nu-7026 and Lung-Neoplasms

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

Our previous data demonstrated that targeting non-homologous end-joining repair (NHEJR) yields a higher radiosensitivity than targeting homologous recombination repair (HRR) to heavy ions using DNA repair gene knockouts (KO) in mouse embryonic fibroblast (MEF). In this study, we determined if combining the use of an NHEJR inhibitor with carbon (C) ion irradiation was more efficient in killing human cancer cells compared with only targeting a HRR inhibitor.. The TP53-null human non-small cell lung cancer cell line H1299 was used for testing the radiosensitizing effect of NHEJR-related DNA-dependent protein kinase (DNA-PK) inhibitor NU7026, HRR-related Rad51 inhibitor B02, or both to C ion irradiation using colony forming assays. The mechanism underlying the inhibitor radiosensitization was determined by flow cytometry after H2AX phosphorylation staining. HRR-related Rad54-KO, NHEJR-related Lig4-KO, and wild-type TP53-KO MEF were also included to confirm the suppressing effect specificity of these inhibitors.. NU7026 showed significant sensitizing effect to C ion irradiation in a concentration-dependent manner. In contrast, B02 showed a slight sensitizing effect to C ion irradiation. The addition of NU7026 significantly increased H2AX phosphorylation after C ion and x-ray irradiations in H1299 cells, but not B02. NU7026 had no effect on radiosensitivity to Lig4-KO MEF and B02 had no effect on radiosensitivity to Rad54-KO MEF in both irradiations.. These results suggest that inhibitors targeting the NHEJR pathway could significantly enhance radiosensitivity of human cancer cells to C ion irradiation, rather than targeting the HRR pathway.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromones; DNA End-Joining Repair; Heavy Ion Radiotherapy; Humans; Lung Neoplasms; Morpholines; Radiation Tolerance; Radiation-Sensitizing Agents

Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand break repair (SSBR) contribute to the determination of sensitivity to IR. A crucial protein in BER/SSBR is DNA polymerase β (polβ). Aberrant polβ expression is commonly found in human tumors and leads to inhibition of BER. Here, we show that truncated polβ variant (polβ-Δ)-expressing cells depend on homologous recombination (HR) for survival after IR, indicating that a considerable fraction of polβ-Δ-induced lesions are subject to repair by HR. Increased sensitization was found not to result from involvement in DNA-dependent protein kinase-dependent nonhomologous end joining, the other major double-strand break repair pathway. Caffeine and the ATM inhibitor Ku55933 cause polβ-Δ-dependent radiosensitization. Consistent with the observed HR dependence and the known HR-modulating activity of ATM, polβ-Δ-expressing cells showed increased radiosensitization after BRCA2 knockdown that is absent under ATM-inhibited conditions. Our data suggest that treatment with HR modulators is a promising therapeutic strategy for exploiting defects in the BER/SSBR pathway in human tumors.

Topics: Ataxia Telangiectasia Mutated Proteins; Blotting, Western; BRCA2 Protein; Cell Cycle Proteins; Chromones; DNA Damage; DNA Polymerase beta; DNA Repair; DNA-Binding Proteins; Humans; Lung Neoplasms; Morpholines; Protein Serine-Threonine Kinases; Radiation Tolerance; Radiation, Ionizing; Recombination, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Proteins

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