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Title: Development of amino acid depleting enzyme for the treatment of cancer
Authors: Chong, Hiu Chi
Advisors: Leung, Y. C. Thomas (ABCT)
Keywords: Enzymes -- Analysis.
Cancer -- Treatment.
Issue Date: 2015
Publisher: The Hong Kong Polytechnic University
Abstract: Gastric and colorectal cancers are the top five most commonly diagnosed cancers around the world.These cancers are usually asymptomatic in the early stage and essentially,early detection can be achieved merely by screening program.Nevertheless,poor prognosis represents a major problem in advanced stage gastric cancer and metastatic colorectal cancer.It is therefore of paramount importance to develop new therapeutics for these cancers.Metabolic reprogramming has been proposed as one of the hallmarks of cancer.Differential requirement of various nutrients such as amino acids has been observed in cancer cells. The best known example of amino acid depleting enzyme is the pegylated bacterial asparaginase (Oncaspar),which has been approved by the US FDA as the firstline treatment for acute lymphoblastic leukemia (ALL) in children.Elevated requirement of arginine has been observed in different types of cancer and thus, a novel arginine depleting enzyme,engineered Bacillus caldovelox arginase (BCA) was developed in this study.Plasmid vector carrying the BCA gene was transformed into E. coli cells for the overexpression,which is mediated by IPTG induction.The thermostable BCA protein with a 6xHis-tag fused to its C-terminus was purified through a simple two-step purification scheme,with heat treatment followed by metal affinity column chromatography.In our structure-based rational drug design,a unique cysteine residue was strategically placed on the surface of the BCA molecule to facilitate site-specific pegylation. Characterization of both the native and the pegylated BCA were performed.Briefly,the major proportion of native BCA exists in hexameric structure while monomeric structure of BCA presents in tiny amount as determined by gel filtration column.The size of BCA increased from 33275.25 Da to about 55 kDa after pegylation.Pegylation do not significantly alter the secondary structure of BCA and comparable enzyme activity was observed for both native and pegylated BCA.The enzymatic activity of BCA was found specific to arginine when tested against 40 different amino acids.The stability of BCA was further improved by pegylation for which 90% activity could be retained for 32 months of storage.The in vitro anti-cancer effects of BCA were tested on three gastric cancer(MKN45, BCG823 and AGS) and two colorectal cancer (HCT-15 and HCT116) cell lines.All the cell lines lacking the expression of OTC were found to be vulnerable to the BCA treatment and arrested in different phases (G2/M and S) of cell cycle while four out of the five cell lines tested were found to undergo apoptotic cell death.Pharmacological study revealed that the circulating half-life of BCA was extended by 12-fold after pegylation while an intraperitoneal injection of this pegylated BCA in mice reduced the serum arginine to an undetectable level for 5-6 days.Importantly,efficacy studies showed that the pegylated BCA effectively suppressed the growth of tumor xenograft in mice and its efficacy is comparable to that of 5-FU.Furthermore,synergistic effect was observed when the pegylated BCA was tested in combination with 5-FU.These promising results warrant the pegylated BCA for further study and cancer patients might benefit from this novel treatment strategy in the future.
Description: PolyU Library Call No.: [THS] LG51 .H577P ABCT 2015 Chong
ix, 257 pages :color illustrations
Rights: All rights reserved.
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