The coupling of p-aminophenyl 2-acetamido-2-deoxy-3-O-beta-D- galactopyranosyl-beta-D-galactopyranoside (gal-beta 1,3-galNAc) to bovine serum albumin (BSA) was achieved by using 1,2-diethoxycyclobutene-3,4-dione (squaric acid diester) as a new coupling reagent. Two selective consequential steps afforded the desired neoglycoprotein: reaction of the p-aminophenyl group of gal-beta 1,3-galNAc with squaric acid diester gave the corresponding squaric acid amide ester, which was transformed into the BSA conjugate by coupling with the lysyl epsilon-amino group of BSA through formation of a squaric acid 1,2-bisamide. The experimental conditions for the reactions and the optimization of average were performed by using p-anisidine as model substance, the methyl group substituting for the carbohydrate part of a p-aminophenylglycoside. Neoglycoproteins have proven to be valuable tools for lectin detection. To evaluate the properties of this type of probe, the obtained neoglycoprotein with the histochemically crucial T-antigen structure was used for glycocytological and glycohistochemical studies. Three cultured human tumor cell lines and tissue sections from human breast carcinomas were chosen. Its efficiency was similar in comparison to measurements with a probe, derived by diazotization with the p-aminophenyl glycosides of gal-beta 1,3-galNAc and already shown to be a reliable marker for lectin localization in tissue sections and cultured cells.

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The toxic galactoside-specific lectin from mistletoe, a component of proprietary extracts with unproven efficacy in oncology, exhibits capacity to trigger enhanced secretion of proinflammatory cytokines at low doses (ng/ml or ng/kg body weight) and reductions of cell viability with increasing concentrations. To infer any tumor selectivity of this activity, cytofluorimetric and cell growth assays with a variety of established human tumor cell lines were performed. Only quantitative changes were apparent, and the toxicity against tumor cells was within the range of that of the tested fibroblast preparations from 5 donors. No indication for any tumor selectivity was observed. In kinetic studies with 8 sarcoma and 4 melanoma lines, this evidence for quantitative variability of the response in interindividual comparison was further underscored. At 50 pg lectin/ml x 10(5) cells, even a growth-stimulatory impact was noted in 5 of 12 tested cases. To mimic in vivo conditions with presence of cytokine-secreting inflammatory and stromal cells, exposure to the lectin was extended to histotypic cultures established from 30 cases of surgically removed tumor. As salient result, 5 specimens from 4 of the 8 tested tumor classes responded with a significant increase of [3H]-thymidine incorporation relative to controls during the culture period of 72 hours, when the lectin was present at a concentration in the described immunomodulatory range (1 ng/ml). A relation of this activity to the extent of the actual proliferative status of the reactive samples could not be delineated. Therefore, a non-negligible percentage of the established tumor cell lines (e.g., 3 from 8 sarcoma lines) can be markedly stimulated by the lectin at a very low dose and with dependence on the cell type. Furthermore, the feasibility to elicit a significant growth enhancement is likewise documented for human tumor explants in 16.6% of the examined cases. In view of the uncontrolled application of lectin-containing extracts in alternative/complementary medicine, the presented results on unquestionably adverse lectin-dependent effects in two culture systems call for rigorous examination of the clinical safety of this unconventional, scientifically entirely experimental treatment modality.

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