Abstract P5-10-06: Arginine- depleting enzyme, pegylated arginase, isolated from beef-liver tissue induces growth inhibition, apoptosis, cell cycle arrest and inhibits induced mammary tumors: A promising strategy for human breast cancer treatment in vitro and in vivo
Authors: Yahiya Islam , Ali Asmaa , Ali Omnia , Sultan Ahmed 
Published: March 2022

Arginine auxotrophy occurs in certain tumor types, renders tumors vulnerable to treatment with arginine-degrading enzymes, and leads to their rapid demise. Arginine is a semi-essential amino acid but is essential for rapidly proliferating cells. Thus, Arginine deprivation can be exploited as a potential targeted therapy for the treatment of various cancers. Arginase, an enzyme of the urea cycle, converts L-arginine to L-ornithine, which is the precursor of polyamines that are essential components of cell proliferation. However, Arginine deprivation and Arginase clinical implications in human breast cancer have not been fully elucidated. The objective of the current study was to investigate the potential role of the Arginine degrading enzyme, Arginase on two breast cell lines that have different differentiation patterns, Luminal A breast cancer (T-47D), and TNBC (MDA-MB-231), respectively. Herein, Arginase was extracted and purified from the beef liver and its specific activity was 0.2 IU/mg protein yielded 49% compared to the crude beef liver Arginase. The purified Arginase is covalently bound via a succinamide propionic acid (SPA) linker to polyethylene glycol (PEG) of molecular weight 5,000 to enhance the half-life time compared to the native enzyme with a maximum specific activity between 37~55°C and pH 7~9. The potential influence of extracted Peg-Arginase was examined on cell proliferation, clonogenic assays, morphological alteration, and oncogenic signaling pathways of STAT3, β-catenin, and miR-23a. Our data showed that Peg-Arginase treatment displayed a significant inhibition in cell proliferation and viability in a dose-dependent manner in both breast cancer cell lines compared to control after 48h. Furthermore, mechanistic studies showed that Peg-Arginase induced apoptosis that was associated with cell morphological changes, histone release from fragmented DNA, caspase-3 activation, and cell cycle arrest at S and/or G2/M phases in a dose-. dependent manner after 48h. Moreover, Peg-Arginase down-regulated miR-23a, STAT3, β-catenin, cyclin D1, and mutant p53 levels as well as increased p21and Bax, pro-apoptotic signal, in both breast cancer cells as determined by qRT-PCR and western blot analysis. In vivo, the effects of native- and Peg-Arginase enzymes were examined on MNU-induced mammary tumors in Albino-Wister rats. Our data showed that injecting 400 IU of native- or peg- Arginase twice per week for two weeks significantly inhibited the growth of MNU-induced mammary tumors by 58.8±4.1 % for peg-Arginase compared to 19.7±3.5 % for native-Arginase. Furthermore, the results also revealed a significant reduction in activities of ALT, AST, and ALP in the Peg-Arginase group compared to MNU untreated or native-Arginase groups, suggesting that Peg-Arginase may eliminate the hepatotoxic effect of the MNU. Furthermore, Peg-Arginase treatment reduced the localization and expression of oncogenic β-catenin in the nucleus compared to MNU-untreated and native-Arginase groups. All in all, our results suggested that the arginine-depleting enzyme, Peg-Arginase, exerted an anticancer effect against both luminal A and triple-negative breast cancer cell lines, through cell proliferation inhibition and apoptosis induction, which might provide a promising alternative strategy targeting oncometabolite in human breast cancer.

Citation Format: Islam Yahiya, Asmaa Ali, Omnia Ali, Ahmed Sultan. Arginine- depleting enzyme, pegylated arginase, isolated from beef-liver tissue induces growth inhibition, apoptosis, cell cycle arrest and inhibits induced mammary tumors: A promising strategy for human breast cancer treatment in vitro and in vivo [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-06.