Involvement of tumor necrosis factor-alpha in the upregulation of CXCR4 expression in gastric cancer induced by Helicobacter pylori.
BACKGROUND
H. pylori, whose infection increases tumor invasiveness and metastasis, is generally labelled as the strongest risk factor for the development of gastric cancer. It appears not to be a coincidence that there is also an overexpression of CXCR4 and an obvious involvement in gastric cancer metastasis. The aim of this study attempts to investigate and further to establish a link between them. With H. pylori being a potent inducer of TNF-alpha, whether TNF-alpha, a tumor promoter, is involved in the induction of CXCR4 expression by H. pylori was also under research in this study.


METHODS
Expression of CXCR4, TNF-alpha, IL-6 and IL-1 beta mRNA was determined by real-time PCR. CXCR4 protein expression was detected by Western blotting. Concentrations of TNF-alpha, IL-6 and IL-1 beta in cell culture supernatants were measured using the Quantikine Elisa kit. To abrogate TNF-alpha expression in HGC27 cells, TNF-alpha RNAi plasmid was used to transfect them.


RESULTS
Levels of CXCR4 and TNF-alpha mRNA were significantly higher in H. pylori-positive gastric cancers (n = 19) compared to H. pylori-negative ones (n = 15). A subsequently Spearman's rank correlation test showed there was a positive correlation between the level of CXCR4 mRNA and that of TNF-alpha in 34 primary gastric cancers. Other results followed: Expression of CXCR4 and TNF-alpha was upregulated in gastric cancer cell MKN45 and HGC27 after infection with H. pylori 26695 (cag PAI+ ) or Tx30a (cag PAI- ); The induction of CXCR4 expression by H. pylori was inhibited significantly by a neutralizing TNF-alpha antibody, infliximab; CXCR4 expression was upregulated in MKN45 cells after treatment with exogenous TNF-alpha or co-culture with macrophage, and was downregulated in HGC27 cells after transfection with TNF-alpha RNAi plasmid. There was a significant increase in the migration of MKN45 cells treated with H. pylori 26695, and a strong inhibition when AMD 3100, a CXCR4 antagonist, or infliximab, was added.


CONCLUSIONS
Our findings demonstrated that H. pylori upregulates CXCR4 expression in gastric cancer through TNF-alpha.
Zhao
Chenghai
C
Department of Pathophysiology, College of Basic Medical Science, China Medical University, Shenyang, China. zhaochenghai2005@yahoo.com.cn
Lu
Xiaomei
X
Bu
Xianmin
X
Zhang
Ning
N
Wang
Wei
W
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D016481:Helicobacter Infections / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D016480:Helicobacter pylori / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000382:microbiology* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D053583:Interleukin-1beta; D015850:Interleukin-6; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019718:Receptors, CXCR4; D014409:Tumor Necrosis Factor-alpha