Inhibition of human breast cancer metastasis in nude mice by synthetic glycoamines.

We have examined the effect of synthetic low molecular weight glycoamine analogues on the metastasis of MDA-MB-435 human breast carcinoma xenografts growing in the mammary fat pads of nude mice. Initial in vitro screening of a panel of synthetic glycoamines was performed using a clonogenic growth assay in 0.9% agarose. Eight of nine compounds manifested a significant dose-dependent inhibition of colony formation by MDA-MB-435 cells in 0.9% agarose. The relative activity ranks of the compounds, based on ID50S independently determined for each synthetic glycoamine analogue, identified N-(1-deoxy-D-lactulos-1-yl)-L-leucine (Lac-L-Leu), N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu), N-(1-deoxy-D-fructos-1-yl)-L-phenylalanine, and N-(1-deoxy-D-fructos-1-yl)-L-leucine as the most effective inhibitors of colony formation. Two separate experimental treatment protocols were used to examine the effect of selected synthetic glycoamines on human breast cancer growth and metastasis in athymic nude mice. Group A mice were treated intraperitoneally daily from day 2 after injection of the breast cancer cells until the end of the experiment (17 weeks). In group B, the mice were untreated until the mean tumor diameter was 10 mm, at which time daily i.p. treatment began. After 7 days, the primary tumors were resected, and the mice were treated for an additional 4 weeks (a total of 5 weeks of treatment). The synthetic glycoamines did not have significant antitumor effects, and there was no difference in the tumor incidence or tumor growth rates in mice treated continuously with synthetic glycoamines or PBS. The significant antimetastatic activity of synthetic glycoamines was detected in both experimental treatment protocols. In mice continuously treated with synthetic glycoamines according to protocol A, the incidence of metastasis was decreased 4.6-fold (P = 0.014) and 2.7-fold (P = 0.031) in mice treated with Fru-D-Leu and Lac-L-Leu, respectively. In mice in protocol B, the incidence of pulmonary metastasis was decreased 1.9-fold (P = 0.069) and 2.5-fold (P = 0.042) in mice treated with Fru-D-Leu and Lac-L-Leu, respectively. Correspondingly, the average number of spontaneous pulmonary metastases was reduced from 37 in control mice to 0.2 (P = 0.005) and 0.9 (P < 0.02) in mice treated according to the protocol A with Fru-D-Leu and Lac-L-Leu, respectively. Treatment of mice with N-(1-deoxy-D-fructos-1-yl)-L-leucine did not have significant antimetastatic effects, and no reduction in metastasis incidence or number was noted in mice treated with this synthetic glycoamine analogue. The treated animals had no apparent toxicity from chronic daily injection (up to 17 weeks of treatment) of synthetic glycoamines, and no obvious pathology was noted in the histological slides of the livers, kidneys, or spleens of the treated mice. Therefore, we have identified two synthetic glycoamines (Fru-D-Leu and Lac-L-Leu) that were the effective inhibitors of spontaneous human breast cancer metastasis in nude mice. Potential mechanisms for antimetastatic activity of synthetic glycoamines may include the inhibition of beta-galectin-mediated homotypic cancer cell aggregation and induction of apoptosis in target cells.

Effects of Thomsen-Friedenreich antigen-specific peptide P-30 on beta-galactoside-mediated homotypic aggregation and adhesion to the endothelium of MDA-MB-435 human breast carcinoma cells.

Both the ability of malignant cells to form multicellular aggregates via homotypic or heterotypic aggregation and their adhesion to the endothelium are important if not critical during early stages of cancer metastasis. The tumor-associated carbohydrate Thomsen-Friedenreich antigen (T antigen) and beta-galactoside binding lectins (galectins) have been implicated in tumor cell adhesion and tissue invasion. In this study, we demonstrate the involvement of T antigen in both homotypic aggregation of MDA-MB-435 human breast carcinoma cells and their adhesion to the endothelium. The T antigen-specific peptide P-30 (HGRFILPWWYAFSPS) selected from a bacteriophage display library was able to inhibit spontaneous homotypic aggregation of MDA-MB-435 cells up to 74% in a dose-dependent manner. Because T antigen has beta-galactose as a terminal sugar, the expression profile of beta-galactoside-binding lectins (galectins) in MDA-MB-435 cells was studied. Our data indicated the abundant expression of [35S]methionine/cysteine-labeled galectin-1 and galectin-3 in this cell line, which suggested possible interactions between galectins and T antigen. As revealed by laser confocal microscopy, both galectin-1 and galectin-3 also participate in the adhesion of the MDA-MB-435 cells to the endothelium. We observed the clustering of galectin-3 on endothelial cells at the sites of the contact with tumor cells, consistent with its possible interaction with T antigen on cancer cells The galectin-1 signal, however, strongly accumulated at the sites of cell-cell contacts predominantly on tumor cells. The T antigen-specific P-30 significantly (50%) inhibited this adhesion, which indicated that T antigen participates in the adhesion of MDA-MB-435 breast cancer cells to the endothelium. The ability of synthetic P-30 to inhibit both the spontaneous homotypic aggregation of MDA-MB-435 cells and their adhesion to the endothelium (>70 and 50%, respectively) suggests its potential functional significance for antiadhesive therapy of cancer metastasis.

