Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line.
Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line.
复制标题
DOI：
10.1186/1472-6882-13-271
复制
发表时间：
2013-10-20
影响因子：
--
通讯作者：
Sekaran SD
中科院分区：
文献类型：
--
作者：
Lee SH;Jaganath IB;Manikam R;Sekaran SD
肺癌是世界上发病率和死亡率最高的恶性肿瘤之一，每年新发病例160万例，死亡140万例。由于多药耐药性的有害发展，预后仍然很差，导致不到15%的肺癌患者达到五年生存期。我们以前已经表明，叶下珠诱导细胞凋亡与其抗肿瘤作用。在目前的研究中，我们的目的是确定叶下珠所利用的信号通路发挥其抗转移活性。应用肿瘤10通路报告基因芯片技术筛选叶下珠对肺癌细胞株A549的影响通路，以发挥其抗肿瘤转移的作用。然后用蛋白质印迹法、细胞周期分析、酶谱技术和基于细胞的人总iNOS ELISA测定来确认来自该阵列的结果。双向凝胶电泳分析叶下珠处理后A549细胞蛋白质的差异表达。观察到叶下珠通过抑制Raf蛋白而抑制ERK 1/2途径引起抗转移活性。抑制该途径导致MMP 2、MMP 7和MMP 9表达的抑制，从而阻止A549转移。叶下珠还通过抑制HIF-1α导致VEGF和iNOS表达减少来抑制缺氧通路。蛋白质组学分析揭示了参与转移过程的叶下珠下调的一些蛋白质，包括侵袭和移动蛋白（细胞骨架蛋白），转录蛋白（增殖细胞核抗原，锌指蛋白），抗凋亡蛋白（Bcl 2）和各种糖酵解酶。在测试的四种叶下珠属物种中，P. urinaria显示出最大的抗转移活性。叶下珠通过抑制ERK 1/2和缺氧途径抑制A549转移，这些途径导致抑制A549侵袭和迁移的各种关键蛋白。
Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities. Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus. Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity. Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549 invasion and migration.