Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
PLoS One. 2010 Sep 22;5(9):e12895. doi: 10.1371/journal.pone.0012895.
Resistance to anoikis, apoptosis triggered by a loss of cellular adhesion to the underlying extracellular matrix, is a hallmark of metastatic cancer. Previously we have shown re-establishment of CXCL12 expression in colorectal carcinoma cells inhibits metastasis by enhancing anoikis sensitivity. The objective of these studies was to define the signaling mechanisms regulating CXCL12-mediated anoikis.
METHODOLOGY/PRINCIPAL FINDINGS: Adhesion, examined by crystal violet staining, immunofluorescence microscopy, and immunoblot analysis indicated decreased focal adhesion signaling corresponding with loss of adhesion in cells constitutively simulated by CXCL12. Loss of adhesion was inhibited by pertussis toxin treatment, indicating CXCL12 regulating anoikis through G(αi)-protein coupled receptors. Non-adherent HCT116 and HT29 colorectal carcinoma cells expressing CXCL12 exhibited enhanced anoikis sensitivity by propidium iodide staining, caspase activity assays, and immunoblot compared to GFP control cells. CXCL12 producing carcinomas cultured on poly-HEMA displayed heightened Bim and loss of Mcl-1 and Bcl-2 preceding cytochrome c release, and caspase-9 activation. RNAi knockdown of Bim reversed anoikis sensitivity of CXCL12-expressing cells and fostered increased soft-agar foci formation and hepatic tumors in an orthotopic mouse model of metastasis.
CONCLUSIONS/SIGNIFICANCE: These data indicate CXCL12 provides a barrier to metastasis by increasing anoikis via activation of a Bim-mediated intrinsic apoptotic pathway. These results underscore the importance of retaining CXCL12 expression to sensitize colorectal carcinomas to anoikis and minimize tumor progression.
抗失巢凋亡，即细胞失去与细胞外基质黏附而触发的细胞凋亡，是转移性癌症的一个特征。我们之前已经表明，在结直肠癌细胞中重新建立 CXCL12 的表达通过增强失巢凋亡敏感性来抑制转移。这些研究的目的是确定调节 CXCL12 介导的失巢凋亡的信号机制。
方法/主要发现：通过结晶紫染色、免疫荧光显微镜和免疫印迹分析检查黏附，表明 CXCL12 持续模拟的细胞黏附丧失与焦点黏附信号减少相对应。百日咳毒素处理抑制黏附丧失，表明 CXCL12 通过 G（αi）-蛋白偶联受体调节失巢凋亡。表达 CXCL12 的非黏附性 HCT116 和 HT29 结直肠癌细胞通过碘化丙啶染色、半胱天冬酶活性测定和免疫印迹与 GFP 对照细胞相比，表现出增强的失巢凋亡敏感性。在聚-HEMA 上培养的 CXCL12 产生的癌显示出更高的 Bim 和 Mcl-1 和 Bcl-2 的丧失，随后是细胞色素 c 释放和 caspase-9 激活。Bim 的 RNAi 敲低逆转了 CXCL12 表达细胞的失巢凋亡敏感性，并促进了 CXCL12 表达细胞在原位小鼠转移模型中形成更多的软琼脂焦点和肝肿瘤。
结论/意义：这些数据表明，CXCL12 通过激活 Bim 介导的内在凋亡途径增加失巢凋亡，为转移提供了一个屏障。这些结果强调了保留 CXCL12 表达以增强结直肠癌细胞对失巢凋亡的敏感性并最小化肿瘤进展的重要性。