The role of Thomsen-Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium.

Interactions of metastatic cancer cells with vasculatory endothelium are critical during early stages of cancer metastasis. Understanding the molecular underpinnings of these interactions is essential for the development of new efficacious cancer therapies. Here we demonstrate that cancer-associated carbohydrate T antigen plays a leading role in docking breast and prostate cancer cells onto endothelium by specifically interacting with endothelium-expressed beta-galactoside-binding protein, galectin-3. Importantly, T antigen-bearing glycoproteins are also capable of mobilizing galectin-3 to the surface of endothelial cells, thus priming them for harboring metastatic cancer cells. The T antigen-mediated, tumor-endothelial cell interactions could be efficiently disrupted using synthetic compounds either mimicking or masking this carbohydrate structure. High efficiency of T antigen-mimicking and T antigen-masking inhibitors of tumor cell adhesion warrants their further development into antiadhesive cancer therapeutics.

Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries.

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.

The blood group antigens (BGA)-related glycoepitopes. A key structural determinant in immunogenesis and cancer pathogenesis.

This overview has been focused on the functional and pathophysiological aspects of blood group antigens (BGA)-related glycodeterminants. It has been postulated that in a broad range of histogenetically different tissues and organs BGA-related glycoepitopes are expressed on the cell surface at definite stages of cell differentiation during embryogenesis, organogenesis, tissue repair, regeneration, remodeling and maturation when 'sorting-out' behaviour of one homotypic cell population from heterotypic assemblage of cells occurs. In this event the BGA-related glycoepitopes, if being expressed on the cell surface, play a role of key structural determinants in cell-cell recognition, association and aggregation. This mechanisms has been discussed in relation to immunogenesis regarding of antigen presentation, self-non-self discrimination, positive and negative selection during thymic education. It is postulated that the appearance of the BGA-related glycoepitopes on the cell membrane is a consequence of the association of MHC and peptides, with subsequent elimination of cells carring high density of BGA-related glycoepitopes on their surface. In cancer it has been considered as a key mechanism of phenotypic divergence of tumor cells, immunoselection, tumor progression and metastasis.

Role of rpoS regulon in resistance to oxidative stress and near-UV radiation in delta oxyR suppressor mutants of Escherichia coli.

Escherichia coli delta oxyR mutants are hyper-sensitive to oxidative agents but this sensitivity is reversed to hyper-resistance in delta oxyR suppressor strains (delta oxyRsup; Greenberg, J.T. and Demple, B. 1988. EMBO J. 7:2611-2618). Also, delta oxyR mutants have increased mutation rates that are also reversed in delta oxyRsup. We now report that the rpoS regulon may have a role in determining hyper-resistance and loss of hyper-mutability of delta oxyRsup. Delta oxyRsup cells were also resistant to near-ultraviolet radiation (near-UV) and survived longer in stationary phase than delta oxyR cells. In delta oxyRsup cells elevated beta-galactosidase expression from a rpoS::lacZ promoter fusion and significant overproduction of RpoS protein was observed. These increases were accompanied by substantial elevation in transcription of rpoS-dependent genes as determined by beta-galactosidase expression from katE::lacZ, dps::lacZ, and xthA::lacZ promoters. Catalase HPI and HPII activities were also increased. When rpoS::Tn10 was transduced into delta oxyRsup, phenotypes switched back to hyper-sensitive, hyper-mutable and reduced catalases I and II. Individual delta oxyR colonies exhibited significant clonal variability in beta-galactosidase expression from rpoS::lacZ promoter. These results provide further evidence of the functional and regulatory overlap between two major anti-oxidant defense systems of bacteria.

Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamine analogs.

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells.

Apoptosis amd metastasis: a superior resistance of metastatic cancer cells to programmed cell death.

