Human Recombinant Arginase I (Co)-PEG5000 [HuArgI (Co)-PEG5000]-Induced Arginine Depletion Leads to Autophagy-Dependent Cell Death in Prostate Cancer Cells

Abstract

Prostate cancer ranks as the second most frequently diagnosed malignancy in men and represents the fifth highest cause of cancer-related mortality globally. While conventional treatments like surgery and radiotherapy reliably cure patients, advanced and recurrent prostate cancer often necessitates alternative therapies. Emerging treatments targeting cancer cell metabolism show promise, particularly those focusing on amino acid deprivation, such as arginine, specifically in tumor types that downregulate argininosuccinate synthetase 1 (ASS1) and ornithine transcarbamylase (OTC) expression—urea cycle enzymes important for the synthesis of arginine—rendering those tumor types auxotrophic for arginine. In this study, we report the use of a pegylated recombinant human arginase I coupled to cobalt, [HuArgI (Co)-PEG5000], to induce arginine depletion in two prostate cancer cell lines: DU-145 and PC-3. Our objective was to investigate the cytotoxic effects of the [HuArgI (Co)-PEG5000]-induced arginine deprivation, characterize the extent of arginine auxotrophy in these cell lines, and elucidate the underlying mechanisms of cell death induced by arginine deprivation. This was tested through a series of proliferation inhibition assays, analysis of ASS1 and OTC expression on flow cytometry and western blots, as well as examining the type of cell death through Annexin V/PI staining. Autophagy activation was assessed through a CYTO-ID autophagosome detection kit on flow cytometry and the impact of autophagy on cell cytotoxicity was investigated after its inhibition through cytotoxicity assays. Our findings reveal that [HuArgI (Co)-PEG5000]-induced arginine deprivation exhibited cytotoxicity in both cell lines, with the earliest effect observed at 24 hours post-treatment and maximal cell death at 120 hours post-treatment. The addition of excess exogenous L-citrulline rescued cell viability in cells, suggesting partial arginine auxotrophy. Analysis of ASS1 and OTC expression confirms partial arginine auxotrophy and suggests a possible feedback loop through which expression patterns change depending on the availability of extracellular arginine and L-citrulline. Analysis of cell death mechanisms revealed negative Annexin V staining, indicating a possible non-apoptotic pathway. Furthermore, autophagy activation was evident following arginine deprivation and the inhibition of this autophagy using chloroquine reduced the cytotoxic effects, underscoring the role of autophagy in cell death induced by arginine deprivation. Our results, in conclusion, demonstrate the potential of arginine deprivation by [HuArgI (Co)-PEG5000] as a potent and selective treatment for prostate cancer, and highlight the involvement of autophagy in this process.