大腸桿菌內化加速腸道上皮細胞分裂週期之機制

Abstract

背景：結腸直腸癌 (colorectal cancer, CRC) 的發生率在台灣已經連續十年蟬聯十大癌症的第一位。先前研究中發現，含有毒性因子與內源性質體之大腸桿菌 (Escherichia coli) LI60C3具有促進小鼠結直腸癌腫瘤生長之能力，而LI60C3內化至人類大腸癌細胞株Caco-2後會導致細胞週期加速。然而關於細菌如何致癌或加速細胞週期之詳細機制目前尚不清楚。目的：探討腸癌模式小鼠結腸上皮細胞分離之大腸桿菌加速細胞週期之機制。方法：利用西方墨點法及免疫螢光染色分析細胞在內吞細菌後E-cadherin/β-catenin與PI3K/Akt訊息路徑之變化。利用MitoSox assay檢測細菌及其毒性因子對細胞內活性氧化物質 (Reactive oxygen species, ROS) 之影響。利用PCR方法觀察細胞在內吞細菌後細胞自噬之情形。利用流式細胞技術觀察細菌及其毒性因子與內源性質體造成細胞週期之變化。利用aldehyde reactive probe (ARP) 檢測細胞在內吞細菌後DNA損傷之情形。結果：將LI60C3細菌與Caco-2細胞共培養後會促使細胞E-cadherin的切割並造成β-catenin進入細胞核。此外，細菌感染細胞後會增加Akt蛋白表現量，並且在給予PI3K及Akt之抑制劑後會抑制細菌造成的細胞週期加速之現象。而細菌的感染也會透過HtrA之絲氨酸蛋白酶活性促使細胞產生ROS進而加速細胞週期。然而，細菌及其毒性因子HtrA並不會造成DNA損傷。細菌與細胞共培養後會抑制細胞自噬相關基因irgm及lc3B之轉錄，進而降低細胞自噬的功能；以siRNA降低lc3B表現量會增加ROS含量及加速細胞週期，而以質體轉染使細胞過度表達lc3B表現量則會降低ROS含量及減緩細胞週期。細菌之內源性質體及該質體上之基因mbeA會促使細胞產生ROS，但不會加速細胞週期或造成DNA損傷。結論：大腸桿菌LI60C3會透過活化E-cadherin/β-catenin與PI3K/Akt訊息路徑促進Caco-2細胞週期之加速。LI60C3也會藉由抑制細胞自噬使ROS累積，導致細胞週期加速，可能進而促進癌化。

Background: The incidence of colorectal cancer (CRC) had been the highest among various types of cancers for 10 years in Taiwan. Previous studies showed that Escherichia coli strain LI60C3 with virulence factors and endogenous plasmid had the ability to promote tumor growth in AOM/DSS mice, and to induce Caco-2 cell cycle acceleration. However, the detailed mechanism of bacteria-elicited epithelial cell cycle acceleration and tumor formation remains unknown. Aim：To investigate the mechanisms through which internalized E. coli strain LI60C3 promote epithelial cell cycle acceleration. Methods: Western blotting and immunofluorescence were used to analyze the changes of E-cadherin/β-catenin and PI3K/Akt signaling pathways in Caco-2 cells after exposure to E. coli. The effect of E. coli and its virulence factors on reactive oxygen species (ROS) in Caco-2 cells were examined by MitoSox assay. PCR was used to determine the autophagy in Caco-2 cells after E. coli infection. The effect of E. coli and its virulence factors on cell cycle in Caco-2 cells were examined by flow cytometry. Aldehyde reactive probe was used to detect the DNA damage of Caco-2 cells after endocytosis of E. coli. Results: Increased expression of cleaved E-cadherin and nuclear translocation of β-catenin were observed in Caco-2 cells after exposure to E. coli strain LI60C3. In addition, coculture of E. coli and Caco-2 cells also increased the expression of Akt; cell cycle acceleration of Caco-2 cells induced by E. coli was inhibited by PI3k/Akt inhibitor. E. coli infection increased ROS production in Caco-2 cells and resulted in cell cycle acceleration; importantly, the serine protease activity of the virulence factor HtrA of E. coli was necessary for ROS generation. However, E. coli and its virulence factor HtrA had no effect on DNA damage. The mRNA expression of autophagy-related genes including irgm and lc3b as well as autophagic activity were decreased after E. coli infection; lc3b knockdown by gene silencing increased ROS levels and accelerated cell cycle, whereas lc3b overexpression by plasmid transfection decreased ROS levels and decelerated cell cycle in Caco-2 cells after exposure to E. coli. The endogenous plasmid of E. coli and its gene mbeA promoted ROS generation, but did not accelerate cell cycle nor enhance DNA damage. Conclusion: The mouse colonocyte internalized E. coli strain LI60C3 induced Caco-2 cell cycle acceleration via E-cadherin/β-catenin and PI3K/Akt signaling. Furthermore, LI60C3 also inhibited autophagy and caused ROS accumulation, which resulted in epithelial cell cycle acceleration and may be involved in tumorigenesis.