Abstract:
Gliobastoma (Grade IV Astrocytoma) is an aggressive, malignant brain neoplasm and is the most recurrent malignant tumor in adult humans. Existing therapies for glioblastoma (GBM) include, among others, neurosurgery, chemotherapy, radiotherapy and systemic antineoplastic therapy. However, high mortality and low survival rates are associated with this disease largely due to poor prognosis, poor understanding of the nature of the disease and the lack of efficient therapies. Hence, more selective and efficient approaches for targeting GBM are urgently needed. In this study we attempt to take advantage of the potential arginine auxotrophy of GBM cells to selectively target them using recombinant human Arginase I cobalt [HuArgI(Co)] coupled with polyethylene glycol 5000 [HuArgI (Co)-PEG5000]. The human form of the enzyme namely human L-arginase I (hArgI) uses two manganese ions as cofactors. To improve the catalytic activity of the enzyme, 2 Cobalt ions (Co2+) have been used to substitute the manganese ions in the active site of the enzyme. The resulting HuArgI (Co) was conjugated to polyethylene glycol to generate the pegylated, Cobalt-substituted recombinant human arginase [HuArgI (Co) PEG5000]. Cytotoxicity of HuArgI (Co)-PEG5000 was tested on a panel of 9 human
GBM cell lines using a proliferation inhibition assay. HuArgI (Co)-PEG5000- induced arginine depletion was cytotoxic to all the GBM cell lines tested with IC50 values ranging between 128 to 985 pM and a percent cell kill at highest concentration > 75%. Addition of excess L-citrulline failed to rescue GBM cells from arginine depletion-induced cytotoxicity indicating complete arginine auxotrophy of GBM cells. Time course analysis revealed that peak toxicity is observed after an incubation time of 48 to 72 h. Sensitivity of GBM cells to HuArgI
(Co)-PEG5000-induced arginine depletion as well as the impact of addition of Lcitrulline on cell sensitivity, were dependent on expression levels of argininosuccinate synthetase-1 (ASS-1), the enzyme whose activity leads to the synthesis of arginine in cells. Cell cycle analysis showed that arginine depletion, in GBM cells, does not induce cell cycle arrest and confirm the cytotoxic response of these cells to arginine deprivation. AnnexinV/PI and caspase staining showed an increase in the percentage of cells staining positively with annexinV and negatively with PI, in one of the cell lines tested, an indication that HuArgI (Co)-PEG5000- mediated arginine depletion may induce apoptotic cell death in GBM cells. In this study we demonstrate that GBM cells are auxotrophic for arginine and are, subsequently, sensitive to the HuArgI (Co)-PEG5000-mediated arginine depletion. Arginine depletion is therefore, a potential novel, targeted therapy for GBM.
