Targeted knockdown of death-associated protein kinase expression induces TRAIL-mediated apoptosis in human endometrial adenocarcinoma cells
Targeted knockdown of death-associated protein kinase expression induces TRAIL-mediated apoptosis in human endometrial adenocarcinoma cells
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DOI：
10.3892/ijo_00000668
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发表时间：
2010-07-01
影响因子：
5.2
通讯作者：
Yukawa, Kazunori
中科院分区：
文献类型：
--
作者：
Bai, Tao;Tanaka, Tetsuji;Yukawa, Kazunori
死亡相关蛋白激酶（DAPK）是一种丝氨酸/苏氨酸激酶，参与细胞凋亡和肿瘤抑制的调节。我们的前期研究发现DAPK蛋白在分化的子宫内膜腺癌细胞中有高水平的表达。为了阐明DAPK在人子宫内膜腺癌中的作用，我们使用特异性小干扰RNA（siRNA）下调了HHUA细胞（一种分化良好的子宫内膜腺癌细胞系）中内源性DAPK的表达。内源性DAPK表达的抑制触发了HHUA细胞的凋亡，如流式细胞术分析中亚G1 DNA含量的增加所证明的。由DAPK siRNA转染诱导的细胞凋亡是半胱天冬酶依赖性的，其特征在于半胱天冬酶-3、-8和-9的激活。RNA酶保护试验检测到更高水平的肿瘤坏死因子相关凋亡诱导配体（TRAIL），DR 4和DR 5的转录DAPK siRNA转染的HHUA细胞比对照siRNA转染的细胞。与这些发现一致，酶联免疫吸附测定显示DAPK siRNA转染显着增加了细胞中TRAIL蛋白的分泌。用重组人TRAIL蛋白的处理剂量依赖性地抑制HHUA细胞的细胞活力。目前的研究结果表明，下调内源性DAPK的表达在HHUA细胞诱导半胱天冬酶依赖性凋亡，可能通过增加TRAIL，DR 4和DR 5信号，从而表明DAPK的表达是必不可少的HHUA细胞存活。因此，内源性DAPK mRNA可能是分子靶向抗癌治疗的潜在候选者。
Death-associated protein kinase (DAPK) is a serine/threonine kinase that participates in the modulation of apoptosis and tumor suppression. Our previous study revealed high levels of DAPK protein expression in differentiated endometrial adenocarcinoma cells. To clarify the role of DAPK in human endometrial adenocarcinomas, we down-regulated endogenous DAPK expression in HHUA cells, a well-differentiated endometrial adenocarcinoma cell line, using specific small-interfering RNAs (siRNAs). The suppression of endogenous DAPK expression triggered apoptosis in HHUA cells, as evidenced by an increase in the sub-G1 DNA content in flow cytometric analyses. The apoptosis induced by the DAPK siRNA transfections was caspase-dependent, as characterized by the activations of caspase-3, -8 and -9. RNase protection assays detected higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 transcripts in the DAPK siRNA-transfected HHUA cells than in the control siRNA-transfected cells. Consistent with these findings, enzyme-linked immunosorbent assays revealed that the DAPK siRNA transfections significantly increased the secretion of TRAIL protein from the cells. Treatment with recombinant human TRAIL protein dose-dependently suppressed the cell viability of HHUA cells. The present findings reveal that down-regulation of endogenous DAPK expression in HHUA cells induces caspase-dependent apoptosis, possibly through increased TRAIL, DR4 and DR5 signaling, thereby suggesting that DAPK expression is essential for HHUA cell survival. Consequently, endogenous DAPK mRNA may represent a potential candidate for molecularly targeted anticancer therapies.