. The plates were returned to 37°C, 5% CO2 for 4–5 h. The media was carefully removed from each well and 150 µl of 37°C DMSO was added. The plates were gently agitated until the color reaction was uniform and the OD540 was determined using a microplate reader. SigmaPlot 2000 software was used for data analysis. Media only treated cells served as the indicator of 100% cell viability.
To determine whether continuous exposure to neoglycans is required for growth inhibition, ARP-1 cells were exposed to 320 µg/ml and 32 µg/ml neoglycans for 5, 15, 30 and 60 min. The cells were washed twice in complete media, plated in triplicate and incubated at 37°C, 5% CO2 for 72 h. A control was included in which the cells were treated with the neoglycans for the usual 72 h. Cell viability was determined by MTT assay as described above.
Apoptosis Determination.
ARP-1 cells and MDA-MB-231 cells were treated with media only, 32 µg/ml GAG chains and 32 µg/ml neoglycans. The ARP-1 cells were harvested after a 48 h incubation period and the MDA-MB-231 cells were harvested after a 96 h incubation period. The induction of apoptosis was determined by staining with fluorescein isothiocyanate-labeled Annexin V (Annexin V-FITC, CALTAG Laboratories, Burlingame, CA) following the manufacturer’s instructions. Just before analysis 0.5 mg/ml propidium iodide (PI, Sigma) in PBS, pH 7.4 was added. Flow cytometry was performed with a Becton-Dickinson (Mountain View, CA) FACScan using Cellquest 1.2 software. Annexin V-FITC positive, PI negative cells were considered apoptotic.
To evaluate DNA ladder formation, a late event of apoptosis, TUNEL assays were performed after treatment of ARP-1 and MDA-MB-231 cells as described above. ARP-1 and MDA-MB-231 cells were harvested after a 48 h or a 120 h incubation period, respectively. Apoptotic cells were identified by TdT-mediated dUTP nick end labeling (TUNEL) assay using the Roche Molecular Biochemicals Fluorescein In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. The cells were analyzed by FACscan using Cellquest 1.2 software.
Treatment of Established MDA-MB-231 Tumors with CS and neoCS.
BALB/c nu/nu mice were anesthetized by i.p. administration of a mixture of ketamine (42.8 mg/ml), xylazine (8.6 mg/ml) and acepromazine (1.4 mg/ml) at a dose of 1.8–2.0 ml/kg. MDA-MB-231 cells (1 x 106) were injected at two s.c. sites on each mouse. The tumors were allowed to grow for two weeks before treatment. Once established, the tumors were injected with a single 1.6 mg dose of CS or neoCS in a 50 µl total volume. Immediately after injection, a caliper electrode was positioned on two sides of the tumor with contact on the skin. An electrical pulse was applied to the tumor through the electrodes with a BTS EC830 power supply (Genetronics, Inc., San Diego, CA). At the indicated time points the tumors were measured with calipers and tumor volumes calculated according to the equation: V = (L x W2) x 0.5. The results reflect two separate experiments in which one tumor was treated with CS and the second tumor on the same animal was treated with neoCS. In the first experiment n = 8 tumors in each treatment group and in the second experiment n = 7 tumors/group, therefore the results include 15 tumors total in each treatment group.
SDS-PAGE Analysis of Neoglycans.
GAG chains and neoglycans were separated by SDS-PAGE through 14% gels. The gels were first stained for the presence of protein with 0.25% Coomassie Blue for 3 h and destained in methanol:H2O (1:1 v/v) and 10% glacial acetic acid overnight. The gels were subsequently stained with 0.1% alcian blue, 3% glacial acetic acid for 15 min and destained in H2O. Alcian blue specifically stains GAG chains and not protein. Rainbow molecular weight markers (Amersham Pharmacia Biotech, Piscataway, NJ) serve as size standards on each gel. However, because of the inherent differences in protein and GAG chain structures, the markers indicate only an estimation of GAG chain size.
