Inhibition of induced autophagy increases apoptosis of Nara-H cells
Inhibition of induced autophagy increases apoptosis of Nara-H cells
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DOI：
10.3892/ijo.2011.1148
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发表时间：
2011-12-01
影响因子：
5.2
通讯作者：
Yamamoto, Tetsuji
中科院分区：
文献类型：
--
作者：
Nakamura, Osamu;Hitora, Toshiaki;Yamamoto, Tetsuji
mTOR信号通路的抑制促进自噬的起始。然而，最近的研究表明，自噬是癌细胞遭受抗肿瘤药物的自我防御机制，并且阻断自噬可以触发凋亡。在这里，我们研究了mTOR抑制剂坦罗莫司对恶性纤维组织细胞瘤（MFH）细胞系Nara-H细胞的影响，以及抑制自噬对这些MFH细胞诱导凋亡的影响。在Nara-H细胞中，我们使用CellTiter 96（R）AQueous One Solution细胞增殖测定法检测了替西罗莫司治疗对细胞增殖的影响，并使用基于Western印迹的测定法检测了替西罗莫司治疗对mTOR途径组分磷酸化和自噬的影响。此外，我们研究了坦罗莫司与或不与3-甲基腺嘌呤（3-MA）诱导细胞凋亡的影响，使用荧光显微镜分析。在Nara-H细胞中，替西罗莫司处理抑制细胞增殖，抑制mTOR通路组分的磷酸化，并诱导自噬，如通过LC-3 II表达所评估的。此外，用替西罗莫司和3-MA的组合处理诱导Nara-H细胞的凋亡。显然，自噬和mTOR的同时抑制诱导了Nara-H细胞的凋亡，因为自噬的抑制阻止了细胞保护自己免受mTOR抑制的影响。因此，包括mTOR抑制剂和自噬抑制剂（分别为替西罗莫司和3-MA）的组合疗法可以通过诱导肿瘤细胞中的凋亡来有效治疗MFH。
Inhibition of the mTOR signaling pathway promotes initiation of autophagy. However, recent studies indicate that autophagy is a self-defense mechanism of cancer cells that are subjected to anti-tumor agents and that blocking autophagy can trigger apoptosis. Here, we examined the effects of an mTOR inhibitor, temsirolimus, on a malignant fibrous histiocytoma (MFH) cell line, Nara-H cells, and the effect of suppressing autophagy on the induction of apoptosis in these MFH cells. In Nara-H cells, we examined the effects of temsirolimus treatment on cell proliferation using the CellTiter 96 (R) AQueous One Solution Cell Proliferation Assay and on phosphorylation of mTOR pathway components and autophagy using Western blot-based assays. Furthermore, we examined the effects of temsirolimus with or without 3-methyladenine (3-MA) on induction of apoptosis using fluorescent microscopic analysis. In Nara-H cells, temsirolimus treatment inhibited cell proliferation, suppressed phosphorylation of mTOR pathway components, and induced autophagy as assessed by LC-3 II expression. Moreover, treatment with a combination of temsirolimus and 3-MA induced apoptosis in Nara-H cells. Apparently, simultaneous inhibition of autophagy and mTOR induced apoptosis in Nara-H cells because inhibition of autophagy prevented the cells from protecting themselves from the effects of the inhibition of mTOR. Therefore, a combination therapy that includes an mTOR inhibitor and an autophagy inhibitor (temsirolimus and 3-MA, respectively) may effectively treat MFH by inducing apoptosis in tumor cells.