gms | German Medical Science

Objective: Here we investigated whether the anti-neoplastic effect of carnosine on glioblastoma (GBM) cell growth can be enhanced in combination with bafilomycin A1 (an inhibitor of late autophagy) and rapamycin which is known to induce autophagy in GBM.

Methods: GBM cells from the lines U87 and T98G were exposed for 48 hours to bafilomycin A1 (Baf; 100 nM) or rapamycin (Rap; 100 nM) and in combination with carnosine (Car; 50 mM). Cell viability was determined by measuring the amount of ATP in cell lysates. Expression of LC3B II and p62 as markers for autophagy were quantified by western blotting.

Results: Both, U87 and T98G glioblastoma cells significantly responded to exposition to Car with a loss of viability (60.1±8.3% and 58.4±10.2%, respectively). In the presence of Baf viability was reduced to 36.6±8.8% (U87) and to 23.4±4.4% (T98G). Combining Baf with Car revealed no synergistic effect (U87: 39.8±8.7%; T98G: 23.2±4.4%). Rap reduced viability to 87.9±14.4% (U87) and to 71.4±10.8% (T98G) and in combination with Car to 65.6±4.3% (U87) and to 48.6±12.6% (T98G). Immunoblotting revealed a reduced expression of LC3B II in the presence of Car in U87 (44.1±28.6%) and an enhanced expression in T98G (146.8±14.9%). Baf induced LC3B II expression in both lines (U87: 410.2±60.2%; T98G: 1656.2±177.6%). In combination with Car expression was further increased in T98G (2172.5±614.3%) but not in U87 (442.2±60.4%). Incubation in Rap attenuated expression of LC3B II in U87 (54.5±19.9%) and augmented it in T98G (307.3±112.3%). Combining Rap with Car decreased LCB II protein levels relative to treatment with Rap alone (U87: 34.2±18.9%; T98G: 242.2±100.0%). The relative amount of p62 was not altered in the presence of Car (U87: 94.9±25.2%; T98G: 104.9±13.3%) but was enhanced in the presence of Baf (U87: 286.7±46.2%; T98G: 447.7±141.1%). Combining Car with Baf, p62 expression was further increased to 363.6±37.5% (U87) and to 513.2±91.6% (T98G). Rap attenuated p62 expression in both lines (U87: 79.9±37.3%; T98G: 60.0±3.2%) with no additional effect of Car (U87: 75.5±1.9%; T98G: 56.1±10.2%).

Conclusion: With regard to LC3B II expression carnosine mimics the effect of rapamycin after 48 hour exposure whereas this is not the case with regard to p62. The autophagy inhibitor bafilomycin A1 as well as the inducer rapamycin both reduce viability but there appears to be no synergistic effects of carnosine in combination with the compounds.