title: Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.
abstract: Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University , Taipei, Taiwan.|Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California , Los Angeles, California 90033, United States.
authors.forename: Tzyy-Harn
authors.initials: TH
authors.lastname: Yeh
authors.affiliation: Department of Metabolic Research, Beckman Research Institute, City of Hope , Duarte, California 91010, United States.
authors.forename: Yun-Ru
authors.initials: YR
authors.lastname: Chen
authors.affiliation: Department of Metabolic Research, Beckman Research Institute, City of Hope , Duarte, California 91010, United States.|Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University , Tainan, Taiwan.
authors.forename: Szu-Ying
authors.initials: SY
authors.lastname: Chen
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California , Los Angeles, California 90033, United States.
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
authors.affiliation: Department of Metabolic Research, Beckman Research Institute, City of Hope , Duarte, California 91010, United States.
authors.forename: David K
authors.initials: DK
authors.lastname: Ann
authors.affiliation: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California , Los Angeles, California 90033, United States.
authors.forename: Jennica L
authors.initials: JL
authors.lastname: Zaro
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University , Taipei, Taiwan.|Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University , Taipei, Taiwan.|Department of Pharmacy, National Taiwan University Hospital , Taipei, Taiwan.
authors.forename: Li-Jiuan
authors.initials: LJ
authors.lastname: Shen
mesh_terms: D000818:Animals; D001124:Argininosuccinate Synthase / Q000378:metabolism; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D005260:Female; D006801:Humans; D006867:Hydrolases / Q000008:administration & dosage* / Q000737:chemistry / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D009374:Neoplasms, Experimental / Q000188:drug therapy*; D011092:Polyethylene Glycols / Q000627:therapeutic use*; D023041:Xenograft Model Antitumor Assays
chemical_list: D057846:Cell-Penetrating Peptides; D011092:Polyethylene Glycols; D006867:Hydrolases; C512527:ADI PEG20; C019723:arginine deiminase; D001124:Argininosuccinate Synthase
keywords: arginine deiminase resistance; argininosuccinate synthetase; hypoxia; pH-sensitive peptide; tumor targeting