Human Recombinant Arginase I (Co)-PEG5000-Induced Arginine Depletion of Breast Cancer Cell Lines Causes Autophagy Mediated Cell Death

In this study, we showed for the first time that arginine deprivation induced by a recombinant human arginase drug induces selective cytotoxicity mediated by autophagy in distinct human breast cancer cell lines. Breast cancer remains the most prevalent, life-threatening cancer in women, posing a dire global burden. As of 2022, 2.3 million women were diagnosed with breast cancer, 670,000 of which died. New selective, targeted therapeutics have been on the rise in the past decade. Arginine-depleting enzymes, such as [HuArgI(Co)-PEG5000], have been showing promising in vitro results in the treatment of auxotrophic Acute Myeloid Leukemia (AML), glioblastoma, pancreatic, and ovarian cancer. This study’s major aim is to examine the potency of the pegylated form of Arginase I enzyme, [HuArgI(Co)-PEG5000], in the selective treatment of three breast cancer cell lines: MDA-MB-231, MCF-7, and UACC-2087. Our cytotoxicity assay results show that all three cell lines were sensitive to the human arginase drug up to 120 hrs post-treatment. Addition of excess amounts of L-citrulline, a precursor of arginine, led to rescue of all three cell lines indicating that these cells do express ASS1 and are, therefore, partially auxotrophic for arginine. Flow cytometry results validated this as all three cell lines were positive for ASS1 expression. In attempt to investigate autophagy activation, both CytoID flow cytometry staining and western blots of LC3-I/LC3-II were conducted. All three cell lines showed sustained activation of autophagy lasting up to 120 hours following arginine deprivation. The impact of autophagy on cell cytotoxicity of [HuArgI(Co)-PEG5000]-induced arginine deprivation was then assessed through autophagy inhibition using the downstream autophagy inhibitor chloroquine (CQ). Inhibition of autophagy decreased sensitivity to arginase-induced cytotoxicity in all cell lines, and hence reversed cell death. This implies that arginase-induced cell death is autophagy-mediated. Overall, these findings suggest that [HuArgI(Co)-PEG5000] is a potent therapeutic agent for breast cancer, leveraging the autophagy pathway to selectively induce cytotoxicity in ASS1-expressing breast cancer cells. Further investigation into this therapeutic strategy could pave the way for new, effective treatments for breast cancer.