Arginine Is a Novel Drug Target for Arginine Decarboxylase in Human Colorectal Cancer Cells.

Colorectal cancer (CRC) has been proven to be highly reliant on arginine availability. Limiting arginine-rich foods or treating patients with arginine-depleting enzymes arginine deiminase (ADI) or arginase can suppress colon cancer. However, arginase and ADI are not the best drug candidates for CRC. Ornithine, the product of arginase, can enhance the supply of polyamine, which favors CRC cell growth, while citrulline, the product of ADI, faces the problem of arginine recycling due to the overexpression of argininosuccinate synthetase (ASS). Biosynthetic arginine decarboxylase (ADC), an enzyme that catalyzes the conversion of arginine to agmatine and carbon dioxide, may be a better choice as it combines both arginine depletion and suppression of intracellular polyamine synthesis via its product agmatine. ADC has anti-tumor potential yet has received much less attention than the other two arginine-depleting enzymes. In order to gain a better understanding of ADC, the preparation and the anti-cancer properties of this enzyme were explored in this study. When tested in vitro, ADC inhibited the proliferation of three colorectal cancer cell lines regardless of their ASS cellular expression. In contrast, ADC had a lesser cytotoxic effect on the human foreskin fibroblasts and rat primary hepatocytes. Further in vitro studies revealed that ADC induced S and G2/M phase cell-cycle arrest and apoptosis in HCT116 and LoVo cells. ADC-induced apoptosis in HCT116 cells followed the mitochondrial apoptotic pathway and was caspase-3-dependent. With all results obtained, we suggest that arginine is a potential target for treating colorectal cancer with ADC, and the anti-cancer properties of ADC should be more deeply investigated in the future.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.