Assessment of Radiobiological α/ß Ratio in Lung Cancer and Fibroblast Cell Lines Using Viability Assays.

BACKGROUND/AIM: Altered fractionation is an area of intense clinical research in radiation oncology. Estimation of the α/ß ratio of individual carcinomas after establishment of primary cell cultures from tumor biopsies may prove of importance in the individualization of radiotherapy schemes. MATERIALS AND METHODS: Here we proposed a simple method to estimate the α/ß ratio in cultured cell lines (two lung carcinomas: A549 and H1299; one lung fibroblast cell line: MRC5), using viability assays. RESULTS: For the A549 cell line, the α/ß ratio ranged from 14-25 Gy, for H1299 from 11-43 Gy and for the MRC5 fibroblast cell line this was far lower, ranging from 0.69 to 6 Gy. The α/ß ratio decreased when extracted from comparisons of lower dose per fraction schemes. CONCLUSION: The α/ß ratio of a cell line can be easily defined after simple viability/dose fractionation experiments.

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

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-Foxn1(nu) 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.