Tumor cells - even if non-auxotrophic - are often highly sensitive to arginine deficiency. We hypothesized that arginine deprivation therapy (ADT) if combined with irradiation could be a new treatment strategy for glioblastoma (GBM) patients since systemic ADT is independent of local penetration and diffusion limitations. A proof-of-principle in vitro study was performed with ADT been mimicked by application of recombinant human arginase or arginine-free diets. ADT inhibited 2-D growth and cell cycle progression, and reduced growth recovery after completion of treatment in four different GBM cell line models. Cells were less susceptible to ADT alone in the presence of citrulline and in a 3-D environment. Migration and 3-D invasion were not unfavourably affected. However, ADT caused a significant radiosensitization which was more pronounced in a GBM cell model with p53 loss of function as compared to its p53- wildtype counterpart. The synergistic effect was independent of basic and induced argininosuccinate synthase (ASS) or argininosuccinate lyase (ASL) protein expression and not abrogated by the presence of citrulline. The radiosensitizing potential was maintained or even more distinguishable in a 3-D environment as verified in p53-knockdown and p53-wildtype U87-MG cells via a 60-day spheroid control probability assay. While the underlying mechanism is still ambiguous, the observation of ADT-induced radiosensitization is of great clinical interest, in particular for patients with GBM showing high radioresistance and/or p53 loss of function.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

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.

It is shown that reduction of beta,D-galactosyl-containing carbohydrate determinants ofglycoconjugates on theplasmatic membrane of segmentonuclear neutrophills of peripheral blood under type 1 diabetes mellitus (DM) is correlated with changes in aggregation of these cells and may cause their functional disorder. Changes in the parameters of ricin-induced neutrophil activation after inhibition of the phosphatidylinositol-3'-kinase (PI-3'-kinase) enzyme with wortmannin indicated that the functional state ofpolymorphonuclear leukocytes is mediated by signaling pathways in which PI-3'-kinase is involved. Thus, PI-3'-kinase-dependent signal networks are involved in the processes of signal transduction through galactosyl-containing glycoprotein receptors into neutrophilic leukocytes. Inertness of the intensity formation in time ofneutrophil granulocyte cell response on RCA-induced translocation of the p85alpha regulatory subunit of PI-3'-kinase from the cytosolic to the membrane fraction under type 1 DM is a consequence of changes in the number or structure of plasmatic galactosyl-containing glycoprotein receptors. The revealed changes may be etiologic premise of diabetic complications and chronic diseases that impair the functional condition of patients with type 1 DM.