Upregulation of BNIP3 by 5-aza-2′-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death
Upregulation of BNIP3 by 5-aza-2′-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death
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DOI：
10.1007/s00535-005-1576-1
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发表时间：
2005-05-01
影响因子：
6.3
通讯作者：
Imai, K
中科院分区：
文献类型：
--
作者：
Abe, T;Toyota, M;Imai, K
背景。胰腺癌细胞通常表现出对缺氧介导的细胞凋亡的抵抗力，但这种抵抗力背后的分子机制仍不清楚。因此，本研究的目的是检查表观遗传基因改变在抵抗胰腺癌细胞缺氧介导的细胞凋亡中的作用。方法。使用逆转录聚合酶链反应 (RT-PCR) 检测一组胰腺癌细胞系中与缺氧介导的细胞凋亡相关的 5 个基因（PUMA、Caspase-8 [CASP8]、APAF-1、BNIP3 和 BNIP3L）的表达。使用常氧或低氧条件下孵育的细胞裂解物通过蛋白质印迹分析检查蛋白质表达。使用亚硫酸氢盐-PCR 和测序确定基因的甲基化状态。使用流式细胞术测定凋亡细胞的百分比。结果。在常氧条件下，所测试的 12 种胰腺癌细胞系中 BNIP-3 基因的表达有所不同，其中 50% 根本没有 BNIP3 表达，而其他 4 种基因的表达在所有 12 种细胞系中都很容易检测到。 BNIP-3基因转录起始位点周围区域的CpG岛的DNA甲基化与其沉默密切相关。甲基转移酶抑制剂 5-aza-deoxycytidine (5-aza-dC) 可以恢复 BNIP3 的表达，缺氧介导的胰腺癌细胞死亡也是如此。结论。 BNIP3 的表达在一些胰腺癌细胞中通过其 CpG 岛的甲基化而被沉默。使用甲基转移酶抑制剂对 BNIP3 进行去甲基化，恢复该基因的表达并诱导缺氧介导的细胞死亡。因此，BNIP-3可能是旨在治疗胰腺癌的新疗法的有用靶标。
Background. Pancreatic cancer cells often show resistance to hypoxia-mediated apoptosis, but the molecular mechanism underlying that resistance remains unknown. The purpose of the present study, therefore, was to examine the role of epigenetic gene alteration in the resistance to hypoxia-mediated apoptosis among pancreatic cancer cells. Methods. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of five genes associated with hypoxia-mediated apoptosis (PUMA, Caspase-8 [CASP8], APAF-1, BNIP3, and BNIP3L) in a panel of pancreatic cancer cell lines. Protein expression was examined by Western blot analysis, using lysates from cells incubated under normoxic or hypoxic conditions. The methylation status of the genes was determined using bisulfite-PCR and sequencing. The percentages of cells that were apoptotic were determined using flow cytometry. Results. Under normoxic conditions, the expression of the BNIP-3 gene varied among the 12 pancreatic cancer cell lines tested, with 50% of them showing no BNIP3 expression at all, whereas expression of the other four genes was readily detected in all 12 cell lines. DNA methylation of BNIP-3's CpG island in the region around the transcription start site of the gene was closely associated with its silencing. The expression of BNIP3 was restored by the methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), as was the hypoxia-mediated pancreatic cancer cell death. Conclusions. BNIP3 expression is silenced in some pancreatic cancer cells by the methylation of its CpG island. Demethylation of BNIP3, using a methyltransferase inhibitor, restores the gene's expression and induces hypoxia-mediated cell death. BNIP-3 may thus be a useful target for new therapies aimed at treating pancreatic cancer.