RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells
RAF Inhibition Overcomes Resistance to TRAIL-Induced Apoptosis in Melanoma Cells
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DOI：
10.1038/jid.2013.347
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发表时间：
2014-02-01
影响因子：
6.5
通讯作者：
Eberle, Juergen
中科院分区：
文献类型：
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作者：
Berger, Anja;Quast, Sandra-Annika;Eberle, Juergen
突变的BRAF代表黑色素瘤中的关键致癌基因，并且选择性抑制剂已被批准用于黑色素瘤治疗。然而，在黑色素瘤细胞中RAF抑制的分子后果在很大程度上仍然难以捉摸。在这里，我们研究了泛RAF抑制剂L-779，450的作用，该抑制剂在BRAF突变和野生型黑素瘤细胞系中均抑制细胞增殖。它还与死亡配体肿瘤坏死因子相关的凋亡诱导配体（TRAIL）组合增强凋亡，并克服黑素瘤细胞中的TRAIL抗性。增强的细胞凋亡与线粒体途径的激活一致，通过线粒体膜电位的丧失和细胞色素c、Smac（第二线粒体源性半胱天冬酶激活剂）和凋亡诱导因子（AIF）的释放来观察。随后，caspase-9和-3被激活。L-779，450/TRAIL诱导的细胞凋亡可被Bcl-2过表达所阻止，并依赖于Bax。因此，通过Box构象变化证明了单独L-779，450对Bax的激活，而巴克没有被激活。此外，仅BH 3蛋白Bim响应于L-779，450而上调。Smac、Bax和Bim在这种情况下的重要作用通过小干扰RNA（siRNA）介导的敲除实验得到证实。L-779，450还导致形态学变化，表明自噬，自噬标志物轻链3-II（LC 3-II）证实了自噬。L-779，450的促凋亡作用可以解释RAF抑制的抗肿瘤作用，并且在评价RAF抑制剂用于黑色素瘤治疗时可以考虑。
Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Box conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy.