Development of new antitumor drugs and apoptotic signal transudation - Application to feline leukemia and canine lymphoma -

临床广泛采用抗癌药物与放射联合治疗实体瘤细胞来增强细胞杀伤。1-（3- c -乙基-β- d -核糖-戊呋喃基）胞嘧啶（ECyd）和2-氯脱氧腺苷（Cl-Ade）是近年来通过抑制RNA合成诱导细胞死亡的抗癌药物。在这项研究中，我们研究了这些抗癌药物或/和电离辐射是否暴露于人胃腺癌MKN45细胞、人白血病细胞系MOLT-4细胞和犬淋巴瘤细胞系CL-1诱导细胞凋亡。在MOLT-4细胞中，Cl-Ade处理后Fas表达和caspase 8/3活化导致凋亡细胞死亡，随后出现凋亡细胞死亡。2Cl-Ade和ECyd可诱导犬淋巴瘤细胞CL-1细胞凋亡。0.1 μM ECyd处理MKN45细胞1 h。20 Gy辐照后，荧光显微镜下观察细胞的凋亡和肿胀，并结合碘化丙啶染色和电镜下观察细胞的形态学变化。单用ECyd处理细胞时，只有5%的细胞出现凋亡或坏死。当细胞单独暴露在X射线下时，出现约5%的凋亡细胞和约45%的肿胀细胞。然而，当细胞暴露在0.1 μM ECyd的X射线下时，凋亡细胞约占总细胞数的25%，肿胀细胞约占总细胞数的10%。用Ac-DEVD-CHO （caspase 3抑制剂）或TPCK （chymotrypsin样蛋白酶抑制剂）处理ECyd和X照射诱导的细胞凋亡明显减少。上述结果提示，ECyd和X射线共同作用诱导的细胞凋亡与caspase 3和凝乳胰蛋白酶样蛋白酶有关。本研究证明，ECyd和X射线共同处理MKN45细胞激活了与caspase 3和糜凝胰蛋白酶样蛋白酶相关的g2期凋亡信号。这些数据可能为开发放射联合抗癌药物治疗实体瘤的临床治疗提供有用的信息。少

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-ribo-pentofuranosyl) cytosine (ECyd) and 2-chrolo-deoxyadenosine (Cl-Ade) was newly developed as anticancer drugs to induce cell death by inhibiting RNA synthesis. In this study, we examined whether the exposure of these anticancer drugs or /and ionizing radiation to human gastric adenocarcinoma MKN45 cells, human lekemia cell line MOLT-4 cells induce apoptosis and canine lymphoma cell line CL-1. In MOLT-4 cells, Fas expression and caspase 8/3 activation apoptotic cell death was observed after treatment of Cl-Ade, followed by apoptotic cell death. 2Cl-Ade and ECyd showed to induce apoptotic cell death in canine lymphoma cell CL-1 cells. MKN45 cells were treated with 0.1 μM ECyd for 1 h before irradiation. After irradiation with 20 Gy, apoptotic as well as swelling cells were morphologically discriminated by fluorescence microscopy … More 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 0.1 μM ECyd, about 25 % of total 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. Less