PDF(Pubmed)
Abstract：
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.
摘要：
多形性胶质母细胞瘤（GBM）是最常见和侵袭性形式的脑肿瘤，也是最难治疗的癌症类型。针对发育途径的疗法，比如Notch,消除肿瘤神经胶质瘤细胞，但是它们的功效可能受到各种机制的限制。联合方案可以代表具有持久效果的有效疗法的良好机会。我们使用低剂量的γ-分泌酶抑制剂RO4929097（GSI），阻断Notch通路的活性,与白藜芦醇（RSV）结合，我们证明了GBM细胞自噬/凋亡转化的机制。白藜芦醇和GSI组合导致细胞死亡的协同诱导以及自噬通量的阻断，由LC3-II和p62蛋白含量的持续增加证明，由于CDK4的急剧减少，CDK4是溶酶体功能的重要调节因子。组成型活性CDK4突变体的异位过表达，极大地抵消了RSV+GSI诱导的自噬阻断。在RSV+GSI处理的细胞中触发自噬，溶酶体功能受损，导致了系统的崩溃和随后的细胞凋亡。例如,通过结合CDK4突变体和早期自噬抑制剂，3-甲基腺苷酸，取消RSV+GSI诱导的胱天蛋白酶激活。引发半胱天冬酶(半胱天冬酶-8和-9)，在RSV+GSI暴露以及TUNEL阳性细胞的百分比后,诱导效应半胱天冬酶(caspase-3)及其下游底物PARP。此外，促凋亡信号MAPKp38被激活，而促存活MAPKp42/p44信号被抑制。总之,我们建立了CDK4在RSV和GSI诱导的GBM细胞自噬/凋亡转化中的作用。这种新的协同治疗组合，增加自噬体的积累，可能对GBM患者有治疗价值。
