Suzuki Atsushi, Kusakai Gen-ichi, Shimojo Yosuke, Chen Jian, Ogura Tsutomu, Kobayashi Masanobu, Esumi Hiroyasu
Cancer Physiology Project, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan.
J Biol Chem. 2005 Sep 9;280(36):31557-63. doi: 10.1074/jbc.M503714200. Epub 2005 Jul 13.
Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and we have reported involvement of AMP-activated protein kinase-alpha (AMPK-alpha), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-alpha phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-alpha expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-alpha activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-beta (TGF-beta) receptor, we showed an involvement of TGF-beta in hypoxia-induced tolerance. TGF-beta1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1alpha-independent manner, and addition of recombinant TGF-beta suppressed cell death during glucose starvation even under normoxic condition. AMPK-alpha, Akt, and ARK5 were activated by TGF-beta1, and Akt and AMPK-alpha phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-beta receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-beta1 through AMPK-alpha activation and the Akt/ARK5 system.
由于人类肝癌细胞的存活和生长依赖营养物质，尤其是葡萄糖，葡萄糖饥饿会诱导细胞急性死亡。这种细胞死亡在缺氧状态下会显著受到抑制，并且我们已经报道过AMP激活的蛋白激酶α（AMPK-α）、Akt和ARK5参与了缺氧诱导的耐受性。在本研究中，我们调查了人类肝癌细胞系HepG2中缺氧诱导耐受性的机制。当HepG2细胞处于葡萄糖饥饿状态时，ARK5表达被诱导，并且我们发现葡萄糖饥饿会短暂诱导Akt和AMPK-α磷酸化，而缺氧会延长这两种蛋白激酶的磷酸化时间。我们还发现，通过阻断Akt/ARK5系统或抑制AMPK-α表达，缺氧诱导的耐受性会部分被消除，而同时抑制两者则会完全消除耐受性，这表明AMPK-α激活信号通路和Akt/ARK5系统在缺氧诱导的耐受性中发挥独立的关键作用。通过使用特异性抑制I型转化生长因子-β（TGF-β）受体激酶活性的化合物，我们表明TGF-β参与了缺氧诱导的耐受性。缺氧以不依赖缺氧诱导因子-1α的方式诱导TGF-β1 mRNA表达，并且添加重组TGF-β即使在常氧条件下也能抑制葡萄糖饥饿期间的细胞死亡。TGF-β1激活了AMPK-α、Akt和ARK5，并且I型TGF-β受体抑制剂可抑制由缺氧延长的Akt和AMPK-α磷酸化。基于这些发现，我们提出缺氧诱导的肿瘤细胞对葡萄糖饥饿的耐受性是由缺氧诱导的TGF-β1通过AMPK-α激活和Akt/ARK5系统引起的。
