Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy.
Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy.
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自噬反应的抑制使肺癌细胞对放射和化学疗法敏感。
DOI：
10.1038/bjc.2016.202
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发表时间：
2016-07-26
影响因子：
8.8
通讯作者：
Koukourakis MI
中科院分区：
文献类型：
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作者：
Karagounis IV;Kalamida D;Mitrakas A;Pouliliou S;Liousia MV;Giatromanolaki A;Koukourakis MI
细胞对辐射的自噬反应是复杂的。不同的细胞和组织对辐射的反应是不同的，这取决于照射的剂量和照射后的时间。在目前的研究中，我们通过评估相关蛋白的表达，以及相关基因沉默在放射和化疗增敏中的作用，来确定肺癌细胞系对辐射的自噬和溶酶体反应。此外，在体内自噬基因沉默模型中评价了辐射后肿瘤的敏感性。采用A549和H1299细胞系作为体外肿瘤模型。将各种小干扰RNA导入两种细胞系，沉默自身溶酶体基因，并用4 Gy射线照射。用AlamarBlue比色法检测细胞生长反应。免疫印迹和共聚焦显微镜用于表征自身溶酶体通量。另外，将MAP1LC3A基因的小发夹RNA稳定地导入H1299细胞系，并对裸鼠Foxn1nu细胞的放射增敏作用进行了评价。4 Gy射线照射后，A549细胞表现出明显的自噬通量，而自体溶酶体基因(LC3A、LC3B、p62、TFEB和LAMP2A)的转录激活不支持自噬通量，从而导致侵袭体的积累。转录活性和自噬效应在照射后7天恢复。或者，H1299细胞，一种相对耐辐射的细胞系，在早期(在2天内)以自身溶酶体基因的转录激活做出尖锐反应，维持有效的自噬体通量，导致足够的侵袭体清除。随后，我们测试了四个基因(LC3A、LC3B、TFEB和LAMP2A)的沉默，证实了对包括顺铂和紫杉烷在内的各种化疗药物的显著放射增敏和化疗增敏。在小鼠移植瘤中，暴露在辐射中显著降低了肿瘤的生长(P<；0.001)，这在shLC3A-H1299转基因肿瘤中加剧。肺癌细胞在4 Gy射线照射后存活的能力取决于它们维持功能性自噬通量的能力。这种能力的丧失会导致辐射敏感性的增加和对各种化疗药物的敏感性。癌细胞自噬功能的选择性抑制剂可能被证明对根除肺癌很重要。
The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation. A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1nu was evaluated. Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours. The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.