Lai Siew-Li, Wong Pooi-Fong, Lim Teck-Kwang, Lin Qingsong, Mustafa Mohd Rais
Centre of Natural Products & Drug Discovery (CENAR), Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Centre of Natural Products & Drug Discovery (CENAR), Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Phytomedicine. 2015 Jan 15;22(1):203-12. doi: 10.1016/j.phymed.2014.11.016. Epub 2014 Dec 3.
Panduratin A (PA), a cyclohexanyl chalcone from Boesenbergia rotunda (L.) Mansf. was shown to possess anti-angiogenic effects in our previous study. In the present study, the molecular targets and anti-angiogenic mechanisms of PA on human umbilical vein endothelial cells (HUVECs) were identified using an iTRAQ-based quantitative proteomics approach. A total of 263 proteins were found to be differentially regulated in response to treatment with PA. Ingenuity Pathway Analysis revealed that cellular growth and proliferation, protein synthesis, RNA post-transcriptional modification, cellular assembly and organization and cell-to-cell signaling and interaction were the most significantly deregulated molecular and cellular functions in PA-treated HUVECs. PA inhibited the expressions of ARPC2 and CTNND1 that are associated with the formation of actin cytoskeleton, focal adhesion and cellular protrusions. In addition, PA down-regulated CD63, GRB-2, ICAM-2 and STAB-1 that are implicated in adhesion, migration and tube formation of endothelial cells. The differential expressions of three targets, namely, ARPC2, CDK4, and GRB-2 were validated by western blot analyses. Furthermore, PA inhibited G1-S progression, and resulted in G0/G1 arrest in HUVECs. The blockage in cell cycle progression was accompanied with the suppression of mTOR signaling. Treatment of HUVECs with PA resulted in decreased phosphorylation of ribosomal S6 and 4EBP1 proteins, the two downstream effectors of mTOR signaling. We further showed that PA is able to inhibit mTOR signaling induced by VEGF, a potent inducer of angiogenesis. Taken together, by integrating quantitative proteomic approach, we identified protein targets in which PA mediates its anti-angiogenic effects. The present study thus provides mechanistic evidence to the previously reported multifaceted anti-angiogenic effects of PA. Our study further identified mTOR signaling as an important target of PA, and therefore highlights the potential of PA for therapeutic intervention against angiogenesis-related pathogenesis, particularly, metastatic malignancy.
莪术二酮（PA）是一种从圆叶莪术（Boesenbergia rotunda (L.) Mansf.）中提取的环己基查尔酮，在我们之前的研究中显示具有抗血管生成作用。在本研究中，使用基于iTRAQ的定量蛋白质组学方法确定了PA对人脐静脉内皮细胞（HUVECs）的分子靶点和抗血管生成机制。共发现263种蛋白质在PA处理后有差异调节。 Ingenuity通路分析显示，细胞生长和增殖、蛋白质合成、RNA转录后修饰、细胞组装和组织以及细胞间信号传导和相互作用是PA处理的HUVECs中最显著失调的分子和细胞功能。PA抑制了与肌动蛋白细胞骨架形成、粘着斑和细胞突起相关的ARPC2和CTNND1的表达。此外，PA下调了与内皮细胞粘附、迁移和管形成有关的CD63、GRB-2、ICAM-2和STAB-1。通过蛋白质印迹分析验证了三个靶点ARPC2、CDK4和GRB-2的差异表达。此外，PA抑制G1-S期进程，并导致HUVECs停滞在G0/G1期。细胞周期进程的阻滞伴随着mTOR信号传导的抑制。用PA处理HUVECs导致核糖体S6和4EBP1蛋白（mTOR信号传导的两个下游效应器）的磷酸化降低。我们进一步表明，PA能够抑制由血管生成的强效诱导剂VEGF诱导的mTOR信号传导。综上所述，通过整合定量蛋白质组学方法，我们确定了PA介导其抗血管生成作用的蛋白质靶点。本研究因此为先前报道的PA的多方面抗血管生成作用提供了机制证据。我们的研究进一步确定mTOR信号传导是PA的一个重要靶点，因此突出了PA在治疗与血管生成相关的发病机制，特别是转移性恶性肿瘤方面的治疗干预潜力。
