Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl Alcohol Extract Inhibits AGS Cell Helicobacter pylori_CagA+VacA+ Promoted Invasiveness by Down-Regulating Caudal Type Homeobox Transcription Factor and Claudin-2 Expression

Objective: To investigate the effects and possible mechanisms of action of Curcuma wenyujin Y. H. Chen et C. Ling n-Butyl alcohol extract (CWNAE) on repression of human gastric cancer (GC) AGS cell invasion induced by co-culturing with Helicobacter pylori (HP).

Methods: AGS cells were cultured with HP of positive or negative cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) expression (CagA+/- or VacA+/-) and divided into 5 group. Group A was cultured without HP as a control, Group B with HP_CagA+VacA+, Group C with HP_CagA-VacA-, Group D with HP_CagA+VacA+ and CWNAE, and Group E with HP_CagA-VacA- and CWNAE. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and tumor invasion assays, examinations of morphology and ultramicroscopic structures, quantitative real-time polymerase chain reaction and Western blots were performed to measure the effects and uncover the mechanisms behind these effects of HP_CagA+VacA+ and CWNAE on the epithelial-mesenchymal transition (EMT) of AGS cells.

Results: The 10% inhibitory concentration of CWNAE against AGS cells after a 48 h incubation was 19.73±1.30 μg/mL. More AGS cells were elongated after co-culturing with HP_CagA+VacA+ than after culturing with HP_CagA-VacA-. In tumor invasion assays, HP_CagA+VacA+ significantly enhanced the invasiveness of AGS cells compared to the other experimental groups (all P value <0.05), and this effect was inhibited by CWNAE. Treatment with CWNAE normalized tight junctions and reduced the number of pseudopodia of AGS cells co-cultured with HP_CagA+VacA+. HP_CagA+VacA+ up-regulated zincfinger ebox binding homeobox 1 (ZEB1) in AGS cells after co-culturing for 24 h. Expression of caudal type homeobox transcription factor (CDX-2) and claudin-2 was significantly increased by HP_CagA+VacA+ (P<0.05), but not by HP_CagA-VacA-.

Conclusion: HP_CagA+VacA+ promoted the invasiveness of AGS cells through up-regulation of ZEB1 transcription and claudin-2 and CDX-2 expression. CWNAE inhibited these effects of HP_CagA+VacA+ on AGS cells by down-regulating ZEB1 transcription, and CDX-2 and claudin-2 expression.