Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPKMMP-9 pathway
Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPKMMP-9 pathway
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DOI：
10.1093/carcin/bgt332
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发表时间：
2014-02-01
期刊：
影响因子：
4.7
通讯作者：
Medina, Carlos
中科院分区：
文献类型：
--
作者：
Radziwon-Balicka, Aneta;Santos-Martinez, Maria J.;Medina, Carlos
血小板与结肠癌的转移和预后有关，但其潜在的分子机制尚不清楚。我们评价了不同的丝裂原活化蛋白激酶(MAPK)通路在血小板刺激的基质金属蛋白酶-9(MMP9)生成和结肠癌侵袭中的作用。此外，还研究了在血小板肿瘤细胞相互作用过程中释放的蛋白质。为此，用扫描电子显微镜、聚集计量学、流式细胞仪和细胞侵袭室研究了Caco-2和HT29细胞与血小板的相互作用。定量聚合酶链式反应和酶谱检测基质金属蛋白酶-9基因的表达和活性，蛋白质印迹法检测p38MAPK的表达。最后，利用细胞培养中氨基酸稳定同位素标记(SILAC)的蛋白质组学研究了血小板癌细胞相互作用过程中蛋白质的来源。我们发现，血小板促进了两种细胞系中p38MAPK的磷酸化和基质金属蛋白酶-9的上调，从而促进了细胞的侵袭。药物抑制p38MAPK导致基质金属蛋白酶-9和结肠癌细胞侵袭力显著下调。此外，p38MAPK小干扰RNA可阻断血小板刺激的基质金属蛋白酶-9的诱导。SILAC实验表明，血小板反应蛋白1(TSP1)主要由血小板释放，聚集素主要由血小板和癌细胞释放。最后，抑制TSP1和Clusterin可抑制p38MAPK的磷酸化、MMP9活性和血小板刺激的结肠癌侵袭。我们的结果表明，在血小板诱导的结肠癌侵袭过程中，血小板分泌的TSP1和Clusterin促进了基质金属蛋白酶-9的信号调节，这是通过p38MAPK调节通路实现的。这些发现与开发预防和减少结肠腺癌诱导的肿瘤细胞转移的治疗方法有关。
Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelettumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during plateletcancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPKsmall interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.