Development of antitumor drugs to enhance apoptotic cell death and clinical application

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Clinically, the combination treatment of solid tumor cells with an anticancer drug and radiation was widely used to enhance cell killing. 1-(3-C-Ethynyl-β-D-rijbo-pentofuranosyl) cytosine (ECyd) was newly developed as an anticancer drug to induce cell death by inhibiting RNA synthesis. In this study, we examined whether the exposure of human gastric adenocarcinoma MKN45 cells to X rays in the presence of ECyd at the limited concentration ranges with no apoptosis induced apoptotic cell death. MKN45 cells were treated with 0.1μM ECyd for 1 h before irradiation. After irradiation with 20 Gy, apoptotic as well as swelling cells were morphologically discriminated by fluorescence microscopy combined with propidium iodide staining or electron microscopy and were scored. When cells were treated with ECyd alone, only 5% of either apoptotic or necrotic cells appeared. When cells were exposed to X rays alone, about 5% of apoptotic and about 45% of swelling cells appeared. However, when cells were exposed to X rays in the presence of 0.1μM ECyd, about 25% of totali cells was apoptotic cells and about 10% of total cells was swelling cells. Apoptosis induced by treating cells with ECyd and X irradiation was significantly reduced by the treatment with Ac-DEVD-CHO (caspase 3 inhibitor) or TPCK (chymotrypsin-like protease inhibitor). These results suggested that caspase 3 and chymotrypsin-like proteases were responsible for apoptosis induced by co-treatment with ECyd and X rays. In this study, it was demonstrated that co-treatment of MKN45 cells with ECyd and X rays activated G2-phase-linked apoptotic signaling associated with caspase 3 and chymotrypsin like protease. These data may provide useful information to develop clinical treatment of radiation combined with anticancer drugs for solid tumors.