lewis-y-antigen and Colonic-Neoplasms

Cancer-homing toxins are a group of man-made cytotoxic molecules targeting cancer cells. In the past decade they have demonstrated potential as cancer therapeutics. These molecules contain a toxin, natural or usually derivatized, connected to a cancer-homing module, such as a monoclonal antibody or growth factor or their derivatives. Various cancer-homing toxins have been designed and tested in cell-lines, animal-models and clinical trials. We review some of these data and discuss ways to better design cancer-homing toxins in the light of advances in cancer genomics, antibody-engineering techniques and computational algorithms.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, CD19; Antigens, Differentiation, B-Lymphocyte; Antineoplastic Agents; Bacterial Toxins; Cell Adhesion Molecules; Clinical Trials as Topic; Colonic Neoplasms; Drug Resistance, Multiple; ErbB Receptors; Hematologic Neoplasms; Humans; Immunotoxins; Lectins; Lewis Blood Group Antigens; Plant Proteins; Receptors, Growth Factor; Receptors, Interleukin-2; Sialic Acid Binding Ig-like Lectin 2

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antigens, Neoplasm; Carcinoma; Clinical Trials, Phase I as Topic; Colonic Neoplasms; Daunorubicin; Digestive System; Doxorubicin; Drug Design; Gastrointestinal Diseases; Humans; Immunotoxins; Lewis Blood Group Antigens; Mice; Mice, Nude; Neoplasms; Neoplasms, Experimental; Organ Specificity; Rats; Rats, Inbred BN; Rats, Nude; Treatment Failure; Xenograft Model Antitumor Assays

The α 1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α 1,2-fucosyltransferase (FUT) activity. 5'-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site -10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region -91 to -81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5'-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells.

Topics: Binding Sites; Cell Line, Tumor; Colonic Neoplasms; Enzyme Activation; ets-Domain Protein Elk-1; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Transcription Initiation Site; Transcription, Genetic; Transfection

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galβ1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinoembryonic Antigen; Cell Adhesion Molecules; Colon; Colonic Neoplasms; Galectin 3; Gastric Mucosa; Glycosylation; GPI-Linked Proteins; Humans; Intestinal Mucosa; Lectins, C-Type; Lewis Blood Group Antigens; Lewis X Antigen; Mannose; Mucin-1; Peanut Agglutinin; Receptors, Cell Surface

Monoclonal antibodies (mAb) are important tools in the management of tumor disease, and the discovery of antibodies with both specific cancer cell targeting and capacity to enter the cells by internalization are critical to improve the therapeutic efficacy.. Antibody cancer cell targeting and internalization properties of fluoroscein-conjugated mAb made against Lewis Y (BR96) were evaluated quantitatively and qualitatively by means of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM), respectively, on cells from a rat tumor cell line (BN7005-H1D2).. The study demonstrated a specific binding of BR96 to LewisY (LeY) located in the cell membrane and as BR96/LeY immunocomplexes (BR96/LeY) internalized into the cytoplasm. BR96/LeY was internalized into about 15% of the cells, usually distributed throughout the cytoplasm, but also located close to the nuclei. Cytotoxic effects by BR96 were indicated, and CLSM visualized subpopulations containing cells with bound or internalized BR96/LeY that possessed morphologically pyknotic nuclei and disrupted DNA.. The spatial-temporal pattern by BR96 cell targeting and internalization processes of BR96/LeY into the cancer cells expressing LeY was demonstrated by FCM and CLSM. Used together, the FCM and CLSM techniques provide a valuable tool for preclinical analyses of antibody targeting and their capacities as carriers of cytotoxic conjugates for the use in cancer therapy.

Topics: Active Transport, Cell Nucleus; Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Colonic Neoplasms; Cytoplasm; DNA Damage; Drug Carriers; Flow Cytometry; Lewis Blood Group Antigens; Microscopy, Confocal; Rats; Time Factors

The chimeric monoclonal antibody cBR96 conjugated to doxorubicin (cBR96-Dox) is selectively internalized by a wide variety of human carcinomas expressing an extended form of Lewis Y antigen (Le(y)). Endocytosis is followed by cleavage and release of free doxorubicin from the endocytic vesicles and subsequent cytotoxicity. Combination studies with standard anti-cancer agents, undertaken to further increase the potency of this targeted therapy, identified significant synergistic anti-tumor activity of cBR96-Dox and either of the taxanes paclitaxel or docetaxel. Treatment with cBR96-Dox 24 hr prior to paclitaxel resulted in a steady increase in the percentage of G(2) tumor cells and corresponding increase in sensitivity to taxanes. Cell cycle analysis indicated the cBR96-delivered doxorubicin was most effective against S-phase cells, yet cells exposed to even subtoxic levels progressed to and arrested in G(2), at a point of high sensitivity to the anti-tubulin agent paclitaxel. The synergy obtained by staged combination of cBR96-Dox and paclitaxel in vitro was reflected in significant anti-tumor efficacy in vivo against xenograft models of human lung and breast tumors that could not be achieved by either agent alone. The staged combination elicited significant or complete regressions of established human Le(y)-positive tumor xenografts using significantly reduced drug levels. Taken together, these data demonstrate a mechanistic approach to the selective elimination of Le(y)-positive tumors by using targeted doxorubicin followed by taxane treatment.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Colonic Neoplasms; Docetaxel; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Immunotoxins; Inhibitory Concentration 50; Lewis Blood Group Antigens; Lung Neoplasms; Mice; Mice, Nude; Paclitaxel; Taxoids; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Cell surface antigens are contributory factors toward metastatic activity. There have been no detailed studies on changes in cell surface antigens of colon cancer cell lines. To control life-threatening metastasis, it is necessary to evaluate what types of changes in cell surface antigens exert an influence on metastatic activity.. In vivo selection was performed using the human colon cancer-derived cell line KM12SM to obtain variants of metastatic activity. A murine spleen injection-liver metastasis procedure reflecting the latter half of the metastatic process was adopted and repeated four times. Flow cytometric analyses were carried out to detect expression of antigens: Lewis a (Lea), Lewis x (Lex), sialyl Lewis a (sLea), sialyl Lewis x (sLex), E-cadherin, CD44v6, integrin alpha2 (CD49b), integrin alpha3 (CD49c), integrin alpha4 (CD49d), integrin alpha5 (CD49e), and integrin beta1 (CD29).. In vivo selection produced variants with higher metastatic activity. In the original line KM12SM, sLea, E-cadherin, CD49b, CD49c, or CD29 were positive in more than 40% of the cells. After selection, the percentage of cells positive for Lea, sLea, and all examined integrins significantly increased. Lex, sLex, and CD44v6 increased slightly, while E-cadherin decreased slightly.. In vivo selection and flow cytometric analysis revealed that Lea, sLea, CD49b, CD49c, and CD29 appear to be involved in the increase of metastatic activity. The changes of integrin expression in this study suggest that integrins collaborate in the promotion of adhesion to an extracellular matrix.

Topics: Animals; Antigens, CD; Colonic Neoplasms; E-Selectin; Flow Cytometry; Integrin alpha2; Integrin beta1; Lewis Blood Group Antigens; Male; Mice; Mice, Inbred BALB C; Mice, Nude