Background and aim: Significant differences exist between the metabolism and antioxidant requirements of normal and malignant cells. Tumor cells depend on exogenous nutrients in their microenvironment to fulfill the elevated energy requirements and for maintaining appropriate intracellular antioxidant levels. Deprivation of amino acids results in growth inhibition or death of tumor cells by the modulation of various signaling cascades and in some cases redox balance. We have been evaluating potential therapeutic enzymes that degrade critical amino acids required for tumor growth. These engineered human enzymes include one that degrades either L-cysteine and one that degrades methionine.
Experimental procedures: (1) in vitro cell culture experiments to evaluate cell survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays; (2) metabolomics analyses of amino acids and metabolites; (3) analyses of oncogenic cell signaling, reactive oxygen species (ROS) levels, and DNA damage as well as cell cycle changes using flow cytometry; (4) in vivo allograft and xenograft tumor experiments with various cancer cell lines.
Results: Depletion of extracellular L-cys/cystine led to depletion of intracellular L-cys, decreased levels of intracellular glutathione (GSH), and increases in intracellular ROS leading to activation of cellular signaling pathways, oxidative DNA damage, and ultimately cancer cell death. Cyst(e)inase, given i.p., significantly reduced serum levels of L-cys and significantly inhibited tumor growth in vivo of both prostate and pancreatic cancer xenograft and allograft tumor models. Notably, targeting a second antioxidant pathway together with cyst(e)inase (i.e., the thioredoxin pathway) using a thioredoxin reductase inhibitor led to synergistic cancer cell killing and also sensitized tumor cells found to be more resistant to cyst(e)inase alone. These and other studies on the mechanisms associated with the potential anticancer activity of Cyst(e)inase are presented. In addition, we have also studied the potential therapeutic application of a human engineered methionine (L-met) degrading enzyme called methionine gamma lyase (hMGL). Both mechanistic studies as well as in vivo preclinical therapeutic studies demonstrate significant efficacy against several cancers with hMGL.
Conclusion: Depletion of amino acids such as L-cys and L-met using human engineered enzymes offer novel approaches for treating cancer either given alone or more likely in combination with other agents.