Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis.
Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis.
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DOI：
10.3892/ijo.2018.4282
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发表时间：
2018-04
影响因子：
5.2
通讯作者：
Miyazawa K
中科院分区：
文献类型：
--
作者：
Saito Y;Moriya S;Kazama H;Hirasawa K;Miyahara K;Kokuba H;Hino H;Kikuchi H;Takano N;Hiramoto M;Tsukahara K;Miyazawa K
细胞内氨基酸水平的维持对于细胞内稳态是至关重要的。这是通过调节细胞外环境的流入和细胞内资源的再循环来实现的。由于表皮生长因子受体（EGFR）-酪氨酸激酶抑制剂，包括吉非替尼（GEF）已被报道诱导几种癌细胞系的凋亡，在本研究中，我们检查是否GEF的细胞毒性作用进一步增强氨基酸饥饿（AAS）培养条件下。在AAS培养条件下，与单独用GEF或AAS处理的细胞相比，GEF的细胞杀伤作用在EGFR表达细胞系中协同显著，即CAL 27、Detroit 562、A549和PANC-1细胞。向细胞培养基中添加必需氨基酸而非必需氨基酸导致这种明显的细胞毒性被取消。通过siRNA敲低L型氨基酸转运蛋白1（LAT-1）也增强GEF诱导的细胞毒性。因此，细胞内氨基酸库的短缺似乎决定了对GEF的敏感性。值得注意的是，这种增强的细胞毒性不是通过诱导细胞凋亡介导的，而是伴随着明显的自噬诱导。Necrostatin-1（一种受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK-1）的抑制剂）的存在，而不是Z-VAD-favor的存在，减弱了AAS培养条件下GEF的细胞毒性作用。电子显微镜观察发现，在AAS培养条件下，用GEF处理的CAL 27细胞表现出胞质和细胞器肿胀，自噬体和自溶酶体数量增加，但没有染色质凝聚和核碎裂。排除自噬细胞死亡，因为自噬的抑制不会减弱细胞毒性。这些结果强烈提示在AAS培养条件下，响应于GEF诱导坏死性凋亡。然而，我们不能检测到RIPK-1和混合谱系激酶结构域样假激酶（MLKL）的任何磷酸化，以及任何坏死体形成。因此，认为AAS培养条件下GEF增强的细胞毒性作用由非典型坏死性凋亡介导。
The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.
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影响因子：
158.5
作者：
Lynch, TJ;Bell, DW;Haber, DA
通讯作者：
Haber, DA
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21.3
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作者：
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