We studied the response to different external signals leading to apoptosis of several poorly and highly metastatic cell lines employing a murine B16 melanoma experimental metastasis model. We found that highly metastatic cells exhibit a superior survival ability and resistance to apoptosis compared to poorly metastatic cells which would give the former an obvious selective growth advantage during tumor progression. Our results indicate that there is a genetic link between aggressive metastatic phenotype and resistance to apoptosis.

Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms.

Programmed cell death, particularly adhesion-dependent regulation of cell survival and apoptosis, is recognized as one of the main homeostatic mechanisms designed to control cell positioning, eliminate misplaced cells and block metastatic dissemination. Recently we reported that highly metastatic cancer cells exhibit a higher resistance to the programmed cell death compared to their poorly metastatic counterparts (Cancer Lett., 101, 43-51, 1996). However, the molecular and genetic basis for the association of aggressive metastatic phenotype with resistance toward apoptosis remains to be elucidated. Here we extended our investigation on apoptosis and metastasis using a panel of nine murine and human cancer cell lines with different metastatic potential. We examined the relationship of the metastatic ability and the sensitivity to apoptosis as well as determined the status of two major apoptosis execution mechanisms (induction of nuclear Ca2+-dependent endonucleases and activation of ICE-like proteases) in cancer cells with distinct metastatic potential and different sensitivity to apoptosis. We found that high metastatic potential is strictly associated with the increased resistance to apoptosis, diminished level of nuclear Ca2+-dependent endonucleases, and significantly reduced activity of CPP32/Yama death protease. We concluded that high resistance to apoptosis of metastatic cancer cells is associated with and may depend upon the profound deficiency of major apoptosis execution mechanisms.

The preparation and characterization of some Amadori compounds (1-amino-1-deoxy-D-fructose derivatives) derived from a series of aliphatic omega-amino acids.

Amadori compounds (1-amino-1-deoxy-D-fructose derivatives) were prepared by reacting D-glucose with a series of aliphatic amino acids. These include Amadori compounds derived from glycine (1), beta-alanine (2), gamma-amino butyric acid (3), delta-aminovaleric acid (4), epsilon-amino-caproic acid (5) and N alpha-formyl-L-lysine (6). In the FAB mass spectra, molecular-ion clusters as well as fragment ions corresponding to loss of water or CO2 molecules were observed. The 13C NMR spectra indicate that all the compounds are conformationally unstable, but that the predominant form present in solution (D2O) is the beta-pyranose form. The 1H NMR spectra of 1 and 2 indicate a slow rotation around the C-1-C-2 bond, possibly as a result of an intramolecular hydrogen bond involving the carboxyl group. The pK alpha's of all compounds were measured by pH-potentiometric titration in 0.2 M KNO3 solution at 25 degrees C. All compounds showed a decrease in the basicity of their amino groups (in the order of approximately 1.5 of the K alpha value), and 1 and 2 showed a decrease in the basicity of their carboxyl groups (in the order of approximately 0.2) in comparison with that of parent amino acids.

Crystal structure of an Amadori compound, N-(1-deoxy-beta-D-fructopyranos-1-yl)-glycine ("D-fructose-glycine").

The first crystal structure data on an Amadori compound, N-(1-deoxy-beta-D-fructopyranos-1-yl)-glycine, are reported. The space group is P2(1) with Z = 2 and cell parameters a = 7.246(1), b = 10.009(1), c = 7.060(1) A, and beta = 101.085(6) degrees. The structure was solved by direct methods and refined to a final R of 2.9% and Rw of 3.8% for 1385 reflections to give esd's of 0.002 A in bond lengths and 0.2 degree in angles. The conformation of the carbohydrate is the normal 2C5 pyranose chair. Bond lengths and valence angles compare well with average values from a number of pyranose structures. The molecule of the Amadori compound exists in the zwitterion form and has the C-6-O-6-C-2-C-1-N-C-2'-C-1'-O-1' chain in a zig-zag conformation, that is (together with O-2') substantionally planar. All hydroxyl, carboxyl, and ring oxygen atoms, and the secondary ammonium group are involved in hydrogen bonding, which forms a three-dimensional network of two infinite chains that have an ammonium group as a common segment. The shortest intra- and inter-molecular hydrogen bonds involved donors of the pyranosyl moiety and acceptors of the amino acid portion, and vice versa.

Molecular and crystal structure of N-(2-deoxy-D-aldohexos-2-yl)-glycines (Heyns compounds).

