Deciphering molecular mechanisms of arginine deiminase-based therapy - Comparative response analysis in paired human primary and recurrent glioblastomas.

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (» cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations. Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo.

Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50%). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70% killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.