探討細菌內化誘發上皮細胞自噬作用和自由基生成與腸癌之關係

Abstract

背景：腸道菌叢失調(Microbiota dysbiosis)與結腸直腸癌(colorectal cancer, CRC)的發展息息相關。實驗室從發炎性結腸直腸癌模式小鼠的腸道上皮細胞中分離出多種細菌，並利用流式細胞技術(flow cytometry)和癌球體(spheroid)實驗發現其中大腸桿菌LI60C3具有促進細胞增生的能力，但詳細機制尚未明瞭。目的：探討大腸桿菌LI60C3以及其他內化細菌對腸道上皮細胞癌化之影響。方法：從發炎性腸癌模式小鼠的大腸上皮細胞分離出內化細菌，將細菌與人類大腸癌細胞株Caco-2，HT-29和T84共培養。以流式細胞技術和MTT試驗觀察細菌促進細胞增生之現象。利用MitoSOX和H2DCFDA試劑分別檢測細胞粒線體超氧化物生成以及整體活性氧化物質之變化。以西方墨點法觀察蛋白質表現量，並藉由real-time PCR分析基因轉錄量。以cytochrome c試驗檢測菌株是否能自行產生並釋放活性氧化物質。最後，以ARP探針偵測宿主細胞DNA損傷程度。結果：從發炎性腸癌模式小鼠大腸上皮細胞分離之大腸桿菌LI60C3，能促使人類腸道上皮細胞增生以及活性氧化物質生成增加，此現象可藉由抗氧化藥物抑制。單獨或同時剔除毒性因子fimA、fimH和htrA的細菌則無法誘導上皮細胞生成活性氧化物質或促進細胞增生。上皮細胞受LI60C3侵入之後, 除了產生活性氧化物質, 亦出現細胞自噬反應的P62蛋白降解。 大腸桿菌LI60C3感染會抑制細胞自噬相關基因(IRGM和LC3B)和促進活性氧化物質相關基因(DUOX2, DUOX2, GPX2)之轉錄量。利用轉染技術分別促進或抑制細胞自噬相關基因LC3B表現量，會導致細胞活性氧化物質之減少或增加。此外，腸癌模式小鼠腸道分離之糞腸球菌能主動釋放出活性氧化物質，但與慢速葡萄球菌以及陰溝腸桿菌相同，皆無法促使上皮細胞活性氧化物質生成增加，也不影響細胞增生。最後, 大腸桿菌和糞腸球菌皆未造成細胞DNA損傷。結論：從發炎性腸癌模式小鼠分離出的大腸桿菌LI60C3能抑制宿主細胞的自噬作用，造成活性氧化物質堆積，進而促進細胞增生。而同樣從腸癌模式小鼠分離出的糞腸球菌、慢速葡萄球菌和陰溝腸桿菌則無法促使上皮細胞活性氧化物質生成和細胞增生。

Background: Microbiota dysbiosis is related to colorectal cancer (CRC) development. Previous studies showed that E. coli LI60C3, isolated from mouse models of colitis-associated colorectal cancers, could promote epithelial cell proliferation as evidenced by flow cytometry and spheroid culturing; however, the mechanisms remain elusive. Aim: To Investigate the effect of E.coli LI60C3 and other bacteria isolated from mouse colonocytes on epithelial cell carcinogenesis. Methods: Internalized bacteria were isolated from mouse colonocytes. Caco-2, HT-29 and T84 cells were apically exposed to the isolated bacteria, and cell cycle rates were examined by flow cytometry with Ki67 and propidium iodide staining and cell viability measured using an MTT assay. Mitochondrial superoxide and intracellular reactive oxygen species (ROS) were examined by MitoSOX and H2DCFDA assays, respectively. Protein expression was detected by Western blots, and mRNA levels measured using real-time PCR. The ROS release by bacteria was evaluated by cytochrome c assay. ARP probe was used to detect DNA damage level. Results: The mouse internalized Escherichia coli LI60C3 accelerated cell cycle rates and increased cell viability of Caco-2, HT-29 and T84 cells, which could be inhibited by antioxidants. E. coli LI60C3 with single or triple deletion of fimA, fimH and htrA failed to induce ROS synthesis or cell hyperproliferation. E. coli LI60C3 infection not only triggered ROS generation but also induce autophagic function of P62 protein degradatation in Caco-2 cells. Moreover, LI60C3 infection inhibited the expression of autophagy-related genes (IRGM and LC3B) but increased the expression of ROS-related genes (DUOX2, DUOX2, GPX2). Overexpression and knockdown LC3B by plasmid transfection altered the level of intracellular ROS. Although mouse-isolated Enterococcus faecalis released ROS, they did not increase the level of epithelial ROS nor promoted cell hyperproliferation, which is similar to Staphylococcus lentus and Enterobacter cloacae. No sign of epithelial DNA damage was observed after exposure to E. coli or E. faecalis. Conclusion: E. coli LI60C3 infection caused intracellular ROS accumulation through autophagy inhibition, which resulted in epithelial proliferation. However, other mouse-isolated bacteria such as E. faecalis, S. lentus and E. cloacae did not trigger intracellular ROS synthesis nor promoted epithelial proliferation.