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Abstract
Purpose: To determine the efficacy of a caffeine derivative 1-methylxanthine (1-MTX) in increasing radiosensitivity of cancer cells and elucidate the underlying mechanisms in vitro.
Materials and methods: RKO human colorectal cancer cells carrying wild type protein 53 kDa (p53) were incubated with 3 mM 1-MTX for 30 min, exposed to 4 Gy ionizing radiation, and further incubated with 1-MTX for three days. The clonogenic cell death was determined, and the cell cycle distribution and apoptosis were studied with flow cytometry at different times after irradiation. The DNA double strand break (DNA DSB) was examined using phosphorylated Histone2A (γ-H2AX) foci formation, and the expression/activity of checkpoint 2 kinase (Chk2), cell division cycle 25 (Cdc25) phosphatase and cyclin B1/Cdc2 kinase were also investigated using western blotting and in vitro kinase assays.
Results: The treatment with 3 mM 1-MTX increased the radiation-induced clonogenic and apoptotic cell death. The radiation-induced phosphorylation of Chk2 and Cdc25c and the radiation-induced increase in the cyclin B1/Cdc2 kinas activity were little affected by 1-MTX. The radiation-induced G2/M arrest was only slightly shortened and the expression of radiation-induced γ-H2AX was markedly prolonged by 1-MTX.
Conclusions: 1-MTX significantly increased the radiosensitivity of RKO human colorectal cancer cells carrying wild type p53 mainly by inhibiting the repair of radiation-induced DNA DSB without causing significant alteration in radiation-induced G2/M arrest. Such a radiosensitization occurred at 1-MTX concentrations almost non-toxic to the target tumor cells.
| Abbreviations | ||
| 1-MTX | = | 1-methylxanthine |
| Caffeine | = | 1,3,7-trimethylxanthine |
| DNA DSB | = | DNA double-strand break |
| ATM | = | ataxia telangiectasia mutated |
| PI3K | = | phosphoinositide-3 kinase |
| ATR | = | ATM and Rad3-related |
| NHEJ | = | nonhomologous end joining |
| HRR | = | homologous recombination repair |
| PBS | = | phosphate buffered saline |
| EDTA | = | ethylene diamine tetra acetic acid |
| SDS-PAGE | = | sodium dodecyl sulfate-poly acrylamide gel electrophoresis |
| PVDF | = | polyvinylidene difluoride |
| DTT | = | dithiothreitol |
| FITC | = | Fluorescein isothiocyanate |
| Chk1 | = | checkpoint kinase 1 |
| Chk2 | = | checkpoint kinase 2 |
| Cdc25 | = | cell division cycle 25 |
| p53 | = | protein 53 kDa |
| p21 | = | cyclin-dependent kinase inhibitor 1A (Cip1) |
| γ-H2AX | = | phosphorylated histone2A (serine 139) |
| DAPI | = | 4′,6-diamidino-2-phenylindole |
| Abbreviations | ||
| 1-MTX | = | 1-methylxanthine |
| Caffeine | = | 1,3,7-trimethylxanthine |
| DNA DSB | = | DNA double-strand break |
| ATM | = | ataxia telangiectasia mutated |
| PI3K | = | phosphoinositide-3 kinase |
| ATR | = | ATM and Rad3-related |
| NHEJ | = | nonhomologous end joining |
| HRR | = | homologous recombination repair |
| PBS | = | phosphate buffered saline |
| EDTA | = | ethylene diamine tetra acetic acid |
| SDS-PAGE | = | sodium dodecyl sulfate-poly acrylamide gel electrophoresis |
| PVDF | = | polyvinylidene difluoride |
| DTT | = | dithiothreitol |
| FITC | = | Fluorescein isothiocyanate |
| Chk1 | = | checkpoint kinase 1 |
| Chk2 | = | checkpoint kinase 2 |
| Cdc25 | = | cell division cycle 25 |
| p53 | = | protein 53 kDa |
| p21 | = | cyclin-dependent kinase inhibitor 1A (Cip1) |
| γ-H2AX | = | phosphorylated histone2A (serine 139) |
| DAPI | = | 4′,6-diamidino-2-phenylindole |
