Combination of Solamargine and Metformin Strengthens IGFBP1 Gene Expression Through Inactivation of Stat3 and Reciprocal Interaction Between FOXO3a and SP1
Combination of Solamargine and Metformin Strengthens IGFBP1 Gene Expression Through Inactivation of Stat3 and Reciprocal Interaction Between FOXO3a and SP1
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Solamargine 和二甲双胍的组合通过 Stat3 失活以及 FOXO3a 和 SP1 之间的相互作用增强 IGFBP1 基因表达
DOI：
10.1159/000484383
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发表时间：
2017
影响因子：
--
通讯作者：
Hann Swei Sunny
中科院分区：
文献类型：
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作者：
Tang Qing;Zheng Fang;Wu JingJing;QianXiao;Li Liuning;Hann Swei Sunny
背景/目的：Solamargine是一种来自传统植物的天然光化学成分，已被证明具有抗癌特性。我们以前发现，solamargine抑制非小细胞肺癌（NSCLC）细胞的生长通过抑制前列腺素E2（PGE 2）受体EP 4基因和调节下游信号通路。然而，这背后的详细机制，特别是与二甲双胍（一种已知的AMPK激活剂）联合使用，仍有待确定。研究方法：分别使用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物（MTT）和比色5-溴-2-脱氧尿苷（BrdU）ELISA方法测量细胞活力。Western blot和免疫组化检测信号转导和转录激活因子3（Stat 3）、SP1、叉头框O3 a（FOXO 3a）和胰岛素样生长因子（IGF）-IGF结合蛋白1（IGFBP 1）的磷酸化和蛋白表达。采用定量真实的时间PCR（qRT-PCR）检测IGFBP 1 mRNA的表达。通过siRNA程序检测FOXO 3a和IGFBP 1的沉默。通过瞬时转染试验进行SP1、FOXO 3a和IGFBP 1的外源表达。使用Secrete-PairTM Dual Luminescence Assay Kit测试IGFBP 1的启动子活性。使用异种移植肿瘤模型进一步测试澳洲茄边碱与二甲双胍组合的体内作用。结果：我们进一步证实了澳洲茄边碱在其他非小细胞肺癌细胞系中抑制生长并诱导细胞周期阻滞。通过基于机制的方法，我们发现，solamargine降低Stat 3的磷酸化;此外，solamargine诱导FOXO 3a，而降低SP1蛋白水平;所有这些都在Stat 3基因过表达的细胞中被废除。有趣的是，FOXO 3a和SP1之间存在相互作用。此外，solamargine增加IGFBP 1的mRNA，蛋白质表达和启动子活性，这在SP1过表达或FOXO 3a基因沉默的细胞中没有观察到。最后，通过siRNA阻断IGFBP 1表达阻断了solamargine对细胞生长抑制的作用。更重要的是，solamargine和二甲双胍的组合具有协同作用。在体内也观察到类似的结果。结论：我们的研究结果表明，茄边碱增加IGFBP 1基因的表达，通过失活的Stat 3，随后的调节和相互作用的FOXO 3a和SP1在体外和体内。这最终导致抑制人肺癌细胞的生长。此外，这是在该过程中solamargine与二甲双胍组合的协同作用。本研究揭示了茄边碱联合二甲双胍抗肺癌作用的新机制，并提出了一种潜在的新的肺癌相关治疗方法。
Background/Aims: Solamargine, one natural photochemical component from traditional plants, has been shown to have anti-cancers properties. We previously showed that solamargine inhibited the growth of non-small-cell lung cancer (NSCLC) cells through suppression of prostaglandin E2 (PGE2) receptor EP4 gene and regulation of downstream signaling pathways. However, the detailed mechanism underlying this, especially in combination of metformin, a known AMPK activator, still remained to be determined. Methods: Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colorimetric 5-bromo-2-deoxyuridine (BrdU) ELISA methods, respectively. Western blot analysis and immunohistochemistry were performed to examine the phosphorylation and protein expressions of signal transducer and activator of transcription 3 (Stat3), SP1, forkhead box O3a (FOXO3a), and insulin-like growth factor (IGF)-IGF binding protein 1 (IGFBP1). The expression of IGFBP1 mRNA was measured by quantitative real time PCR (qRT-PCR). Silencing of FOXO3a and IGFBP1 were examined by siRNA procedures. Exogenously expression of SP1, FOXO3a, and IGFBP1 were carried out by transient transfection assays. The promoter activity of IGFBP1 was tested using Secrete-PairTM Dual Luminescence Assay Kit. A xenografted tumor model was used to further test the effect of solamargine in combining with metformin in vivo. Results: We further demonstrated that solamargine inhibited growth and induced cell cycle arrest in other NSCLC cell lines. Through mechanism-based approaches, we showed that solamargine decreased the phosphorylation of Stat3; In addition, solamargine induced FOXO3a, whereas reduced SP1 protein levels; all of which were abrogated in cells with overexpressed Stat3 gene. Interestingly, there is interaction between FOXO3a and SP1. Moreover, solamargine increased mRNA, protein expression and promoter activity of IGFBP1, which was not observed in cells with overexpressed SP1 or with silenced FOXO3a genes. Finally, ablation of IGFBP1 expression by siRNA blocked the effect of solamargine on cell growth inhibition. More importantly, there was a synergy of combination of solamargine and metformin. Similar findings were also observed in vivo. Conclusion: Our results show that solamargine increases IGFBP1 gene expression through inactivation of Stat3, followed by regulation and reciprocal interaction of FOXO3a and SP1 in vitro and in vivo. This ultimately leads to suppression of human lung cancer cell growth. Moreover, this is a synergy of solamargine in combination with metformin in this process. This study unravels a novel mechanism underlying the anti-lung cancer effects of solamargine in combination of metformin, and suggests a potential new lung cancer associated therapy.