title: Synthesis and Evaluation of a CBZ-AAN-Dox Prodrug and its in vitro Effects on SiHa Cervical Cancer Cells Under Hypoxic Conditions.
abstract: Although doxorubicin (Dox) is widely used in clinical treatment for solid tumors, it causes many side-effects such as heart and kidney damage, bone marrow suppression, and drug resistance. Legumain is a lysosomal protease that is elevated and associated with an invasive and metastatic phenotype in a number of solid tumors. In this study, we designed and synthesized a Dox prodrug, N-benzyloxycarbonyl-Ala-Ala-Asn-Doxorubicin (CBZ-AAN-Dox), with 94% purity. Single substrate kinetic assays demonstrated hLegumain-specific enzymatic cleavage and activation of the prodrug in vitro, and this enzymatic cleavage of the prodrug substrate was more sensitive in acidic conditions, releasing more than 70% of Dox after 24 h. Treatment of tumor cells with our prodrug demonstrated a much higher IC50 value, significantly enhanced uptake of the prodrug, and considerably less cellular toxicity compared to Dox treatment alone. Our study presents a novel prodrug, CBZ-AAN-Dox, to potentially increase both the safety and efficacy of clinical treatment of tumors by exploiting the tumor's innate expression of legumain.
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.|Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02129, USA.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Clayman
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Shao
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Manshan
authors.initials: M
authors.lastname: Xiao
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Xuyan
authors.initials: X
authors.lastname: Tian
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Wuyu
authors.initials: W
authors.lastname: Fu
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Caiyun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Bibo
authors.initials: B
authors.lastname: Ruan
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou, 510060, China.
authors.forename: Pengjun
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Biomedicine Research and Development Center of Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, 510632, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Biomedicine Research and Development Center of Jinan University, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Guangzhou, 510632, China.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Centre Laboratory, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Rui
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D060326:Chemistry Techniques, Synthetic; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000494:pharmacology; D011355:Prodrugs / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000473:pathology; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D004317:Doxorubicin; D009842:Oligopeptides; D011355:Prodrugs; D000099240:Asparaginyl Endopeptidase; C000609000:N-benzyloxycarbonyl-alanyl-alanyl-aspartyl-doxorubicin
keywords: N-benzyloxycarbonyl-Ala-Ala-Asn-Doxorubicin; SiHa cells; asparagine endopeptidase; doxorubicin; hypoxia; legumain; prodrug