Tu Chengyi, Xu Robert, Koleti Meghana, Zoldan Janet
Department of Biomedical Engineering, University of Texas at Austin, United States.
Department of Biomedical Engineering, University of Texas at Austin, United States.
Stem Cell Res. 2017 Aug;23:182-187. doi: 10.1016/j.scr.2017.07.019. Epub 2017 Jul 22.
Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome.
抑制糖原合酶激酶3（GSK3）是激活Wnt信号通路以实现多能干细胞（PSC）分化的广泛应用策略。然而，尽管GSK3广泛参与其他细胞活动，如胰岛素信号传导和细胞生长/存活调节，但除了上调Wnt信号通路外，这种抑制对PSC的影响鲜有研究。在此，我们描述了GSK3抑制（如Chir99021和LY2090314）与各种通常无毒的含硫醇抗氧化剂（如N - 乙酰半胱氨酸，NAC）之间一种前所未知的协同作用，该作用可诱导人诱导多能干细胞（iPSC）凋亡。单独使用Chir99021或抗氧化剂均未诱导显著凋亡，而它们联合处理则导致快速且广泛的凋亡，3小时内可观察到显著的半胱天冬酶3活性，24小时后细胞活力下降超过90%。我们用多个独立的iPSC系、各种基于硫醇的抗氧化剂和不同的GSK3抑制剂证实了这一现象的普遍性。从机制上讲，我们证明雷帕霉素处理可大幅减少细胞死亡，提示雷帕霉素哺乳动物靶点（mTOR）的关键作用。还发现Akt失调部分促成细胞凋亡，但并非主要原因。此外，在小鼠胚胎干细胞中未检测到这种协同促凋亡作用，但在另一种人类细胞系：乳腺癌细胞系（MDA - MB - 231）中存在。鉴于GSK3抑制在生物医学研究中的广泛应用：从iPSC分化到癌症干预和神经疾病治疗，研究人员可利用或避免这种协同作用以改善实验或临床结果。
