Xu Li-Hong, Deng Chang-Sheng, Zhu You-Qing, Liu Shi-Quan, Liu Dong-Zhou
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Donghu Road 169, Wuhan 430071, Hubei Province China.
World J Gastroenterol. 2003 Jun;9(6):1241-5. doi: 10.3748/wjg.v9.i6.1241.
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent. Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.
SW480 cells were cultured in the regular condition and incubated with different levels of agents. Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.
(1) SW480 cells were not sensitive to TRAIL, with IC(50)>1 mg/L(-1) and dose-independent cytotoxicity. (2) SW480 cells were sensitive to doxorubicin at a certain degree, with dose-dependent cytotoxicity and IC(50)=65.25+/-3.48 micromol/L(-1). (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC(50) of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 microg/L(-1)), combined with subtoxic doxorubicin (0.86 micromol/L(-1)), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+/-2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82+/-1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P>0.05).
SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）已被报道可特异性诱导癌细胞凋亡，尽管只有一小部分细胞系对其敏感。体外对TRAIL无反应的细胞系在与另一种抗癌药物联合使用时据说更易发生凋亡。一般来说，影响药物敏感性的因素涉及许多凋亡相关蛋白，包括p53。野生型p53基因的表达被认为是肿瘤细胞对化疗产生反应的重要前提。本研究旨在探讨TRAIL对结肠癌细胞系SW480的细胞杀伤作用、其与阿霉素的协同作用及可能机制。
SW480细胞在常规条件下培养，并与不同浓度的药物孵育。在相差显微镜下观察处理后这些细胞的形态变化，并用1天微量培养四氮唑蓝染料（MTT）法对单独使用TRAIL及与阿霉素联合使用时的细胞毒性进行定量。此外，采用流式细胞术（FCM）和透射电子显微镜检测这些细胞中的凋亡情况。通过蛋白质免疫印迹法检测不同组中根据药物浓度p53蛋白水平的变化。
（1）SW480细胞对TRAIL不敏感，半数抑制浓度（IC50）>1mg/L-1，且细胞毒性与剂量无关。（2）SW480细胞对阿霉素有一定程度的敏感性，细胞毒性呈剂量依赖性，IC50=65.25±3.48μmol/L-1。（3）TRAIL可与阿霉素协同有效杀伤SW480细胞，表现为阿霉素对这些细胞的杀伤作用增强。当阿霉素与TRAIL联合使用时，其对SW480细胞的IC50急剧降低。（4）亚毒性浓度的TRAIL（100μg/L-1）与亚毒性浓度的阿霉素（0.86μmol/L-1）联合使用可充分杀伤SW480细胞。MTT法检测的细胞毒性达到80.12±2.67%，显著高于单独使用TRAIL或阿霉素时，P值分别为0.006和0.003。这种杀伤作用部分归因于凋亡。相差显微镜下可见大量凋亡细胞、FCM检测细胞凋亡率为76.82±1.93%以及透射电子显微镜观察到典型的凋亡形态均证实了这一点。联合处理后凋亡增加与p53蛋白水平无关（P>0.05）。
SW480细胞对TRAIL不敏感，但TRAIL可与较低浓度的阿霉素协同有效诱导凋亡。p53蛋白状态不参与协同凋亡机制。这提示TRAIL与阿霉素联合应用对结肠癌具有潜在的治疗适用性。
