XiaoYi Zhang1,2, Chao Li1,2, Yuan Xie3,4, Dingyu Chen1,5, XiaoFeng He1,5, Yan Zhao1,5, LiYa Bao6, Qingrong Wang1,5, JianJiang Zhou7,8,9. 1. Key Laboratory of Endemic and Ethnic Diseases (Guizhou Medical University), Ministry of Education, Guiyang, China. 2. Guiyang Maternity and Child Health Care Hospital, Guiyang, Guizhou Province, China. 3. Key Laboratory of Endemic and Ethnic Diseases (Guizhou Medical University), Ministry of Education, Guiyang, China. xieyuan@gmc.edu.cn. 4. Key Laboratory of Medical Molecular Biology (Guizhou Medical University), No. 9, Beijing Road, Guiyang, 550004, China. xieyuan@gmc.edu.cn. 5. Key Laboratory of Medical Molecular Biology (Guizhou Medical University), No. 9, Beijing Road, Guiyang, 550004, China. 6. Affiliated Hospital, Guiyang Medical University, No. 9, Beijing Road, Guiyang, 550004, China. 7. Key Laboratory of Endemic and Ethnic Diseases (Guizhou Medical University), Ministry of Education, Guiyang, China. jianjiangzhou@sina.cn. 8. Key Laboratory of Medical Molecular Biology (Guizhou Medical University), No. 9, Beijing Road, Guiyang, 550004, China. jianjiangzhou@sina.cn. 9. Affiliated Hospital, Guiyang Medical University, No. 9, Beijing Road, Guiyang, 550004, China. jianjiangzhou@sina.cn.
Abstract
OBJECTIVE: The CagA (cytotoxin-related gene A, CagA) protein is an important factor for the pathogenicity of Helicobacter pylori (H. pylori). Although H. pylori has previously been shown to activate the NLRP3 inflammasome, it remains unclear what role CagA plays in this process. In the current study, we aimed to investigate the effect of CagA on NLRP3 activation and how it is linked to gastric cancer cell migration and invasion. METHODS: CagA positive H. pylori strain (Hp/CagA+) and CagA gene knockout mutant (Hp/ΔCagA) infected and the pcDNA3.1/CagA plasmid transfected gastric epithelial cell lines, respectively. The morphological alterations of cells under a microscope; the NLRP3 inflammasome-related markers: NLRP3, caspase-1, and ASC protein levels were detected by Western blot, IL-1β and IL-18 levels were determined by ELISA; cell migration and invasion were determined by transwell assay; and the pyroptosis levels and intracellular ROS were determined by flow cytometry analysis. Then, pretreated with 5 mM NAC for 2 h and subsequently transfected with the pcDNA3.1/CagA plasmid for 48 h, the effects of NAC pretreatment on CagA-induced NLRP3 inflammasome-related markers expression and cell pyroptosis were examined, finally assessed the effect of CagA on migration and invasion in NLRP3-silenced cells. RESULTS: We found that Hp/CagA+ strain infection and pcDNA3.1/CagA vector transfection result in NLRP3 inflammasome activation, generation of intracellular ROS, and increased invasion and migration of gastric cancer cells. Moreover, we found that ROS inhibition via NAC effectively blocks NLRP3 activation and pyroptosis. Silencing of NLRP3 reduces the effects of CagA on gastric cancer cell migration and invasion. CONCLUSION: Our study shows that CagA can promote the invasion and migration of gastric cancer cells by activating NLRP3 inflammasome pathway. These findings provide novel insights into the mechanism of gastric cancer induction by H. pylori.
OBJECTIVE: The CagA (cytotoxin-related gene A, CagA) protein is an important factor for the pathogenicity of Helicobacter pylori (H. pylori). Although H. pylori has previously been shown to activate the NLRP3 inflammasome, it remains unclear what role CagA plays in this process. In the current study, we aimed to investigate the effect of CagA on NLRP3 activation and how it is linked to gastric cancer cell migration and invasion. METHODS: CagA positive H. pylori strain (Hp/CagA+) and CagA gene knockout mutant (Hp/ΔCagA) infected and the pcDNA3.1/CagA plasmid transfected gastric epithelial cell lines, respectively. The morphological alterations of cells under a microscope; the NLRP3 inflammasome-related markers: NLRP3, caspase-1, and ASC protein levels were detected by Western blot, IL-1β and IL-18 levels were determined by ELISA; cell migration and invasion were determined by transwell assay; and the pyroptosis levels and intracellular ROS were determined by flow cytometry analysis. Then, pretreated with 5 mM NAC for 2 h and subsequently transfected with the pcDNA3.1/CagA plasmid for 48 h, the effects of NAC pretreatment on CagA-induced NLRP3 inflammasome-related markers expression and cell pyroptosis were examined, finally assessed the effect of CagA on migration and invasion in NLRP3-silenced cells. RESULTS: We found that Hp/CagA+ strain infection and pcDNA3.1/CagA vector transfection result in NLRP3 inflammasome activation, generation of intracellular ROS, and increased invasion and migration of gastric cancer cells. Moreover, we found that ROS inhibition via NAC effectively blocks NLRP3 activation and pyroptosis. Silencing of NLRP3 reduces the effects of CagA on gastric cancer cell migration and invasion. CONCLUSION: Our study shows that CagA can promote the invasion and migration of gastric cancer cells by activating NLRP3 inflammasome pathway. These findings provide novel insights into the mechanism of gastric cancer induction by H. pylori.
Authors: Haitao Gu; Wensheng Deng; Yi Zhang; Yu Chang; Vishal G Shelat; Kunihiro Tsuchida; Leonardo S Lino-Silva; Zhaowen Wang Journal: Transl Lung Cancer Res Date: 2022-05