Purpose: The development of acquired resistance to arginine deprivation therapy (ADT) is a major barrier to its efficacy. This study aimed to elucidate the possible mechanisms underlying the resistance to ADT. Methods: We applied repeated ADT and established a subline SAS-R9 of the human head and neck squamous cell carcinoma (HNSCC) cells semi-resistant to arginine (Arg) deprivation in vitro. This subline was compared to the parental SAS cell lines for its relative clonogenic proliferation, aggregation, adhesion, and migration capacities. The transcriptomic changes were assessed by RNA sequencing. Signaling pathway alterations were confirmed by RT-PCR and Western blotting. Relative cell radioresistance was analyzed by radiobiological clonogenic survival assay. DNA double-strand break (DSB) repair was assessed by γH2A.X foci analysis. Results: SAS-R9 cells showed higher survival in response to ADT and radiotherapy, elevated clonogenic proliferation rate, cell–cell aggregation, and cell–matrix adhesion, along with increased epithelial–mesenchymal transition (EMT) markers and enhanced DNA DSB repair, potentially related to a more aggressive and therapy-resistant phenotype. Conclusions: While acute ADT has radiosensitizing potential, this new study suggests that long-term, repeated ADT is associated with cell selection and reprogramming, resulting in resistance to radiotherapy-induced DNA damage and higher tumor cell aggressiveness.

Metabolic anticancer therapy based on enzymatic arginine (Arg) deprivation (ADT) is currently being evaluated in clinical trials. The combination of ADT with low doses of the plant cytotoxic analogs of Arg, canavanine (Cav) or indospicine (Isp), have been proposed as being more efficient and selective against malignant cells. The leguminous plant Indigofera spicata contains one of the highest known amounts of Isp. Here we demonstrate for the first time that the Isp-containing ethanolic extract from I. spicata is growth-inhibiting and toxic for cultured human colorectal and ovarian carcinoma cells. The extract reduces the viability of colorectal carcinoma cells two-fold under Arg-deficient conditions and entirely abrogates their residual proliferative potential (growth recovery) after the treatment. Pre-exposure of the extract to recombinant human arginase I (rhARGI) as a therapeutic Arg-depleting agent did not impact the extract’s efficacy. Further development of Isp as a component of combinatorial anticancer metabolic targeting strategies is discussed.