title: Intracellular delivery of recombinant arginine deiminase (rADI) by heparin-binding hemagglutinin adhesion peptide restores sensitivity in rADI-resistant cancer cells.
abstract: Recombinant arginine deiminase (rADI) has been used in clinical trials for arginine-auxotrophic cancers. However, the emergence of rADI resistance, due to the overexpression of argininosuccinate synthetase (AS), has introduced an obstacle in its clinical application. Here, we have proposed a strategy for the intracellular delivery of rADI, which depletes both extracellular and intracellular arginine, to restore the sensitivity of rADI-resistant cancer cells. In this study, the C terminus of heparin-binding hemagglutinin adhesion protein from Mycobacterium tuberculosis (HBHAc), which contains 23 amino acids, was used to deliver rADI into rADI-resistant human breast adenocarcinoma cells (MCF-7). Chemical conjugates (l- and d-HBHAc-SPDP-rADI) and a recombinant fusion protein (rHBHAc-ADI) were produced. l- and d-HBHAc-SPDP-rADI showed a significantly higher cellular uptake of rADI by MCF-7 cells compared to that of rADI alone. Cell viability was significantly decreased in a dose-dependent manner in response to l- and d-HBHAc-SPDP-rADI treatments. In addition, the ratio of intracellular concentration of citrulline to arginine in cells treated with l- and d-HBHAc-SPDP-rADI was significantly increased by 1.4- and 1.7-fold, respectively, compared with that obtained in cells treated with rADI alone (p < 0.001). Similar results were obtained with the recombinant fusion protein rHBHAc-ADI. Our study demonstrates that the increased cellular uptake of rADI by HBHAc modification can restore the sensitivity of rADI treatment in MCF-7 cells. rHBHAc-ADI may represent a novel class of antitumor enzyme with an intracellular mechanism that is independent of AS expression.
authors.affiliation: School of Pharmacy and ‡Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University , Taipei 10050, Taiwan.
authors.forename: Fe-Lin Lin
authors.initials: FL
authors.lastname: Wu
authors.forename: Tzyy-Harn
authors.initials: TH
authors.lastname: Yeh
authors.forename: Ying-Luen
authors.initials: YL
authors.lastname: Chen
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Chiu
authors.forename: Ju-Chen
authors.initials: JC
authors.lastname: Cheng
authors.forename: Ming-Feng
authors.initials: MF
authors.lastname: Wei
authors.forename: Li-Jiuan
authors.initials: LJ
authors.lastname: Shen
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001124:Argininosuccinate Synthase / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D002956:Citrulline / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004705:Endocytosis; D019793:Fluorescein / Q000737:chemistry; D006801:Humans; D006867:Hydrolases / Q000008:administration & dosage*; D037102:Lectins / Q000737:chemistry*; D061986:MCF-7 Cells; D009169:Mycobacterium tuberculosis; D009369:Neoplasms / Q000188:drug therapy; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*
chemical_list: D000596:Amino Acids; D037102:Lectins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C104578:heparin-binding hemagglutinin; D002956:Citrulline; D006867:Hydrolases; C019723:arginine deiminase; D001124:Argininosuccinate Synthase; D019793:Fluorescein