Department of Periodontology, Operative and Preventive Dentistry, University of Bonn, Bonn, Germany.
Department of Prosthodontics, Preclinical Education, and Material Sciences, University of Bonn, Bonn, Germany.
J Cell Physiol. 2019 Dec;234(12):21903-21914. doi: 10.1002/jcp.28754. Epub 2019 Apr 26.
The aim of this study was to get new insights into molecular processes involved in tumor propagation of immortalized oral keratinocytes induced by the keystone pathogen Porphyromonas gingivalis. Cell culture experiments with immortalized OKF6 cells were performed to analyze cellular effects caused by bacterial stimulation focusing on altered gene expression, signaling pathways, proliferation rate, cell viability, migration and invasion behavior, and on the development of antiapoptotic pathways. Gene and protein expression were analyzed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and protein arrays. Trypan blue staining was used to analyze proliferation and viability, transwell assays for cellular migration, Matrigel assays for invasion, and anoikis-assays for evaluating anoikis resistance. Stimulation of OKF6 cells with Porphyromonas gingivalis led to an alteration in the molecular repertoire of proteins which are involved in cell proliferation, epithelial-mesenchymal transition, stem cell formation, migration, invasion, and anoikis resistance. Higher proliferation rates were detected in conjunction with an activation of PI3K/Akt signaling and the mTOR-pathway. Additionally, inhibition of glycogen-synthase-kinase3-β led to stabilization of β-catenin and Snail, which resulted in a switch from predominant E-cadherin to N-cadherin expression and increased expression of the stem cell markers Oct3/4, Sox2, and Nanog. Enhanced biosynthesis and enzyme activity of matrix metalloproteinase-9 was accompanied by elevated invasion behavior. Finally, anoikis resistance was detected in stimulated keratinocytes by decreased apoptosis of nonadherent cells and elevated expression of epidermal growth factor receptor and c-Met. Hence, Porphyromonas gingivalis is able to induce a more aggressive tumor-like phenotype in immortalized oral keratinocytes, thus contributing to enhanced tumor features.
本研究旨在深入了解关键病原体牙龈卟啉单胞菌诱导永生化口腔角质形成细胞肿瘤增殖的分子过程。通过永生化 OKF6 细胞的细胞培养实验，分析细菌刺激引起的细胞效应，重点研究基因表达改变、信号通路、增殖率、细胞活力、迁移和侵袭行为以及抗凋亡途径的发展。使用实时聚合酶链反应、酶联免疫吸附试验、western blot 和蛋白质芯片分析基因和蛋白质表达。使用台盼蓝染色分析增殖和活力，使用 Transwell 测定细胞迁移，使用 Matrigel 测定侵袭，使用 anoikis 测定评估抗 anoikis 能力。牙龈卟啉单胞菌刺激 OKF6 细胞导致参与细胞增殖、上皮-间充质转化、干细胞形成、迁移、侵袭和抗 anoikis 能力的蛋白质分子谱发生改变。检测到更高的增殖率与 PI3K/Akt 信号和 mTOR 途径的激活有关。此外，抑制糖原合成酶激酶-3-β 导致 β-连环蛋白和 Snail 稳定化，从而导致 E-钙黏蛋白表达向 N-钙黏蛋白表达的转变和干细胞标志物 Oct3/4、Sox2 和 Nanog 的表达增加。基质金属蛋白酶-9 的生物合成和酶活性增强伴随着侵袭行为的增强。最后，通过非贴壁细胞凋亡减少和表皮生长因子受体和 c-Met 表达升高，检测到刺激角质形成细胞的抗 anoikis 能力。因此，牙龈卟啉单胞菌能够诱导永生化口腔角质形成细胞产生更具侵袭性的肿瘤样表型，从而增强肿瘤特征。
