title: Enhancement of radiation-induced DNA damage and inhibition of its repair by a novel camptothecin analog.
abstract: A novel camptothecin derivative (TLC388) with higher efficacy and reduced toxicity has been synthesized and tested as a novel chemoradiosensitizing agent. This study investigated the mechanisms of the chemoradiosensitizing effects of TLC388 on H23 human non-small cell lung cancer (NSCLC) cells. Using the TUNEL assay, a significantly higher percentage of apoptotic cells was observed in the group treated with TLC388 plus X-ray radiation than those in groups treated with drug or radiation alone. The sensitizer enhancement ratio (SER) was 1.91. Apoptosis increased with drug concentration and radiation dose, exhibiting dose-dependent pattern. The results suggested that apoptosis could be a main mode of cell death that might underlie the increased chemoradio-sensitization of TLC388. Treatment with 30 nM of TLC388 plus 4 Gy X-ray also produced up to 42% of necrotic cells that were measured by trypan blue exclusion assay, but with TLC388 alone or 4 Gy radiation alone 9.8% or 11.1% necrotic cells were detected, respectively. An immunofluorescent staining method was employed to determine the levels of gamma-H2AX (phosphorylated H2AX, a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes and is phosphorylated by kinases like ATM and ATR in the PI3K pathway, as the first step in recruiting and localizing DNA repair proteins) as a molecular biomarker of DNA double strand breaks (DSBs) in cells treated with TLC388 +/-radiation, or radiation alone. The formation of gamma-H2AX foci was observed after TLC388 or radiation exposure and when the cells were treated with 30 nM TLC388 plus radiation at a dose of 2 Gy, the percentage of cells containing gamma-H2AX foci increased significantly. Even more interesting, a markedly higher percentage (65.4%) of mitotic cells displayed gamma-H2AX foci after treatment with 30 nM TLC388 plus 0.5 Gy radiation, compared to only 5.9% or 26.1% of the M-phase cells treated with 30 nM TLC388 alone or 0.5 Gy radiation alone, respectively. It is suggested that mitotic cells become very sensitive to the production of DSBs after TLC388-radiation combined treatment and the formation of DSBs is strongly suggested to lead to the induction of apoptosis at doses lower than 4 Gy and to some necrosis at doses of 4 Gy or above. TLC388 enhances the production of DSBs and inhibits their repair, which contributes to the elucidation of the mechanisms of chemoradiosensitization of TLC388 and its development as a novel chemoradiosensitizing drug for improved radiotherapy.
authors.affiliation: Radiobiology Laboratory, California Pacific Medical Center Research Institute, San Francisco, CA 94118, USA.
authors.forename: Ge
authors.initials: G
authors.lastname: Huang
authors.forename: Huijuan
authors.initials: H
authors.lastname: Wang
authors.forename: Li-Xi
authors.initials: LX
authors.lastname: Yang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004273:DNA, Neoplasm / Q000187:drug effects / Q000528:radiation effects; D006657:Histones / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy; D010766:Phosphorylation; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D059004:Topoisomerase I Inhibitors
chemical_list: D004273:DNA, Neoplasm; C468783:H2AX protein, human; D006657:Histones; D011838:Radiation-Sensitizing Agents; C549429:TLC 388; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin