Arginine deprivation, growth inhibition and tumour cell death: 2. Enzymatic degradation of arginine in normal and malignant cell cultures

Abstract

Arginase added to culture medium reduced arginine to negligible levels within B6 h, and enzyme activity persisted relatively undiminished for at least 3 days. Human and bovine arginase proved equally effective. The response of normal cells was to enter G1 (G0) arrest, from which most of the cells could be recovered weeks later. In contrast, malignant cell lines treated with unpegylated or pegylated enzyme resulted in cell death on a massive scale within 3 -5 days, with a very low to negligible percentage of cells (o0.01%) being recoverable on restoration with arginine. Although pegylation resulted in a 40% drop in specific activity, arginase was considerably more stable and remained active for b8 days. Arginine decarboxylase caused malignant cell arrest at the same units per millilitre as arginase. Its breakdown product, agmatine, was relatively nontoxic in the presence of arginine, but exacerbated cell death above millimolar concentration in its absence. Although ornithine failed to rescue cells from deprivation, citrulline recovered cells in all cases, although less well in fast-growing tumour cell populations, whereas readdition of arginine failed to work unless a complete medium change was given (because of the persistence of the enzymes in the medium catabolising its destruction). The advantages and disadvantages of these two arginine-catabolising enzymes are discussed, and compared with arginine deiminase.

Arginine deprivation, growth inhibition and tumour cell death: 3. Deficient utilisation of citrulline by malignant cells

“…This potential Achilles' heel in tumour metabolism deserves further exploitation in view of our previous findings (Scott et al, 2000;Campbell, 2001, 2002;Philip et al, 2003). Although melanomas do not exclusively show this argininosuccinate synthetase deficiency, the findings show that most cases of this dangerous and aggressive tumour can be appropriately and quickly selected for treatment.…”

“…However, MEWO can use argininosuccinate, and A375 cells grow well on it, whereas G361 survive although they do not proliferate to any significant extent in its presence. We should also note that normal fibroblasts manage well on citrulline, but do not thrive in the presence of argininosuccinate (Table 1; Philip et al, 2003). However, until more extensive studies are carried out we will not know whether most normal cell cultures can convert citrulline to arginine.…”

“…Having successfully reduced L1210 cells in culture to negligible viability in 3-5 days with arginase (EC 3.5.3.1), citrulline supplementation in the continued presence of the enzyme partially circumvented the arginine deficiency. The rate of conversion became a rate-limiting factor, most noticeably in such fast-growing leukaemia as L1210 (Philip et al, 2003). In a corresponding in vivo study, arginase treatment of DBA/2 mice injected intraperitoneally with 1210 cells increased neither their survival time nor decreased tumour burden, despite plasma arginine falling to B1-2 mM level.…”

“…In a corresponding in vivo study, arginase treatment of DBA/2 mice injected intraperitoneally with 1210 cells increased neither their survival time nor decreased tumour burden, despite plasma arginine falling to B1-2 mM level. Plasma citrulline remained normal, and therefore the tumour cells must have converted citrulline to arginine fast enough to sustain relatively normal tumour growth rate (Wu and Morris, 1998;Philip et al, 2003). This makes it difficult to understand how previous in vivo work with arginase and arginine deiminase could have achieved significant inhibition of tumour growth under similar conditions (Bach and Swaine, 1965;Miyazaki et al, 1990), and begs further analysis of the ability of tumour cells to use citrulline in lieu of arginine (Wheatley and Campbell, 2002).…”

Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

“…Trophozoites use ADI and OCT to actively metabolize arginine for energy, thus depleting arginine from the growth medium. Arginine depletion is known to induce apoptosis in human cell lines [40] and human giardiasis patients show an increased rate of apoptosis of intestinal epithelial cells [23]. This has been suggested to be a major disease mechanism [22].…”