nu-7026 and Adenocarcinoma

The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated γ-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following γ-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent γ-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced γ-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of γ-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the quantity of γ-H2AX determines the radioresistance of cells. The DNA repair pathway is important in mediating radioresistance, and treatment with the DNA-PKcs inhibitor, NU7026 restored the acquired radiation resistance.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Cell Line, Tumor; Chromones; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; Gamma Rays; Gene Expression Regulation, Neoplastic; Histones; Humans; Lung Neoplasms; Morpholines; Nuclear Proteins; Phosphorylation; Radiation Tolerance

We investigated p53-dependent and -independent molecular events associated with cell cycle alteration and cell death in human lung adenocarcinoma A549 cells using cryptolepine, a DNA-damaging agent. After a 24-h treatment, cryptolepine caused an accumulation of p53 at concentrations of 1.25-10 microM and induction of p21(Cip1/WAF1) but only at concentrations up to 5muM. p21(Cip1/WAF1) was also strongly induced by cryptolepine (2.5-5 microM) in cells with p53 largely ablated via small interfering RNA-mediated gene silencing. Cryptolepine induced G1-phase block at 1.25-2.5 microM, S-phase and G2/M-phase block at 2.5-5 microM, and cell death at 10 microM. The dead cells displayed condensed and fragmented nuclei, features of apoptosis. Wortmannin, an inhibitor of ataxia telangiectasia-mutated and DNA-dependent protein kinase (DNA-PK), caused cell cycle arrest at G1 phase without inducing p53 and p21(Cip1/WAF1) expression and cell death. The addition of wortmannin partially prevented cryptolepine-induced expression of p53 and p21(Cip1/WAF1) together with the S-phase block and sensitized cells to induction of cell death. NU7026, a DNA-PK-specific inhibitor, showed neither induction of cell cycle arrest and apoptosis nor the expression of p53 and p21(Cip1/WAF1). The presence of NU7026 caused further reduction of cells in G1 phase induced by cryptolepine at 5 microM without affecting the induction of p53 and p21(Cip1/WAF1) and cell death. This study using the A549 cell as a model demonstrated that cryptolepine selects different molecular pathways to cell cycle checkpoint activation in a dose-specific manner and evokes a wortmannin-sensitive antiapoptosis response.

Topics: Adenocarcinoma; Alkaloids; Androstadienes; Apoptosis; Caffeine; Cell Cycle; Cell Line, Tumor; Chromones; Cyclin-Dependent Kinase Inhibitor p21; Drug Interactions; Gene Expression Regulation; Genes, p53; Humans; Indole Alkaloids; Indoles; Lung Neoplasms; Microscopy, Fluorescence; Morpholines; Quinolines; Wortmannin