Huang Wen Si, Xu Feng Mei, Zeng Qing Zhong, Liu Xiao Hui, Gao Xue Juan, Liu Lang Xia
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, Guangdong, China.
Biomed Environ Sci. 2017 Jul;30(7):473-481. doi: 10.3967/bes2017.063.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.
Nuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.
Previously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.
Taken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）耐药性极大地限制了TRAIL的临床治疗效果。阐明TRAIL耐药的分子机制对于解决这一问题至关重要。
采用细胞核和细胞质蛋白提取及免疫荧光（IF）分析检测H1299细胞中异质性核糖核蛋白K（hnRNPK）定位的变化。使用裂解的半胱天冬酶-3抗体对转染了GFP-hnRNPK、GFP-hnRNPK S284/353A或GFP-hnRNPK S284/353D突变体的细胞中的细胞凋亡进行评估。通过定量RT-PCR检测XIAP的基因表达。
此前，我们报道hnRNPK通过抑制PKC介导的GSK3β磷酸化来拮抗TRAIL诱导的细胞凋亡。在本研究中，我们进一步证明TRAIL处理可诱导H1299细胞中hnRNPK的细胞质积累。定位于细胞质的hnRNPK具有更高的拮抗TRAIL诱导的细胞凋亡的能力。ERK1/2信号抑制剂U0126和ERK磷酸化受体位点突变体（GFP-hnRNPK S284/353A）均可减少TRAIL诱导的hnRNPK的细胞质积累。此外，我们表明XIAP参与了H1299细胞中hnRNPK介导的TRAIL耐药性。
综上所述，这些结果为理解肺腺癌中与TRAIL耐药相关的分子机制提供了新的见解。
