Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy

Abstract

Background: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.Methods: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.Results: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.Conclusions: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.

Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells

“…Because the Akt/mTOR pathway is recognized as a major pathway regulating autophagy [16], we also studied the role of autophagy in the development of radiation resistance in MK-2206-treated cells, especially in case of the SNB19 cell line. To this end, we detected the autophagosomal membrane-bound LC3B protein along with the expression of the p62/sequestome protein, a pleiotropic protein that is consumed during autophagy [31]. As seen in the Additional file 4: Figure S3, IR increased the expression of LC3B-I and LC3B-II proteins in both cell lines.…”

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299

“…For example, CQ derivatives are commonly-used inhibitors of autophagy which are already approved for clinical trials focusing on cancer therapy [17]. Regarding these studies, Toulany et al [18], as well as Karagounis et al [19], reported the radiosensitizing effect of CQ on lung cancer cells as a result of autophagy blockade. Even though both CQ and HCQ can effectively inhibit autophagy, the doses necessary for the appropriate effect in vitro are not consistently achievable in patients, and there is an identified need for new inhibitors with better physicochemical and pharmacokinetic properties.…”

Lys05 induces lysosomal membrane permeabilization and increases radiosensitivity in glioblastoma

“…The transcription factor EB (TFEB) has recently been identified as one of the key regulators of lysosomal biogenesis. A previous study also reported that after irradiation treatment, the expression of TFEB increased significantly . As a result, we assumed that irradiation might increase the lysosomal function in a TFEB‐dependent manner.…”

“…TFEB is a transcription factor that is involved in lysosomal biogenesis . Previous study found that TFEB was upregulated after irradiation treatment, which suggested that lysosomal biogenesis was enhanced . Besides, many studies also illustrated that autophagy was upregulated after irradiation treatment, and the enhancement of lysosomal function—as the most important part of the late stage of autophagy—was in consistent with increased autophagy .…”