阻断对葡萄糖剥夺的存活反应对肿瘤细胞的影响。

Effect on tumor cells of blocking survival response to glucose deprivation.

作者信息

Park Hae-Ryong, Tomida Akihiro, Sato Shigeo, Tsukumo Yoshinori, Yun Jisoo, Yamori Takao, Hayakawa Yoichi, Tsuruo Takashi, Shin-ya Kazuo

机构信息

Laboratory of Chemical Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

出版信息

J Natl Cancer Inst. 2004 Sep 1;96(17):1300-10. doi: 10.1093/jnci/djh243.

DOI:10.1093/jnci/djh243

PMID:15339968

Abstract

BACKGROUND

Glucose deprivation, a feature of poorly vascularized solid tumors, activates the unfolded protein response (UPR), a stress-signaling pathway, in tumor cells. We recently isolated a novel macrocyclic compound, versipelostatin (VST), that inhibits transcription from the promoter of GRP78, a gene that is activated as part of the UPR. We examined the effect of VST on the UPR induced by glucose deprivation or other stressors and on tumor growth in vivo.

METHODS

Human colon cancer HT-29, fibrosarcoma HT1080, and stomach cancer MKN74 cells were cultured in the absence of glucose or in the presence of glucose and a UPR-inducing chemical stressor (the N-glycosylation inhibitor tunicamycin, the calcium ionophore A23187, or the hypoglycemia-mimicking agent 2-deoxyglucose [2DG]). The effect of VST on UPR induction was determined by reverse transcription-polymerase chain reaction and immunoblot analysis of the UPR target genes GRP78 and GRP94; by immunoblot analysis of the UPR transcriptional activators ATF6, XBP1, and ATF4; and by analyzing reporter gene expression in cells transiently transfected with a GRP78 promoter-reporter gene. Cell sensitivity to VST was examined with a colony formation assay and flow cytometry. In vivo antitumor activity of VST was assessed with an MKN74 xenograft model.

RESULTS

VST inhibited expression of UPR target genes in glucose-deprived or 2DG-treated cells but not in cells treated with tunicamycin or A23187. VST also inhibited the production of the UPR transcriptional activators XBP1 and ATF4 during glucose deprivation. The UPR-inhibitory action of VST was seen only in conditions of glucose deprivation and caused selective and massive killing of the glucose-deprived cells. VST alone and in combination with cisplatin statistically significantly (P =.004 and P<.001 for comparisons with untreated control, respectively) inhibited tumor growth of MKN74 xenografts.

CONCLUSION

Disruption of the UPR may provide a novel therapeutic approach to targeting glucose-deprived solid tumors.

摘要

背景

葡萄糖剥夺是血管生成不良的实体瘤的一个特征，它能激活肿瘤细胞中的未折叠蛋白反应（UPR），这是一种应激信号通路。我们最近分离出一种新型大环化合物，即versipelostatin（VST），它能抑制GRP78基因启动子的转录，GRP78基因是作为UPR的一部分而被激活的。我们研究了VST对葡萄糖剥夺或其他应激源诱导的UPR以及对体内肿瘤生长的影响。

方法

将人结肠癌细胞HT - 29、纤维肉瘤细胞HT1080和胃癌细胞MKN74在无葡萄糖的条件下培养，或在有葡萄糖及一种诱导UPR的化学应激源（N - 糖基化抑制剂衣霉素、钙离子载体A23187或模拟低血糖的试剂2 - 脱氧葡萄糖[2DG]）的条件下培养。通过逆转录 - 聚合酶链反应和对UPR靶基因GRP78和GRP94的免疫印迹分析；对UPR转录激活因子ATF6、XBP1和ATF4的免疫印迹分析；以及分析用GRP78启动子 - 报告基因瞬时转染的细胞中的报告基因表达，来确定VST对UPR诱导的影响。用集落形成试验和流式细胞术检测细胞对VST的敏感性。用MKN74异种移植模型评估VST的体内抗肿瘤活性。

结果

VST抑制了葡萄糖剥夺或2DG处理的细胞中UPR靶基因的表达，但对衣霉素或A23187处理的细胞没有抑制作用。VST还抑制了葡萄糖剥夺期间UPR转录激活因子XBP1和ATF4的产生。VST的UPR抑制作用仅在葡萄糖剥夺的条件下出现，并导致对葡萄糖剥夺细胞的选择性大量杀伤。单独使用VST以及与顺铂联合使用在统计学上均显著（与未处理对照相比，P分别为0.004和P<0.001）抑制了MKN74异种移植瘤的生长。