Cytotoxicity of folate-Pseudomonas exotoxin conjugates toward tumor cells. Contribution of translocation domain.
Folate-protein conjugates can be nondestructively delivered into a cell's cytoplasm via folate receptor-mediated endocytosis if (i) the target cells express a folate-binding protein, and (ii) if the folate is linked to its attached protein at a site that does not interfere with receptor recognition. Because such conjugates have been observed to remain in endosomal compartments for extended periods following cellular uptake, we decided to evaluate whether release into the cytoplasm might be expedited by inclusion of a translocation domain in the folate-protein construct. To test this possibility, momordin-folate and truncated Pseudomonas exotoxin-folate conjugates (LysPE38 and Cys-PE35), i.e. protein synthesis inhibitors either lacking or containing the desired translocation domain, respectively, were examined for their abilities to block protein synthesis in a variety of cell types. The translocation competent LysPE38-folate construct was found to kill cells six times more rapidly with 10-fold greater potency than the permeation-incompetent mormordin-folate. Further, cells expressing low levels of folate receptors could only be exterminated by the translocation competent Pseudomonas exotoxin-folate conjugates. When the translocation capability of CysPE35-folate was inactivated by modification of Cys287, the construct also lost most of its cytotoxicity. These data suggest that autocatalysis of transport from an internal vesicular compartment into the cytoplasm can greatly augment the cytotoxicity of a protein toxin entering cells via the folate endocytosis pathway. Because the folate ligand can selectively target a protein conjugate to cancer cells in the presence of normal cells, such translocatable toxin-folate constructs warrant further study as a possible treatment for some malignancies.
Leamon
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Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Pastan
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Low
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D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D001692:Biological Transport; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003603:Cytotoxins / Q000378:metabolism / Q000633:toxicity*; D005098:Exotoxins / Q000378:metabolism / Q000633:toxicity*; D005492:Folic Acid / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000494:pharmacology; D014176:Protein Biosynthesis; D054790:Ribosome Inactivating Proteins, Type 2; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003603:Cytotoxins; D005098:Exotoxins; D005492:Folic Acid; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A