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Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)–dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)–dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest–derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. Significance: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Melanoma has been shown to require arginine for growth, thus providing a potential Achilles' heel for therapeutic exploitation. Our investigations show that arginine depletion, using a recombinant form of human arginase I (rhArg), efficiently inhibits the growth of mammalian melanoma cell lines in vitro. These cell lines are consistently deficient in ornithine transcarbamylase (OTC) expression, correlating with their sensitivity to rhArg. Cell cycle distribution of A375 human melanoma cells treated with rhArg showed a remarkable dual-phase cell cycle arrest in S and G₂/M phases, in contrast to the G₂/M single-phase arrest observed with arginine deiminase (ADI), another arginine-degrading enzyme. rhArg and ADI both induced substantial apoptosis in A375 cells, accompanied by global modulation of cell cycle- and apoptosis-related transcription. Moreover, PEGylated rhArg dramatically inhibited the growth of A375 and B16 melanoma xenografts in vivo. Our results establish for the first time that (PEGylated) rhArg is a promising candidate for effective melanoma treatment, with fewer safety issues than ADI. Insight into the mechanism behind the antiproliferative activity of rhArg could inform us in designing combination therapies for future clinical trials.

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro , and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G 2 /M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

Hepatocellular carcinoma (HCC) is believed to be auxotrophic for arginine through the lack of expression of argininosuccinate synthetase (ASS). The successful use of the arginine-depleting enzyme arginine deiminase (ADI) to treat ASS-deficient tumors has opened up new possibilities for effective cancer therapy. Nevertheless, many ASS-positive HCC cell lines are found to be resistant to ADI treatment, although most require arginine for proliferation. Thus far, an arginine-depleting enzyme for killing ASS-positive tumors has not been reported. Here, we provide direct evidence that recombinant human arginase (rhArg) inhibits ASS-positive HCCs. All the five human HCC cell lines we used were sensitive to rhArg but ADI had virtually no effect on these cells. They all expressed ASS, but not ornithine transcarbamylase (OTC), the enzyme that converts ornithine, the product of degradation of arginine with rhArg, to citrulline, which is converted back to arginine via ASS. Transfection of HCC cells with OTC resulted in resistance to rhArg. Thus, OTC expression alone may be sufficient to induce rhArg resistance in ASS-positive HCC cells. This surprising correlation between the lack of OTC expression and sensitivity of ASS-positive HCC cells shows that OTC-deficient HCCs are sensitive to rhArg-mediated arginine depletion. Therefore, pretreatment tumor gene expression profiling of ASS and OTC could aid in predicting tumor response to arginine depletion with arginine-depleting enzymes. We have also shown that the rhArg native enzyme and the pegylated rhArg (rhArg-peg5,000mw) gave similar anticancer efficacy in vitro. Furthermore, the growth of the OTC-deficient Hep3B tumor cells (ASS-positive and ADI-resistant) in mice was inhibited by treatment with rhArg-peg5,000mw, which is active alone and is synergistic in combination with 5-fluorouracil. Thus, our data suggest that rhArg-peg5,000mw is a novel agent for effective cancer therapy. [Cancer Res 2007;67(1):309–17]

Hepatocellular carcinoma (HCC) is auxotrophic for the semi-essential amino acid arginine, depletion of which leads to tumor death. In humans, arginine is not an essential amino acid since many adult somatic cells can re-synthesize it from other sources, such as citrulline. Enzymes capable of depleting arginine in vitro include the urea cycle enzyme arginase, which is found in abundance in human liver. For over three decades, arginase has not been considered as a potential drug candidate because of its low substrate affinity, short circulatory half-life and sub-optimal enzymatic activity at physiological pH, though its in vitro anti-tumor activities in certain tumors have been amply reported. Arginine deiminase, a bacterial enzyme from Mycoplasma hominus has been shown to induce HCC remission through the mechanism of arginine depletion. We report here an innovative treatment approach for the treatment of locally advanced and metastatic HCC with transhepatic arterial embolisation (TAE) of the liver tumor with lipiodol and gel foam as a means of inducing a leakage of hepatic arginase from the liver into the circulation. Hepatic arginase released into the systemic circulation rapidly depleted plasma arginine. High-dose insulin was included to induce a state of hypoaminoacidaemia to augment arginine depletion. With this protocol, we have treated seven patients with locally advanced and/or metastatic HCC. Five patients achieved arginine depletion, ranging from 0 to 20 microM (normal plasma level 100-120 microM); all had varying degrees of tumor remission in their primary tumors and extra-hepatic sites in the lymph nodes, lungs and bones, suggesting systemic anti-cancer effect of arginine depletion. The two non-responders did not show significant reduction in plasma arginine. Based on our findings, we propose that the urea cycle enzyme, arginase, is a good drug candidate for the treatment of HCC.

Arginase has been shown to inhibit growth of human hepatocellular carcinoma by depletion of arginine. We have studied the effects of the pegylated human recombinant arginase (BCT-100, rhArg-peg5,000MW) on RPMI8226 cells, a multiple myeloma cell line. This study showed that three day exposure of the myeloma cells to pegylated rhArg at a concentration of 0.08 IU/ml and 0.48 IU/ml resulted in growth suppression of 10% and 70% respectively, as compared to untreated control. Cell cycle analysis revealed significant decreases in the proportion of cells in both S- and G2M-phase and a concomitant increase of cells in G1-phase in a time- and concentration- dependent manner. We further studied the mechanisms of cell cycle arrest induced by the pegylated rhArg. The pegylated rhArg inhibited both cyclin-dependent kinases CDK2 and CDK4, enhanced the expression of the CDK inhibitor p21, and reduced the expression of cyclinD1, D2, and E. The level of phosphorylated Rb protein was also found to be significantly decreased. The regulators of cell cycle have thus been revealed as targets of pegylated rhArg for myeloma growth arrest. The pegylated rhArg may serve as a novel antitumor agent for multiple myeloma.

Background Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. Methods The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. Results Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. Conclusion Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Background Recombinant human arginase (rhArg) has been developed for arginine deprivation therapy in cancer, and is currently under clinical investigation. During pre-clinical evaluation, rhArg has exhibited significant anti-proliferative activity in cancer cells deficient in the expression of ornithine carbamoyl transferase (OCT). Interestingly, a variety of cancer cells such as melanoma and prostate cancer deficient in argininosuccinate synthetase (ASS) are sensitive to arginine deprivation by arginine deiminase. In this study, we investigated levels of gene expression of OCT and ASS, and the effects of rhArg in human prostate cancer cells: LNCaP (androgen-dependent), PC-3 and DU-145 (both androgen-independent). Results Quantitative real-time PCR showed minimal to absent gene expression of OCT, but ample expression of ASS expression in all 3 cell lines. Cell viability assay after 72-h exposure of rhArg showed all 3 lines had half maximal inhibitory concentration less than or equal to 0.02 U/ml. Addition of ornithine to cell culture media failed to rescue these cells from rhArg-mediated cytotoxicity. Decreased phosphorylation of 4E-BP1, a downstream effector of mammalian target of rapamycin (mTOR), was noted in DU-145 and PC-3 after exposure to rhArg. Moreover, there was no significant apoptosis induction after arginine deprivation by rhArg in all 3 prostate cancer cell lines. Conclusion rhArg causes significant cytotoxicity in LNCaP, DU-145 and PC-3 prostate cancer cells which all demonstrate decreased OCT expression. Inhibition of mTOR manifested by hypophosphorylation of 4E-BP1 suggests autophagy is involved as alternative cell death mechanism. rhArg demonstrates a promising novel agent for prostate cancer treatment.