Polysaccharide Lyase Digestion and Superdex 200 Column Chromatography of Neoglycans.
CS and neoCS (500 µg each) were speed vacuumed to dryness and resuspended in H2O, 20 mM Tris-HCL, pH 8.0 (lyase buffer), or 150 µU chondroitin ABC lyase (Segagiku, Tokyo, Japan). Digestions were performed at 37°C for 4 h.
Superdex 200 prep grade gel filtration media (Amersham Pharmacia Biotech), which fractionates proteins by size in a range from 10–600 kDa, was equilibrated in 6 M guanidine, 50 mM sodium acetate, pH 5.8 column running buffer. A 95 cm x 2 cm Superdex 200 column was poured and packed at a flow rate of 6 ml/hour. The column was washed with one bed volume of running buffer. A 100 mg preparation of neoCS was digested with 0.3 U of chondroitin ABC lyase in 20 mM Tris, pH 8.0 for 4 h. The digested neoCS was brought 1:1 with column running buffer, boiled for 10 min, cooled to room temperature, loaded on the Superdex 200 column and fractions were collected. Numerous fractions were buffer exchanged into H2O by 5,000 molecular weight cutoff spin column filtration (Millipore) and stored at 4°C.
Statistical Analysis.
The 50% inhibitory concentration (IC50) is defined as the concentration at which 50% of treated cells die within 72 h. Data from triplicate wells and replicate experiments were used jointly to estimate a common IC50 through use of a compound nonlinear degradation model based on the three-parameter logistic. Data from the triplicate wells at each neoglycan concentration were combined by computing means, denoted by Yij for concentration i (i = 1,5) and replicate j (j = 1,2), and these were used as the dose levels in the modeling. The model was defined as
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x = 2 + log10 (dose), if dose > 0, else x = 0.
In this model, the estimate of IC50 is given by 10(b3-2), where b3 represents the estimate of the parameter ß3 obtained by using the NLIN procedure of SAS (The SAS System, Version 8, SAS Institute Inc., Cary NC). Ninety-five percent confidence intervals were based on asymptotic limits for the estimated parameter which were inverse transformed to obtain limits for IC50.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For example, after 72 h of treatment, neoheparin and neoCS (32 µg/ml) produce a 79% and 96% reduction in ARP-1 cell viability, respectively. In comparison, unmodified heparin treatment may slightly enhance cell proliferation and CS has no effect. The 50% inhibitory concentration (IC50) values for neoheparin and neoCS on ARP-1 cells are 21.94 µg/ml (95% confidence interval (CI) of 18.18 to 26.48 µg/ml) and 14.79 µg/ml (95% CI of 12.13 to 18.06 µg/ml), respectively (Table 1)
. In comparison to ARP-1 cells, U266 myeloma cells exhibit a similar sensitivity to neoCS but are less susceptible to treatment with neoheparin. All of the breast cancer cell lines tested including MDA-MB-231 and Hs578t are also sensitive to treatment with the neoglycans (Fig. 1A
and Table 1
).
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and Table 1
). Mitogen activated peripheral blood mononuclear cells (PBMCs) also show a similar sensitivity to both neoglycans (Fig. 1A
and Table 1
). Therefore, the viability of myeloma, breast cancer and normal cell lines and activated PBMCs is reduced by exposure to either neoglycan.
