Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC 50 < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC 50 < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor -deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin -sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin -resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxinresistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin -sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway. [Mol Cancer Ther 2005;4(9):1303 -10]

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Purpose: Colorectal cancer (CRC) is the third most common type of cancer worldwide, and it represents over half of all gastrointestinal cancer deaths. Knowing that cancer cells have a high proliferation rate, they require high amounts of amino acids, including arginine. In addition, several tumor types have been shown to downregulate ASS-1 expression, becoming auxotrophic for arginine. Therefore, Arginine deprivation is one of the promising therapeutic approaches to target cancer cells. This can be achieved through the use of a recombinant human arginase, HuArgI(Co)-PEG5000, an arginine degrading enzyme. Methods: In this present study, the cytotoxic effect of HuArgI(Co)-PEG5000 on CRC cell lines (HT-29, Caco-2, Sw837) is examined though cytotoxicity assays. Wound healing assays, invasion assays, and adhesion assays were also performed to detect the effect on metastasis. Results: Wound healing and invasion assays revealed a decrease in cell migration and invasion after treatment with arginase. Cells that were treated with arginase also showed a decrease in adhesion, which coincided with a decrease in RhoA activation, demonstrated though the use of a FRET biosensor to detect RhoA activation in a single cell assay, and a decrease in MMP-9 expression. Treating cells with both arginase and L-citrulline, which significantly restores intracellular arginine levels, reversed the effect of HuArgI(Co)-PEG5000 on cell viability, migration, and invasion. Conclusion: We can, therefore, conclude that colorectal cancer is partially auxotrophic to arginine and that arginine depletion is a potential selective inhibitory approach for motility and invasion in colon cancer cells.

In this study, we attempt to target Arginine auxotrophy in glioblastoma multiforme (GBM) cells using a pegylated recombinant human Arginase I cobalt [HuArgI (Co)-PEG5000]. We tested and characterized the activity of HuArgI (Co)-PEG5000 on a panel of 9 GBM cell lines and on human fetal glial cells (SVG-p12). HuArgI (Co)-PEG5000 was cytotoxic to all GBM cells tested. SVG-p12 cells were not sensitive demonstrating the selective cytotoxicity of HuArgI (Co)-PEG5000-induced arginine deprivation. Addition of L-citrulline led to the rescue of 6 GBM cell lines but only at concentrations of 11.4 mM, reflecting the extent of arginine auxotrophy in GBM. The ability of L-citrulline to rescue cells was dependent on the expression of argininosuccinate synthetase-1 (ASS1) with the cells that were not rescued by L-citrulline being negative for ASS1 expression. Knocking-down ASS1 reversed the ability of L-citrulline to rescue GBM cells, further illustrating the dependence of arginine auxotrophy on ASS1 expression. Inhibition of autophagy increased cell sensitivity to HuArgI (Co)-PEG5000 indicating that, following arginine deprivation, autophagy plays a protective role in GBM cells. Analysis of the type of cell death revealed a lack of AnnexinV staining and caspase activation in HuArgI (Co)-PEG5000-treated cells, indicating that arginine deprivation induces caspase-independent, non-apoptotic cell death in GBM. We have shown that GBM cells are auxotrophic for arginine and can be selectively targeted using HuArgI (Co)-PEG5000-induced arginine depletion, thus demonstrating that L-Arginine deprivation is a potent and selective potential treatment for GBM.