Arginine Deiminase as an Innovative Anti-Breast Cancer Agent

Institution: University of Southern California
Investigator(s): Wei-Chiang Shen, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6IB-0045 Award: $81,507
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2000)
Innovative treatments for breast cancer are desperately needed because the current mortality rate of this disease in California is the second highest of all female cancers and has not decreased significantly, especially among minority groups, during the last 10 years. In this project, we will explore a new type of anti-tumor agent, which possesses the inhibitory effect on growth of both the blood vessel and tumor cells. In order for a cancerous tumor to proliferate and disseminate, it must first coax a blood supply to grow towards it, a process that is known as tumor angiogenesis. Angiogenesis is a complex sequence of events leading to the formation of new blood vessels from pre-existing blood vessels. Any substance that can act on and inhibit this process is considered anti-angiogenic and potentially inhibitory for solid tumor growth. Breast cancer is an angiogenesis-dependent disease, making the development of angiogenic inhibitors a very promising approach to the treatment of this disease. Our laboratory has demonstrated that a mycoplasma protein, arginine deiminase, can inhibit angiogenesis in several anti-angiogenesis assay systems. In addition, our preliminary studies and those in others laboratories indicate that this protein is effective in inhibiting tumor cell growth both in cell culture systems and in animal models. We hypothesize that the depletion of extracellular arginine by using arginine deiminase will alter the intracellular signals of growth regulation, especially the arginine-dependent synthesis of nitric oxide and polyamines. The decrease of angiogenesis in solid tumors may be a consequence of a change in the regulatory signal balance. In addition, this alteration of regulatory signals may also have a direct inhibitory effect on the proliferation of tumor cells. Recently, we have successfully cloned and produced this protein from mycoplasma genes by using a recombinant DNA technique. Our goal in this application is to establish our hypothesis that this recombinant protein possesses both and angiogenic and and-proliferative activities against breast cancer. To our knowledge, this type of therapeutic agent with a dual action has not been investigated before. Our long-term goal is to develop this recombinant protein into a unique anti-tumor agent for breast cancer treatment.

Final Report (2001)
A bacterial enzyme, arginine deiminase, was cloned from genomic DNA in our laboratory. The recombinant arginine deiminase (rADI) was purified to homogeneity and was investigated as a new type of anti-breast cancer agent due to its potential activity in inhibiting blood vessel growth (anti-angiogenesis) and cell division (anti-proliferation) in laboratory tests. The objectives of this project were to demonstrate the anti-angiogenic and anti-proliferative activities of rADI, and to determine the effects of rADI on nitric oxide (NO) and polyamine synthesis in the cells lining the blood vessels (endothelial cells).

The purified rADI exhibited an inhibitory effect on the migration and the microvascular formation assays of human umbilical vein endothelial (HUVE) cells in culture. It also inhibited the growth of HUVE cells which, at low concentrations of rADI, was reversible indicating that it stopped cells from growing rather than killing them. However, at high concentrations, rADI-induced growth inhibition was irreversible. rADI only moderately reduced the production of NO by endogenous NO synthetase (eNOS) in both HUVE and mammary carcinoma MCF7 cells. However, it completely abolished the activity of NO synthetase (iNOS) induced by cytokines in rat blood-brain barrier (TRBBB) endothelial cells, a recently established endothelial cell line. The growth of MCF7 and TRBBB cells were arginine-dependent, but, interestingly, both were resistant to the treatment of rADI. It was found that the survival of these two types of cells with rADI treatment was due to the presence of citrulline, the product from the deimination of arginine, in the medium. In fact, both cell lines survived in arginine-free medium supplemented with citrulline. Therefore, it was concluded that MCF7 and TRBBB, but not HUVE, cells are capable of intracellularly regenerating arginine from citrulline, most likely via the argininosuccinate synthetase-argininosuccinate lyase pathway. The production of NO in cytokine-treated TRBBB cells was completely abolished by rADI treatment because the intracellular arginine pool is inaccessible for iNOS reaction. On the other hand, rADI has very little effect on the NO production by eNOS in HUVE cells. Furthermore, there was no correlation between the anti-proliferative activity of rADI and the sensitivity of the cells towards difluoromethylornithine (DFMO), an inhibitor of the key enzyme, ornithine decarboxylase, in polyamine synthesis, indicating that the anti-angiogenic activity of rADI is independent of polyamine synthesis in the cells.

Taken together, our results suggest that the anti-angiogenic effect of rADI is most likely due to a regulatory control of NO production, rather than a direct anti-proliferative activity, in endothelial cells.

Recombinant arginine deiminase as a potential anti-angiogenic agent
Periodical:Cancer Letters
Index Medicus: Cancer Lett
Authors: Beloussow K, Wang L, Wu J, Ann D, Shen WC
Yr: 2002 Vol: 183 Nbr: 2 Abs: Pg:155-62