To determine whether the continuous presence of the neoglycan is required for its activity, ARP-1 cells were pulse-treated with neoheparin or neoCS for 5, 15, 30 or 60 min at 37°C, 5% CO2. Cells were washed several times to remove neoglycans, placed in fresh media and, after a 72 h incubation, cell viability was determined by MTT assay. Pulse treatment of ARP-1 cells with 320 µg/ml of neoheparin and neoCS for as short a period as 15 min reduces ARP-1 cell viability by 44% and 92%, respectively (Fig. 1B)
. A 5 min treatment of ARP-1 cells with neoCS reduces cell viability by 42%. At the lower dose of 32 µg/ml, a 60 min pulse with the neoheparin produces a 44% reduction in cell proliferation whereas the same length pulse with neoCS produces a 23% reduction in cell viability (Fig. 1B)
. These results indicate that the neoglycans need not be present continually for a reduction in cancer cell viability, which suggests that the neoglycans are affecting cells directly and not simply neutralizing nutrients within the culture media. Moreover, this suggests that even a transient high concentration of neoglycan at the tumor site in vivo could be effective in eradicating tumor burden.
Neoglycans Induce Apoptosis in Cancer Cells.
To determine whether neoglycan treatment of cells promotes apoptosis, ARP-1 cells and MDA-MB-231 cells were treated with media only or media supplemented with GAG chains or neoglycans. Apoptosis was evaluated by two methods, Annexin V-FITC staining and TUNEL assay. Treatment of the ARP-1 cells for 48 h or MDA-MB-231 cells for 96 h with media only or 32 µg/ml unmodified GAG chains results in no induction of apoptosis. However, when either cell line was treated with neoglycan for the same period of time, cells underwent extensive apoptosis as demonstrated by Annexin V-FITC staining on the cell surface (Fig. 2A)
. For TUNEL assays, ARP-1 and MDA-MB-231 cells were treated with 32 µg/ml of the neoglycans and GAG chains and incubated for 48 and 120 h, respectively. Media only treatment results in no apoptosis, whereas treatment of either cell line with either neoglycan results in apoptosis as shown by the presence of a significant shift in fluorescence (ARP-1 cells) or the presence of a population of brightly stained cells (MDA-MB-231 cells; Fig. 2B
). A slight shift in fluorescence intensity is noted after treatment of the MDA-MB-231 cells with the native GAG chains however, a brightly stained second population of cells that is indicative of apoptosis is not present. Collectively, the results of two separate assay systems demonstrate that the neoglycans, but not unmodified GAG chains, reduce cell viability by inducing apoptosis.
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and Table 1
) and because neoheparin may exhibit the anticoagulant properties of native heparin, we evaluated neoCS for in vivo activity. MDA-MB-231 breast cancer tumors were establish in female BALB/c nu/nu mice by s.c. injection of 1 x 106 cells at two sites on each animal. A single injection of 1.6 mg of CS or neoCS was administered to separate tumors on the same animal. After injections, tumors were electroporated to enhance permeability of the tumor cells. The size of each tumor was determined 48 h later and at several subsequent time points. Results from duplicate experiments demonstrate that a single dose of neoCS completely eradicated the MDA-MB-231 breast tumors in every animal by day 5 (Fig. 3)
. In both experiments only one animal had a neoCS-treated tumor reemerge. The neoCS-treated tumors on the other animals did not recur during the one month experimental period (data not shown).
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. As expected, CS (CS-W and CS-B) is very heterogeneous in size and appears as a large smear covering the entire lane. The CS-L lane and the neoCS-L lane show high molecular weight smears that remain after lyase digestion. Further lyase treatment failed to digest the remaining CS or neoCS (data not shown). Because most of the neoCS was digested, cell viability experiments were performed to determine whether killing activity had been reduced. ARP-1 cells were treated with digested CS and neoCS at a concentration equivalent to 32 µg/ml nondigested material. Interestingly, the neoCS activity is not blocked by lyase digestion (Fig. 4B
, neoCS-L) and the digested neoCS is as active as the undigested controls including neoCS in H2O (neoCS-W) and neoCS in lyase buffer (neoCS-B). The chondroitin ABC lyase used in these experiments did not affect ARP-1 cell proliferation because cells treated with lyase-digested CS grew as well as media only treated cells (Fig. 4B
, compare CS-L and M). These results indicate no detectible protein contamination of the neoglycan preparations and suggest either the high molecular weight neoCS remaining after lyase digestion is active, the small fragments of the neoCS remaining after lyase digestion are active or residual EDAC is present and active.
