BackgroundTemozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ.MethodsUsing the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ.ResultsHere, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis.ConclusionsThe understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

中文翻译：

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PX-866抑制磷脂酰肌醇3-激酶抑制替莫唑胺诱导的自噬并促进胶质母细胞瘤细胞凋亡

背景替莫唑胺 (TMZ) 是最常用的化学治疗剂，用于治疗胶质母细胞瘤 (GBM)，它会对高度增殖的细胞造成显着的 DNA 损伤。我们的观察增加了越来越多的证据，即 TMZ 诱导已知促进细胞存活的应激反应细胞程序，包括自噬。因此，针对这些生存途径可能代表了用 TMZ 治疗后 GBM 的新脆弱性。 方法使用 T98G 人神经胶质瘤细胞系，我们评估了与 TMZ 治疗相关的分子信号传导，使用泛 PI3K 抑制剂 PX-866 的细胞后果，并进行了克隆测定以确定 TMZ 和 PX-866 的连续处理对集落形成的影响。此外，我们还使用皮下 GBM 患者衍生的异种移植 (PDX) 肿瘤来显示相对 LC3 蛋白表达以及生存途径和分子标记之间的相关性，这些标记决定了对 TMZ 的临床反应。结果在这里，我们报告了 TMZ 可以诱导 T98G 神经胶质瘤细胞中的自噬通量。用 TMZ 治疗的 GBM 患者来源的异种移植 (PDX) 肿瘤也显示自噬体标志物 LC3 II 的增加。此外，O6-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 的表达与 PI3K/AKT 活性相关，这表明对 TMZ 具有固有耐药性（MGMT 高）的患者除了 TMZ 外，还将受益于 PI3K/AKT 抑制剂。因此，我们已经确定血脑屏障 (BBB) 渗透性泛 PI3K 抑制剂 PX-866 是自噬通量的早期抑制剂，同时保持其抑制胶质瘤细胞中 PI3K/AKT 信号的能力。最后，由于 TMZ 诱导自噬通量，我们提供了 TMZ 序贯治疗和 PX-866 的证据，而不是联合治疗，作为关闭 GBM 细胞中自噬诱导的存活和增强细胞凋亡的手段.结论 了解 TMZ 如何诱导生存途径，如自噬，可能会提供新的治疗弱点和机会，以使用调节自噬的替代促生存途径的顺序抑制。因此，确定抑制 TMZ 诱导的自噬的其他方法可以提高 TMZ 的功效。而不是联合联合治疗，作为关闭 GBM 细胞中自噬诱导的存活并增强细胞凋亡的一种手段。结论了解 TMZ 如何诱导存活途径，如自噬，可能提供新的治疗弱点和机会使用顺序抑制调节自噬的替代促生存途径。因此，确定抑制 TMZ 诱导的自噬的其他方法可以提高 TMZ 的功效。而不是联合联合治疗，作为关闭 GBM 细胞中自噬诱导的存活并增强细胞凋亡的一种手段。结论了解 TMZ 如何诱导存活途径，如自噬，可能提供新的治疗弱点和机会使用顺序抑制调节自噬的替代促生存途径。因此，确定抑制 TMZ 诱导的自噬的其他方法可以提高 TMZ 的功效。