Arginine deprivation achieved by means of recombinant arginine-degrading enzymes is currently being developed as a novel anticancer enzymotherapy. In this study, we showed that arginine deprivation in vitro profoundly and selectively sensitized human cancer cells of different organ origin to low doses of canavanine, an arginine analogue of plant origin. In sensitive cancer cells arginine starvation led to the activation of caspase-9, caspase-3 and caspase-7, cleavage of reparation enzyme, polyADP ribosyl polymerase, and DNA fragmentation, which are the typical hallmarks of intrinsic apoptosis realized by the mitochondrial pathway. Co-administration of canavanine significantly accelerated and enhanced apoptotic manifestations induced by arginine deprivation. The augmentation of canavanine toxicity for cancer cells was observed when either a formulated arginine-free medium or complete medium supplemented with bovine arginase preparation was used. Cycloheximide efficiently rescued malignant cells from canavanine-induced cytotoxicity under arginine deprivation, suggesting that it results mainly from canavanine incorporation into newly synthesized proteins. Cancer cells sensitive or resistant to arginine deprivation alone were not capable of restoring their proliferation after 24 h of combined treatment, whereas pseudonormal cells retained such ability. Our data suggest that the incorporation of canavanine into anticancer treatment schemes based on artificially created arginine starvation could be a novel strategy in tumor enzymochemotherapy.

Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg-deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.

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The moderate anticancer effect of arginine deprivation in clinical trials has been linked to an induced argininosuccinate synthetase (ASS1) expression in initially ASS1-negative tumors, and ASS1-positive cancers are anticipated as non-responders. Our previous studies indicated that arginine deprivation and low doses of the natural arginine analog canavanine can enhance radioresponse. However, the efficacy of the proposed combination in the presence of extracellular citrulline, the substrate for arginine synthesis by ASS1, remains to be elucidated, in particular for malignant cells with positive and/or inducible ASS1 as in colorectal cancer (CRC). Here, the physiological citrulline concentration of 0.05 mM was insufficient to overcome cell cycle arrest and radiosensitization triggered by arginine deficiency. Hyperphysiological citrulline (0.4 mM) did not entirely compensate for the absence of arginine and significantly decelerated cell cycling. Similar levels of canavanine-induced apoptosis were detected in the absence of arginine regardless of citrulline supplementation both in 2-D and advanced 3-D assays, while normal colon epithelial cells in organoid/colonosphere culture were unaffected. Notably, canavanine tremendously enhanced radiosensitivity of arginine-starved 3-D CRC spheroids even in the presence of hyperphysiological citrulline. We conclude that the novel combinatorial targeting strategy of metabolic-chemo-radiotherapy has great potential for the treatment of malignancies with inducible ASS1 expression.

Increased amino acid requirement of malignant cells is exploited in metabolic antitumor therapy, e.g., enzymotherapies based on arginine or methionine deprivation. However, studies on animal models and clinical trials revealed that solid tumors are much less susceptible to single amino acid starvation than could be expected from the in vitro data. We conducted a comparative analysis of the response of several tumor cell lines to single amino acid starvation in 2-D monolayer versus 3-D spheroid culture. We revealed for the first time that in comparison with monolayer culture tumor cells, spheroids are much less susceptible to the deprivation of individual amino acids (i.e., arginine, leucine, lysine or methionine). Accordingly, even after prolonged (up to 10 days) starvation, spheroid cells could readily resume proliferation when appropriate amino acid was resupplemented. In the case of arginine deprivation, similar apoptosis induction was detected both in 2-D and 3-D culture, suggesting that this process does not determine the level of tumor cell sensitivity to this kind of treatment. It was also observed that spheroids much better mimic the in vivo ability of tumor cells to utilize citrulline as arginine precursor for growth in amino acid deficient environment. We conclude that 3-D spheroid culture better reflects in vivo tumor cell response to single amino acid starvation than 2-D monolayer culture and should be used as an integral model in the studies of this type of antitumor metabolic targeting.

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Ключові слова: голодування за аргініном, метаболіти аргініну, путресцин, спермін, агматин, оксид азоту, орнітин, сечовина. ВСТУПСтворення дефіциту індивідуальних амінокислот в організмі людини є одним із перспективних підходів у сучасній терапії раку. Відомо, що деякі типи пухлин є ауксотрофами за аспарагіном, метіоніном чи аргініном і виявляють підвищену * Ці автори зробили однаковий внесок у виконання роботи.Biol. Stud. 2011: 5(2); 5-16 •