gms | German Medical Science

Objective: The massive infiltration of tumor cells into the surrounding brain tissue and the development of resistance mechanisms towards adjuvant radio- and chemotherapy are two main aspects that need to be overcome, for a successful treatment of Glioblastoma (GBM). In both these processes, calcium-dependent proteases (calpains) could be involved. Calpains are cysteine proteases highly expressed in tumor cells and could therefore serve as potential therapeutic targets in malignancies. This study aims to validate calpains as therapeutic targets in GBM.

Methods: Expression analysis was performed on primary tissue from GBM and gliomas grade II and III (n=20 each) using qPCR. Furthermore, the expression profiles between first manifested and patient-matched recurrent GBM (n=20 patients) was compared and correlated to patient outcome. In addition the role of calpains in several GBM celllines including U87, U217, G28, G112 and in patient derived primary GBM cells was investigated. To this end, cells were treated with 50 µM PD 150606, a specific calpain inhibitor, alone or in combination with the chemotherapeutic Temozolomide (TMZ).

Results: Calpains 1 and 2 were found to be up to six-fold up-regulated in GBM compared to gliomas grade II and III. In addition to a correlation between calpain expression and malignancy, it was also shown that calpains are over-expressed in recurrent GBM tissue compared to patient matched first manifested GBM tissue. The observed trend was further investigated in order to discover the role of calpains especially in manifestation of recurrent GBMs. Therefore calpains were inhibited in established GBM cell lines as well as in patient derived GBM cells by treatment with 50 µM PD 150606. Calpain inhibition resulted in an increased sensitivity towards TMZ in these cells, therefore suggesting a synergistic effect between TMZ and calpain inhibition. The underlying mechanism of cell death, induced by combined treatment, was identified as necroptosis by western blotting of different markers associated with apoptosis, necroptosis and autophagy, respectively.

Conclusion: In order to optimize therapy for GBM patients the molecular mechanisms of chemo and radio-resistance have to be identified and overcome. Calpains are shown to play a role in the cellular response towards TMZ and their inhibition sensitizes GBM cells to TMZ, suggesting that co-treatment of GBMs provides a better outcome than the standard chemotherapy.