Abstract：
OBJECTIVE: Further investigation on the mechanism of action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in NSCLC would shed light on the understanding of TRAIL resistance and provide new clues for the counter-strategy.
BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3β (GSK3β) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway.
OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3β during the treatment of TRAIL.
METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3β. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis.
RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3β in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3β-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3β knockdown was blocked by the combined interference of ITCH.
CONCLUSIONS: These results suggested that GSK3β/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3β/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.
BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3β (GSK3β) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway.
OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3β during the treatment of TRAIL.
METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3β. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis.
RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3β in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3β-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3β knockdown was blocked by the combined interference of ITCH.
CONCLUSIONS: These results suggested that GSK3β/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3β/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.
摘要：
目的：进一步研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)在非小细胞肺癌中的作用机制,将有助于对TRAIL耐药的认识,并为制定相应的治疗策略提供新的线索。
背景：细胞FLICE抑制蛋白（c-FLIP）是TRAIL诱导的细胞凋亡的关键抑制剂。我们先前的研究表明，糖原合酶激酶3β（GSK3β）正调节人肺腺癌细胞中c-FLIP的表达。同时,其他研究报道，c-FLIP通过蛋白酶体途径被HECT型E3连接酶ITCH（发痒的E3泛素蛋白连接酶）降解。
目的：我们将探讨ITCH在TRAIL治疗过程中是否参与GSK3β阳性控制的c-FLIP的表达调控。
方法：使用人肺腺癌细胞稳定过表达和敲低GSK3β。采用实时定量PCR(qRT-PCR)检测基因mRNA的表达水平。蛋白质印迹分析用于检测蛋白质水平的蛋白质表达。ITCH的siRNA用于敲低其表达。TRAIL处理用于引起细胞凋亡。
结果：在本研究中，我们已经证实了肺腺癌细胞中c-FLIP的ITCH蛋白降解和GSK3β下调ITCH表达。此外，ITCH沉默逆转了细胞中GSK3β敲低引起的c-FLIP蛋白的下调。因此，TRAIL诱导的GSK3β敲低诱导的细胞凋亡被ITCH的联合干扰阻断。
结论：这些结果表明，GSK3β/ITCH轴调节c-FLIP的稳定性，并影响TRAIL诱导的细胞凋亡。一起来看,我们的研究揭示了GSK3β/ITCH/c-FLIP轴可以抵消TRAIL诱导的人肺腺癌细胞凋亡。
背景：细胞FLICE抑制蛋白（c-FLIP）是TRAIL诱导的细胞凋亡的关键抑制剂。我们先前的研究表明，糖原合酶激酶3β（GSK3β）正调节人肺腺癌细胞中c-FLIP的表达。同时,其他研究报道，c-FLIP通过蛋白酶体途径被HECT型E3连接酶ITCH（发痒的E3泛素蛋白连接酶）降解。
目的：我们将探讨ITCH在TRAIL治疗过程中是否参与GSK3β阳性控制的c-FLIP的表达调控。
方法：使用人肺腺癌细胞稳定过表达和敲低GSK3β。采用实时定量PCR(qRT-PCR)检测基因mRNA的表达水平。蛋白质印迹分析用于检测蛋白质水平的蛋白质表达。ITCH的siRNA用于敲低其表达。TRAIL处理用于引起细胞凋亡。
结果：在本研究中，我们已经证实了肺腺癌细胞中c-FLIP的ITCH蛋白降解和GSK3β下调ITCH表达。此外，ITCH沉默逆转了细胞中GSK3β敲低引起的c-FLIP蛋白的下调。因此，TRAIL诱导的GSK3β敲低诱导的细胞凋亡被ITCH的联合干扰阻断。
结论：这些结果表明，GSK3β/ITCH轴调节c-FLIP的稳定性，并影响TRAIL诱导的细胞凋亡。一起来看,我们的研究揭示了GSK3β/ITCH/c-FLIP轴可以抵消TRAIL诱导的人肺腺癌细胞凋亡。