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. The 66 kDa band that is evident in fractions 35–41 is BSA that is included as a carrier in the lyase reagent. The GAG concentration of each of these fractions is too low to determine by carbazole reaction although SDS-PAGE analysis of equal volumes of the fractions suggests similar concentrations in the GAG chain-containing fractions. To examine the activity of the individual fractions, ARP-1 cells were treated with equal volumes of fractions 25–42, incubated at 37°C, 5% CO2 for 72 h and cell viability was determined by MTT assay. The results indicate that the fractions vary greatly in their activity, with the most activity contained within fractions 32–37 (Fig. 5B)
. Interestingly, a series of fractions very high in activity are clustered around fraction 33. Because free EDAC is less than 0.2 kDa in size, it would not be present within the high molecular weight fractions where the activity resides. Thus, these results demonstrate that a contaminant is not producing activity and confirm that the chondroitin ABC lyase-resistant high molecular weight neoCS specifically reduces cell viability.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EDAC conjugation is a two-step chemical reaction that is widely used to conjugate molecules containing carboxyl and amino groups (37 , 38) . In the first step of the reaction, EDAC couples to a carboxyl group forming an activated O-acylisourea derivative. Subsequent attack by an amino group produces an amide linkage between the carboxyl and amino group-containing molecules and displaces a urea derivative of EDAC. Heparin and CS contain a free carboxyl group on the number 6 carbon of the glucuronic acid component of the GAG chains. Heparin that has been isolated from porcine intestinal mucosa contains a limited number of N-nonsubstituted glucosamine residues and therefore a limited number of free amino groups (39) . Heparin and CS used for the production of neoglycans may contain free amino groups on covalently bound amino acids remaining from conventional isolation procedures (40 , 41) . Therefore, EDAC modification of heparin and CS could produce molecules that are composed of GAG chains coupled together producing GAG complexes, perhaps similar to the multimeric array of heparan sulfate found on syndecan-1. However, because there are abundant carboxyl groups and limited amino groups present on GAG chains, the EDAC reaction may not go to completion. The result of the incomplete EDAC reaction would be the O-acylisourea derivative produced in the first step of the reaction (42) . Indeed, preliminary 1H-NMR spectra analysis of neoCS compared with native CS or EDAC 1H-NMR spectra reveals the presence of EDAC bound to the GAG chains at approximately a 1:1 ratio of EDAC to GAG disaccharide suggesting the presence of an O-acylisourea derivative. Other signals are present but have not been identified however, contamination with free EDAC or free GAG chains has been excluded (data not shown). The 1H-NMR analysis also supports the conclusion that there is little if any protein contamination and there are probably few unsubstituted amino groups present on the GAG chains because GAG bound to GAG was not detected as a major product. On the basis of the chemistry of the EDAC reaction, one would expect the production of GAG bound to GAG if contaminating amino acids were bound to the GAG chains or if unsubstituted amino groups on the GAG chains were available. Analysis of the active neoCS fractions isolated after lyase digestion and size exclusion chromatography should reveal the specific structure of the active neoCS. Interestingly, O-acylisourea derivatives of highly sulfated GAG chains such as heparin and CS are reminiscent of suramin, a polysulfated naphthylurea compound that also exhibits anticancer properties (43) .
EDAC modification of GAG chains produces molecules with strikingly different activities as compared with the native GAG chains. Treatment of cell lines with native heparin and CS does not reduce cell growth and in fact enhances cell growth in some cases (Fig. 1)
. Heparin and CS do not induce apoptosis in ARP-1 and MDA-MB-231 cancer cell lines (Fig. 2)
. In great contrast to heparin and CS, neoheparin and neoCS severely reduce cell viability in normal and cancer cell lines and induce apoptosis in the ARP-1 myeloma cell line and the MDA-MB-231 breast cancer cell line. Interestingly, the activity of the neoglycans is not inhibited by the addition of excessive amounts of GAG chains. Treating ARP-1 cells with 10-fold greater concentrations of native GAG chains does not block the killing affect of the neoglycans (data not shown), a result that highlights the different activities of GAG chains and neoglycans. The contrasting functions of the native CS and the neoCS are also evident in the results of in vivo experiments. NeoCS eradicates breast cancer tumor burden in nude mice whereas CS has no detectable effect (Fig. 3)
.
