Table_1_Periodontal Pathogens Promote Oral Squamous Cell Carcinoma by Regulating ATR and NLRP3 Inflammasome.doc

Periodontitis is closely related to oral cancer, but the molecular mechanism of periodontal pathogens involved in the occurrence and development of oral cancer is still inconclusive. Here, we demonstrate that, in vitro, the cell proliferation ability and S phase cells of the periodontitis group (colonized by Porphyromonas gingivalis and Fusobacterium nucleatum, P+) significantly increased, but the G1 cells were obviously reduced. The animal models with an in situ oral squamous cell carcinoma (OSCC) and periodontitis-associated bacteria treatment were constructed, and micro-CT showed that the alveolar bone resorption of mice in the P+ group (75.3 ± 4.0 μm) increased by about 53% compared with that in the control group (48.8 ± 1.3 μm). The tumor mass and tumor growth rate in the P+ group were all higher than those in the blank control group. Hematoxylin–eosin (H&E) staining of isolated tumor tissues showed that large-scale flaky necrosis was found in the tumor tissue of the P+ group, with lots of damaged vascular profile and cell debris. Immunohistochemistry (IHC) of isolated tumor tissues showed that the expression of Ki67 and the positive rate of cyclin D1 were significantly higher in tumor tissues of the P+ group. The qRT-PCR results of the expression of inflammatory cytokines in oral cancer showed that periodontitis-associated bacteria significantly upregulated interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-18, apoptosis-associated speck-like protein containing a CARD (ASC) (up to six times), and caspase-1 (up to four times), but it downregulated nuclear factor (NF)-κB, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and IL-1β (less than 0.5 times). In addition, the volume of spleen tissue and the number of CD4+ T cells, CD8+ T cells, and CD206+ macrophages in the P+ group increased significantly. IHC and Western blotting in tumor tissues showed that expression levels of γ-H2AX, p-ATR, RPA32, CHK1, and RAD51 were upregulated, and the phosphorylation level of CHK1 (p-chk1) was downregulated. Together, we identify that the periodontitis-related bacteria could promote tumor growth and proliferation, initiate the overexpressed NLRP3, and activate upstream signal molecules of ATR-CHK1. It is expected to develop a new molecular mechanism between periodontitis-related bacteria and OSCC.

牙周炎与口腔癌密切相关，但牙周病原体参与口腔癌发生发展的分子机制仍不明确。本研究证实，体外实验中，定植牙龈卟啉单胞菌（Porphyromonas gingivalis）与具核梭杆菌（Fusobacterium nucleatum）的牙周炎模型组（记为P+组）的细胞增殖能力及S期细胞占比显著升高，而G1期细胞占比则明显降低。本研究构建了携带原位口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）并定植牙周炎相关细菌的动物模型；显微计算机断层扫描（micro-CT）结果显示，P+组小鼠的牙槽骨吸收量为(75.3±4.0)μm，较对照组的(48.8±1.3)μm升高约53%。P+组的肿瘤质量与肿瘤生长速率均显著高于空白对照组。对分离获取的肿瘤组织进行苏木精-伊红（H&E）染色后发现，P+组肿瘤组织中可见大范围片状坏死，伴随大量受损血管轮廓与细胞碎片。对分离肿瘤组织开展免疫组织化学（IHC）检测结果显示，P+组肿瘤组织中Ki67的表达水平以及细胞周期蛋白D1（cyclin D1）的阳性率均显著升高。口腔癌炎症细胞因子表达的实时定量聚合酶链反应（qRT-PCR）检测结果显示，牙周炎相关细菌可显著上调白细胞介素（IL）-6、肿瘤坏死因子（TNF）-α、IL-18、含半胱天冬酶募集结构域的凋亡相关斑点样蛋白（ASC）（表达量上调至对照组的6倍）以及半胱天冬氨酸蛋白酶-1（caspase-1，表达量上调至对照组的4倍）的表达水平，但可下调核因子κB（NF-κB）、核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）以及IL-1β的表达（表达量不足对照组的0.5倍）。此外，P+组小鼠的脾脏组织体积以及CD4+ T细胞、CD8+ T细胞与CD206+巨噬细胞的数量均显著升高。对肿瘤组织开展免疫组织化学与蛋白质印迹（Western blotting）检测结果显示，γ-H2AX、p-ATR、RPA32、CHK1及RAD51的表达水平均上调，而检查点激酶1（CHK1）的磷酸化水平（p-chk1）则下调。综上，本研究证实牙周炎相关细菌可促进肿瘤生长与增殖，引发NLRP3过度表达，并激活ATR-CHK1通路的上游信号分子；该研究有望阐明牙周炎相关细菌与口腔鳞状细胞癌之间的新型分子机制。