The contribution of autophagy in glioblastoma multiforme cells treated with human recombinant arginase I-induced arginine deprivation. (c2018)

Abstract

Glioblastoma multiforme (GBM) is one of the most aggressive and common types of brain cancer. It is associated with a dismal prognosis, thus excluding the possibility of surgical resection. Moreover, it is resistant to traditional chemotherapies and radiotherapies. All this results in a poor survival rate in GBM patients, which doesn’t exceed 1 year. Therefore, there is a need for more selective and effective approaches, such as the use of human recombinant arginase I to selectively target cells that are auxotrophic to arginine. However, the mechanism of cell death due to human recombinant arginase I-induced arginine deprivation is still poorly understood, hence the need for further investigation. In this study, we attempt to investigate the mechanism of cell death following arginine deprivation and in particular the contribution of autophagy. In order to study the effect of apoptosis on GBM cells treated with HuArgI (Co)-PEG5000, Z-VAD, a pan-caspase inhibitor was used. Following the coincubation of SF cells with Z-VAD and HuArgI (Co)-PEG5000, failure of Z-VAD to decrease arginine depletion-induced cell death was shown. Hence demonstrating that apoptosis does not contribute to arginase induced cell death. Furthermore, the contribution of autophagy to arginine depletion-induced cytotoxicity in GBM cells was tested by incubating SF and U87 cells with either HuArgI (Co)-PEG5000 alone and in combination with an autophagy inhibitor (chloroquine or 3- methyladenine) at different time points. Cell death decreased when autophagy was inhibited in SF cells at late time points, thus proving its role in arginine depletion induced cell death. To further study the contribution of autophagy, several autophagy markers were considered. Protein analysis through western blots was conducted to check for the conversion/activation of LC3-I to LC3-II and for the activation of mtTOR to PmTOR. The activation of LC3-I peaked in control conditions (chloroquine treatment), considerably decreased in cells treated with the highest arginase concentration, then increased again in cells treated with both arginase at its highest concentration and chloroquine. mTOR is active in control cells, while at highest arginase concentration there is an inactivation of mTOR resulting in the activation of autophagy. To conclude, we have excluded the contribution of apoptosis in GBM cells. And instead demonstrated the contribution of autophagy to arginase depletion induced cytotoxicity, by checking for autophagy molecular markers, and proving the contribution of such markers by doing cytotoxicity assays and showing the suppression of cell death when autophagy is inhibited.