An important characteristic of any therapeutic agent is effectiveness at an achievable and safe dosage. The cell culture studies reported here demonstrate that varying but high concentrations of neoglycan are required for a 50% reduction in cell viability in vitro (Table 1)
. However, chondroitin ABC lyase-digested neoCS reduces cell viability as effectively as undigested neoCS (Fig. 4B)
despite the obvious reduction in total GAG content (Fig. 4A)
. This result suggests that neoCS is a mixture of active and nonactive molecules. Evaluation of the Superdex 200-purified neoCS fractions confirms this finding. Some neoCS fractions did not reduce cell viability whereas others did even at concentrations too low to determine. Therefore, the neoglycan preparations contain nonactive components and future studies using purified active neoglycan should demonstrate improved IC50 values and lower therapeutic dose requirements. In terms of safety, the neoglycans inhibit the growth of several normal cell lines and activated primary PBMCs in vitro (Fig. 1A)
. However, treatment of established breast cancer tumors in nude mice abolishes tumors without obvious toxicity to surrounding tissue or the whole animal. Therefore, the effect of neoglycans on normal cells growing in vitro may be an artifact of rapidly dividing laboratory cultures. Moreover, a safe and effective dose of neoCS is achievable in nude mice.
Importantly, neoglycans act on the target cells in an expeditious manner. Pulse treatment of cells with neoglycans (32 µg/ml) for 1 h resulted in a reduction in ARP-1 cell viability whereas at a higher concentration (320 µg/ml) a pulse as short as 5–15 min resulted in a 42–92% reduction in cell viability (Fig. 1B)
. This finding suggests that the mechanism of activity of the neoglycans involves direct interaction with the cells as opposed to an indirect means of killing such as depletion of essential components in the media. The ability to produce an effect quickly and irreversibly is beneficial because in vivo the neoglycans may be cleared rapidly or metabolized.
In conclusion, we report the initial description of novel anticancer therapeutic agents called neoglycans. The mechanism of antitumor activity is wholly unknown, although it is likely that the neoglycans interact with the cells because a short exposure to neoheparin or neoCS irreversibly kills cells. The isolation of active neoCS fractions provides the foundation for future studies of the structure and function of neoglycans and for the optimization of neoglycan activities. Exploitation of the structural and functional diversity of GAG chains and the neoglycans made from them represents an opportunity for development of a new class of anticancer agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by DOD Grants DAMD17-99-1-9066 and DAMD17-99-1-9064 and NIH Grant CA55819. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Slot 517, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205. Phone: (501) 686-6413; Fax: (501) 686-5168; E-mail: sandersonralphd{at}uams.edu
3 The abbreviations used are: GAG, glycosaminoglycan; EDAC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; CS, chondroitin sulfate; IC50, 50% inhibitory concentration; PBMC, peripheral blood mononuclear cell; MTT, 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; PI, propidium iodide.
Received 7/16/01. Accepted 4/12/02.
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) or 1.6 mg neoCS (
) followed by electroporation of the tumors. On the indicated days after treatment, tumor size was measured and tumor volume calculated. Bars indicate means ± SD of two experiments. The results represent a total of 15 tumors treated with neoCS. However, for the CS control group n = 15 on days 0, 2 and 5, n = 14 on days 7 and 9 and n = 13 beginning on day 13. The reduction in control-treated tumor numbers is because of mouse morbidity.
