A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis

: Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors and the hardest type of cancer to treat. Therapies targeting developmental pathways, such as Notch, eliminate neoplastic glioma cells, but their efficacy can be limited by various mechanisms. Combination regimens may represent a good opportunity for effective therapies with durable effects. We used low doses of the γ-secretase inhibitor RO4929097 (GSI), to block the Notch pathway activity, in combination with Resveratrol (RSV) and we evidenced the mechanisms of autophagy/apoptosis transition in GBM cells. Resveratrol and GSI combination results in the synergistic induction of cell death together with the block of the autophagic flux evidenced by a sustained increase of LC3-II and p62 protein content, due to the dramatic reduction of CDK4, an important regulator of lysosomal function. The ectopic overexpression of the constitutive active CDK4 mutant, greatly counteracted the RSV+GSI induced block of the autophagy. Triggering autophagy in RSV+GSI-treated cells, which have impaired lysosomal function, caused the collapse of the system and a following apoptosis. For instance, by combining the CDK4 mutant as well as the early stage autophagy inhibitor, 3-methyladenina, abolished the RSV+GSI induced caspases activation. The initiator caspases (caspases-8 and -9), effector caspase (caspase-3) and its downstream substrate PARP were induced after RSV+GSI exposure as well as the percentage of the TUNEL positive cells. Moreover, the pro-apoptotic signaling MAPK p38 was activated while the pro-survival MAPK p42/p44 signaling was inhibited. In short, we establish the role of CDK4 in the regulation of autophagy/apoptosis transition induced by RSV and GSI in GBM cells. This new synergistic therapeutic combination, increasing the accumulation of autophagosomes, may have therapeutic value for GBM patients.