gms | German Medical Science

Objective: Calcium-dependent proteases (calpains) are involved in GBM cell infiltration and in therapy resistance. Calpains are cysteine proteases, naturally inhibited by Calpastatins, are highly expressed in tumor cells, and could serve as potential therapeutic targets in malignancies. Our study aims to target the calpain/Calpastatin system for therapeutic purposes in GBM.

Methods: Calpain expression profiles between first manifested and patient-matched recurrent GBM (n=20 patients) were correlated to patient outcome. The role of calpains in several GBM cell lines and in patient derived primary GBM cells was investigated and the activity of calpains was determined by fluorescence analysis in correlation to expression levels of calpastatin. To inhibit calpains, cells were treated with 50 µM PD 150606, a specific calpain inhibitor, either alone or in combination with Temozolomide (TMZ). Using shRNAs, calpain/calpastatin was knocked down in GBM cell lines to investigate its role in TMZ therapy.

Results: Calpains are over-expressed in recurrent GBM tissue compared to patient matched primary GBM tissue (11/19 for Calpain 1; 16/19 for Calpain 2). Calpain activities wereanalyzed in GBM cell lines and in patient derived GBM cells under TMZ therapy conditions andinhibited Calpains by treatment with 50 µM PD 150606. An additive effect of co-treatment with PD150606/TMZ was observed. Only those cells were sensitized to TMZ treatment that exerted cellular calpain activity, suggesting that calpain activity is part of the intrinsic TMZ resistance of GBM cells. Sensitization of Calpain inhibitor treated cells to TMZ was concomitant with enhanced apoptosis, as revealed by increased Caspase-3 activity in responding GBM cells. Moreover, knockdown of calpain 1 or 2 in U251N cells led to an increased Caspase-3 activity under TMZ treatment. A172 cells treated with TMZ showed an increased level of dead cells when calpain 2 was knocked down. Knockdown of calpastatin, the endogenous calpain inhibitor, reversed that effect.

Conclusion: To optimize GBM therapy, several molecular mechanisms of radio- and chemotherapy resistance have to be overcome. Here we show that inhibition of Calpains facilitates TMZ induced apoptosis of GBM cells, suggesting that a co-treatment with TMZ and Calpain inhibitor in GBMs could provide a better outcome than the standard TMZ chemotherapy.