Heyns compounds, 2-carboxymethylamino-2-deoxy-D-glucose (1), -mannose (2), and -galactose (3), were prepared by N-carboxymethylation of the corresponding hexosamines and 1 was also prepared via the reaction of D-fructose with glycine. Both 1 and 3 crystallize from aqueous solutions as zwitterions in the alpha-pyranose form and in the 4C1 conformation. Crystalline 1 is nearly isostructural to N-acetylglucosamine, forming stacks of molecules with infinite chains of homodromic hydrogen bonds along the stacks. For both 1 and 3, all hydroxyl, ammonium, and carboxyl groups are involved in intermolecular hydrogen-bonding, and an intramolecular hydrogen bond in 3 is formed via interaction of the ammonium and carboxyl groups. 1H and 13C NMR spectra (D2O solutions) indicate that all of the compounds are conformationally unstable, and that the major form present in D2O solution at 25 degrees C is the 4C1 alpha-pyranose form, with the 4C1 beta-pyranose form present in lesser amounts. In addition, for solutions of 2 and 3, considerable amounts of alpha- and beta-furanose forms are present and exist in conformations favorable for a cis-relationship between the carboxymethylammonium and anomeric hydroxyl groups.

The blood group antigen-related glycoepitopes: key structural determinants in immunogenesis and AIDS pathogenesis.

This overview will focus on the functional and pathophysiological aspects of blood group antigen (BGA)-related glycodeterminants with regard to immunogenesis and AIDS pathogenesis. It has been postulated that in a broad range of histogenetically different tissues and organs, BGA-related glycoepitopes are expressed on the cell surface at definite stages of cell differentiation. These glycoepitopes are expressed during embryogenesis, organogenesis, tissue repair, regeneration, remodelling and maturation when 'sorting-out' of one homotypic cell population from a heterotypic assemblage of cells occurs (1). In this event, the BGA-related glycoepitopes, if being expressed on the cell surface, play roles of key structural determinants in cell-cell recognition, association and aggregation. This mechanism will be discussed in relation to immunogenesis with regard to antigen presentation, self-non-self discrimination, and positive and negative selection during thymic education. It is postulated that the appearance of BGA-related glycoepitopes on the cell membrane is a consequence of the association of major histocompatibility complex antigens (MHC) and peptides, with the subsequent elimination of cells carrying a high density of BGA-related glycoepitopes on their surface. After human immunodeficiency virus (HIV) glycoproteins are glycosylated by host cell glycosyltransferases, the virus may use the BGA-related glycodeterminants as ligands and/or receptors for expansion to a spectrum of target cells during AIDS development and generalization of the infection throughout the body. We will review the experimental evidence that supports the concept that HIV uses an alternative to the gp120/CD4 ligand/receptor system, and that the alternative mechanism is probably carbohydrate-mediated in nature.

Tetraiodothyroacetic acid (tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid.

CONTEXT: Tetraiodothyroacetic acid (tetrac) blocks angiogenic and tumor cell proliferation actions of thyroid hormone initiated at the cell surface hormone receptor on integrin alphavbeta3. Tetrac also inhibits angiogenesis initiated by vascular endothelial growth factor and basic fibroblast growth factor. OBJECTIVE: We tested antiangiogenic and antiproliferative efficacy of tetrac and tetrac nanoparticles (tetrac NP) against human medullary thyroid carcinoma (h-MTC) implants in the chick chorioallantoic membrane (CAM) and h-MTC xenografts in the nude mouse. DESIGN: h-MTC cells were implanted in the CAM model (n = 8 per group); effects of tetrac and tetrac NP at 1 microg/CAM were determined on tumor angiogenesis and tumor growth after 8 d. h-MTC cells were also implanted sc in nude mice (n = 6 animals per group), and actions on established tumor growth of unmodified tetrac and tetrac NP ip were determined. RESULTS: In the CAM, tetrac and tetrac NP inhibited tumor growth and tumor-associated angiogenesis. In the nude mouse xenograft model, established 450-500 mm(3) h-MTC tumors were reduced in size over 21 d by both tetrac formulations to less than the initial cell mass (100 mm(3)). Tumor tissue hemoglobin content of xenografts decreased by 66% over the course of administration of each drug. RNA microarray and quantitative real-time PCR of tumor cell mRNAs revealed that both tetrac formulations significantly induced antiangiogenic thrombospondin 1 and apoptosis activator gene expression. CONCLUSIONS: Acting via a cell surface receptor, tetrac and tetrac NP inhibit growth of h-MTC cells and associated angiogenesis in CAM and mouse xenograft models.