Resveratrol suppresses NSCLC cell growth, invasion and migration by mediating Wnt/β-catenin pathway via downregulating SIX4 and SPHK2

Resveratrol (RSV) has been found to have a cancer-suppressing effect in a variety of cancers, including non-small cell lung cancer (NSCLC). Studies have shown that sine oculis homeobox 4 (SIX4) and sphingosine kinase 2 (SPHK2) are tumour promoters of NSCLC. However, whether RSV regulates SIX4 and SPHK2 to mediate NSCLC cell functions remains unclear. NSCLC cell functions were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, transwell assay and wound healing assay. Protein expression levels were detected by western blot. SIX4 and SPHK2 mRNA levels in NSCLC tumour tissues were examined using quantitative real-time PCR. In addition, mice xenograft models were built to explore the impact of RSV on NSCLC tumour growth. RSV inhibited NSCLC cell proliferation, invasion and migration, while facilitated apoptosis. SIX4 and SPHK2 were up-regulated in NSCLC tissues and cells, and their expression was reduced by RSV. Knockdown of SIX4 and SPHK2 suppressed NSCLC cell growth, invasion and migration, and the regulation of RSV on NSCLC cell functions could be reversed by SIX4 and SPHK2 overexpression. RSV inactivated Wnt/β-catenin pathway via decreasing SIX4 and SPHK2 levels. In animal experiments, RSV reduced NSCLC tumour growth in vivo. RSV repressed NSCLC malignant process by decreasing SIX4 and SPHK2 levels to restrain the activity of Wnt/β-catenin pathway.

白藜芦醇（Resveratrol, RSV）在包括非小细胞肺癌（non-small cell lung cancer, NSCLC）在内的多种癌症中均被证实具有抑癌效果。已有研究表明，眼缺失同源框4（sine oculis homeobox 4, SIX4）与鞘氨醇激酶2（sphingosine kinase 2, SPHK2）均为NSCLC的肿瘤促发因子，但白藜芦醇是否通过调控SIX4与SPHK2来介导NSCLC细胞功能，目前尚不明确。本研究采用噻唑蓝（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四氮唑溴盐，MTT）实验、5-乙炔基-2′-脱氧尿苷（5-ethynyl-2′-deoxyuridine, EdU）实验、流式细胞术（flow cytometry）、Transwell实验（transwell assay）及划痕愈合实验（wound healing assay）评估NSCLC细胞功能；通过蛋白质免疫印迹（western blot）检测蛋白表达水平；利用实时定量聚合酶链式反应（quantitative real-time PCR）检测NSCLC肿瘤组织中SIX4与SPHK2的mRNA表达水平；此外构建小鼠异种移植模型（mice xenograft models），探究白藜芦醇对NSCLC体内肿瘤生长的影响。实验结果显示，白藜芦醇可抑制NSCLC细胞的增殖、侵袭与迁移，并促进细胞凋亡。SIX4与SPHK2在NSCLC组织及细胞中呈高表达状态，且其表达可被白藜芦醇下调。敲低SIX4与SPHK2可抑制NSCLC细胞的生长、侵袭与迁移，而过表达SIX4与SPHK2则可逆转白藜芦醇对NSCLC细胞功能的调控作用。白藜芦醇可通过降低SIX4与SPHK2的表达水平，失活Wnt/β-连环蛋白通路（Wnt/β-catenin pathway）。动物实验结果表明，白藜芦醇可在体内抑制NSCLC的肿瘤生长。综上，白藜芦醇通过下调SIX4与SPHK2的表达，抑制Wnt/β-连环蛋白通路的活性，从而阻滞NSCLC的恶性进程。