| Author: | Wei, Xinlei |
| Title: | Arginine decarboxylase (ADC) : preparation, expression, purification and test of anti-cancer properties |
| Advisors: | Leung, Y. C.Thomas (ABCT) |
| Degree: | Ph.D. |
| Year: | 2015 |
| Subject: | Arginine Arginine -- Therapeutic use Decarboxylation Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xxx, 322 pages : color illustrations |
| Language: | English |
| Abstract: | Arginine metabolic enzymes are being investigated worldwide as agents
for cancer treatment, because many tumor cells are auxotrophic for arginine.
Biosynthetic arginine decarboxylase (ADC), an enzyme that catalyzes the
conversion of arginine to agmatine and carbon dioxide, possesses anti-tumor
activity yet has received much less attention than the other two argininedepleting
enzymes – arginine deiminase (ADI) and arginase. In order to
gain a better understanding of ADC, the expression, purification and anticancer
properties of this enzyme originates from Escherichia coli were
explored in this project. ADC tagged with 6 histidine residues was expressed in E. coli grown in shake flask and had undergone a single-step affinity chromatographic purification. Typically, around 110 mg of ADC can be purified from E. coli grown in 1 L culture medium. Purified ADC is of around 28.9 ± 2.7 units/mg at 37°C and pH 8.0, and remains relatively stable for at least 6 months when stored at 4°C in darkness. When tested in vitro, ADC inhibits the proliferation of ten cell lines of different human cancer types, with IC50 values ranging from 3.8 to 38.1 μg/ml, yet is relatively safer in a non-tumorous cell line. Further in vitro studies focusing on HCT116 and LoVo colorectal cancer cells indicates that ADC induces S and/or G2/M phase arrest as well as intensive apoptosis in these cells. The ADC-induced apoptosis follows the mitochondrial apoptotic pathway and is caspase-3-dependent in HCT116 cells but not in LoVo cells. Autophagy, surprisingly, is not observed in either cell lines. In fact, the antiproliferation effect of ADC in HCT116 cells is antagonized by the autophagy inhibitor hydroxychloroquine (HCQ). Related to these effects, multiple pathways in HCT116 cells may have been altered by the treatment of ADC, including the inhibition of extracellular regulated protein kinase (ERK) activity and the activation of Akt through phosphorylation. Further drug combination studies suggest that ADC is synergistic with doxorubicin and LY204002 at high doses, while antagonistic to verapamil at all doses. To proceed to in vivo studies, a major challenge for a protein drug is the extension of its blood circulation half-life. To tackle this problem, ADC fused with an albumin binding domain (ABD) has been tested in this project. Having the specific activity of ADC almost fully retained, ADCABD is even more potent than ADC in vitro but fails to prolong the arginine-depletion effect in vivo. The Bacillus caldovelox arginase (BCA)- ABD fusion protein (BHA), however, decreases serum arginine to an undetectable level for a much longer period (24 h) than that when using native BCA (2 h), thus proves the feasibility of the ABD fusion strategy. With all results obtained, we suggest that ADC has the potential to be a competent drug material due to its simple production process, satisfactory stability, as well as the broad anti-cancer spectrum with high efficacy. Therefore, ADC is worthy to be more deeply investigated in the future. |
| Rights: | All rights reserved |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| b28157680.pdf | For All Users | 5.14 MB | Adobe PDF | View/Open |
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