Time-course analysis of DNA damage response-related genes after in vitro radiation in H460 and H1229 lung cancer cell lines

Radiation is the most useful treatment modality for cancer patients. It initiates a series of signal cascades such as DNA damage response (DDR) signaling for repairing damaged DNA, arresting the cell cycle, and inducing cell death. Until now, few genes have been found to be regulated by radiation, which explains the molecular mechanisms of cellular responses to radiation. Although the transcriptional changes caused by radiation have been widely investigated, little is known about the direct evidence for the transcriptional control of DDR-related genes. Here, we examined the radiosensitivity of two non-small cell lung cancer cell lines (H460 and H1299), which have different p53 status. We monitored the time-dependent changes of 24 DDR-related gene expressions via microarray analysis. Based on the basal expression levels and temporal patterns, we further classified 24 DDR-related genes into four subgroups. Then, we also addressed the protein levels of several DDR-related genes such as TopBP1, Chk1 and Chk2, confirming the results of microarray analysis. Together, these results indicate that the expression patterns of DDR-related genes are associated with radiosensitivity and with the p53 statuses of H460 and H1299, which adds to the understanding of the complex biological responses to radiation.

miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFkappaB1

The activation of nuclear factor-kappa B1 (NFkappaB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkappaB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in gamma-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFkappaB1. Overexpression of miR-9 down-regulated the level of NFkappaB1 in H1299 cells, and the surviving fraction of gamma-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFkappaB1, although there was no canonical target site for let-7g in the NFkappaB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFkappaB1.

Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation

In this study, we have investigated if current cancer therapeutic modalities including hyperthermia and ionizing radiation can increase the expression of NKG2D ligands in human cancer cell lines. The expressions of NKG2D ligands were induced by both heat shock and ionizing radiation in various cell lines including KM12, NCI-H23, HeLa and A375 cells with peaks at 2 h and 9 h after treatment, respectively, although inducibility of each NKG2D ligand was various depending on cell lines. During the induction of NKG2D ligands, heat shock protein 70 was induced by heat shock but not by ionizing radiation. These results were followed by increased susceptibilities to NK cell-mediated cytolysis after treatment with heat shock and ionizing radiation. These results suggest that heat shock and ionizing radiation induce NKG2D ligands and consequently might lead to increased NK cell-mediated cytotoxicity in various cancer cells.

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.