Abstract 3661: The biological activity of H.pylori SlyD in vitro.
Authors: Kang Dan , Gong Yuehua , