globotriaosylceramide and Adenocarcinoma

The B-subunit of the bacterial Shiga toxin (STxB), which is nontoxic and has low immunogenicity, can be used for tumor targeting of breast, colon, and pancreatic cancer. Here, we tested whether human gastric cancers, which are among the most aggressive tumor entities, express the cellular receptor of Shiga toxin, the glycosphingolipid globotriaosylceramide (Gb3/CD77). The majority of cases showed an extensive staining for Gb3 (36/50 cases, 72%), as evidenced on tissue sections of surgically resected specimen. Gb3 expression was detected independent of type (diffuse/intestinal), and was negatively correlated to increasing tumor-node-metastasis stages (P = 0.0385), as well as with markers for senescence. Gb3 expression in nondiseased gastric mucosa was restricted to chief and parietal cells at the bottom of the gastric glands, and was not elevated in endoscopic samples of gastritis (n = 10). Gb3 expression in established cell lines of gastric carcinoma was heterogeneous, with 6 of 10 lines being positive, evidenced by flow cytometry. STxB was taken up rapidly by live Gb3-positive gastric cancer cells, following the intracellular retrograde transport route, avoiding lysosomes and rapidly reaching the Golgi apparatus and the endoplasmic reticulum. Treatment of the Gb3-expressing gastric carcinoma cell line St3051 with STxB coupled to SN38, the active metabolite of the topoisomerase type I inhibitor irinotecan, resulted in >100-fold increased cytotoxicity, as compared with irinotecan alone. No cytotoxicity was observed on gastric cancer cell lines lacking Gb3 expression, demonstrating receptor specificity of the STxB-SN38 compound. Thus, STxB is a highly specific transport vehicle for cytotoxic agents in gastric carcinoma. Mol Cancer Ther; 15(5); 1008-17. ©2016 AACR.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Dose-Response Relationship, Drug; Gastric Mucosa; Gene Expression; Humans; Immunotoxins; Molecular Targeted Therapy; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Shiga Toxins; Stomach Neoplasms; Trihexosylceramides

Shiga toxins (Stxs) are composed of an enzymatically active A subunit (StxA) and a pentameric B subunit (StxB) that preferentially binds to the glycosphingolipid (GSL) globo\\xadtriaosylceramide (Gb3Cer/CD77) and to a reduced extent to globotetraosylceramide (Gb4Cer). The identification of Gb3Cer as a tumor-associated GSL in human pancreatic cancer prompted us to investigate the expression of Gb3Cer and Gb4Cer in 15 human pancreatic ductal adenocarcinoma cell lines derived from primary tumors and liver, ascites, and lymph node metastases. Thin-layer chromatography overlay assays revealed the occurrence of Gb3Cer in all and of Gb4Cer in the majority of cell lines, which largely correlated with transcriptional expression analysis of Gb3Cer and Gb4Cer synthases. Prominent Gb3Cer and Gb4Cer lipoform heterogeneity was based on ceramides carrying predominantly C16:0 and C24:0/C24:1 fatty acids. Stx2-mediated cell injury ranged from extremely high sensitivity (CD(50) of 0.94 pg/ml) to high refractiveness (CD(50) of 5.8 μg/ml) and to virtual resistance portrayed by non-determinable CD(50) values even at the highest Stx2 concentration (10 μg/ml) applied. Importantly, Stx2-mediated cytotoxicity did not correlate with Gb3Cer expression (the preferential Stx receptor), suggesting that the GSL receptor content does not primarily determine cell sensitivity and that other, yet to be delineated, cellular factors might influence the responsiveness of cancer cells.

Topics: Adenocarcinoma; Ascites; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Globosides; Humans; Liver Neoplasms; Lymph Nodes; Shiga Toxin 2; Shiga-Toxigenic Escherichia coli; Trihexosylceramides

Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.. Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.. Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Topics: Adenocarcinoma; Carbohydrate Sequence; Chromatography, Thin Layer; Colonic Neoplasms; Humans; Immunohistochemistry; Molecular Sequence Data; Pancreatic Neoplasms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trihexosylceramides

Porcine and bovine aortic endothelial cells and human colonic adenocarcinoma cells were compared for their susceptibility to the toxic effect of purified Shiga-like toxin IIe (SLT-IIe), measured by the neutral red cytotoxicity assay. Cytotoxicity correlated with toxin binding as indicated by fluorescence activated cell sorter analysis and with the globotriosylceramide (Gb3) and globotetraosylceramide (Gb4) content of cells determined by high pressure liquid chromatography. One line of porcine aortic endothelial cells was 1400-fold more susceptible than the line of bovine aortic endothelial cells that was tested, but a second line of porcine aortic endothelial cells was highly refractory to SLT-IIe. Human colonic adenocarcinoma cells lacked detectable levels of Gb4 and were least susceptible to SLT-IIe.

Topics: Adenocarcinoma; Animals; Aorta; Bacterial Toxins; Cattle; Cell Line; Cell Survival; Cells, Cultured; Chlorocebus aethiops; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cytotoxins; Endothelium, Vascular; Escherichia coli; Flow Cytometry; Globosides; Humans; Receptors, Cell Surface; Shiga Toxin 2; Swine; Trihexosylceramides; Tumor Cells, Cultured; Vero Cells

Differentiated villus intestinal epithelial cells express globotriaosylceramide, the Shiga-like toxin 1 (SLT-1) receptor, and are sensitive to toxin-mediated cytotoxicity, whereas undifferentiated crypt cells neither express Gb3 nor respond to toxin. To investigate if SLT-1 receptors are maturationally regulated in human intestinal cells, we examined the effect of butyrate, a known transcriptional regulator of differentiation genes in many cell types, using cultured colonic cancer-derived epithelial cell lines. Exposure to butyrate increased villus cell marker enzymes such as alkaline phosphatase, sucrase, and lactase, expression of toxin receptors, and sensitivity to SLT-1 in villus-like CaCo-2A and HT-29 cells. These effects were reversibly inhibited by preincubation of CaCo-2A cells with actinomycin D or cycloheximide. Butyrate-treated CaCo-2A cells unable to bind fluoresceinated SLT-1 B subunit were undifferentiated as assessed by alkaline phosphatase activity. HT-29 cells induced to differentiate by another signal, glucose deprivation, upregulated receptor content and response to toxin. Crypt-like T-84 cells responded to butyrate with a modest increase in alkaline phosphatase and toxin binding, but no induction of sucrase or lactase, and no change in sensitivity to toxin. The results demonstrate that expression of SLT-1 toxin receptors and toxin sensitivity are coregulated with cellular differentiation in cultured intestinal cells.

Topics: Adenocarcinoma; Bacterial Toxins; Butyrates; Butyric Acid; Cell Line; Cell Survival; Colonic Neoplasms; Cycloheximide; Dactinomycin; Enterotoxins; Epithelium; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glycolipids; Humans; Kinetics; Receptors, Cell Surface; Shiga Toxin 1; Time Factors; Transcription, Genetic; Trihexosylceramides; Tumor Cells, Cultured