gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) is characterized by a potent resistance against antineoplastic treatment. Although Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for new therapy approaches of various malignant diseases, many glioma cell lines are per se resistant to TRAIL. The purpose of this study was to 1) evaluate the efficiency of combination therapies to reactivate TRAIL sensitivity in GBM and to 2) analyze the molecular signalling events underlying reactivation of apoptosis in GBM.

Methods: A panel of six GBM cell lines were examined for apoptosis sensitivity. Cell death induced by recombinant TRAIL, Gamma-irradiation, chemical inhibitors of Bcl-2/Bcl-xL and proteasome inhibitors MG132 and epoxomicin was quantified by caspase assays and FACS analysis of annexin-V and propidium iodide stained cells. RNA interference was used to knock down potential upstream regulators of TRAIL receptor expression. Differential gene expression profiles of GBM cell lines were obtained with DNA microarrays.

Results: Here we investigated the sensitivity of human GBM cell lines to apoptosis induced by TRAIL [250 ng/ml], and TRAIL in combination with proteasome inhibitors (PIs) [2,5 µM MG132; 50 nM epoxomicin], or Bcl-2/Bcl-xL inhibitors [30 µM HA14-1, 30 µM BH3-I2´] and gamma-irradiation [20 Gy]. The cell lines evinced drastic differences in their sensitivity to these distinct apoptotic stimuli, with only two of them showing significant induction of cell death in response to TRAIL. The combinatory treatments revealed that apoptosis could be efficiently enhanced with the Bcl-2/Bcl-xL inhibitors BH3-I2´ and HA14-1 in three of the six cell lines. Gamma irradiation in combination with TRAIL potentiated cell death in only one of three cell lines. Interestingly, our data show that apoptosis was markedly enhanced by PIs in the TRAIL-sensitive cell lines U87 and U251, and could be potently reactivated in both TRAIL-resistant cell lines U343 and U373. siRNA-mediated knockdown of DR5 followed by combined treatment with MG132 and TRAIL resulted in a significant decrease of DR5 surface expression and cell death. Further studies employing JNK inhibitors in combination with analysis of DR5 expression and cell death revealed that the JNK/c-Jun pathway might play an important role for enhanced TRAIL sensitivity of GBM cells after treatment with PIs.

Conclusions: Combinatorial approaches based on TRAIL or agonistic TRAIL receptor antibodies in conjunction with PIs might be a promising strategy to reactivate apoptosis in TRAIL- and therapy-resistant GBMs in the future.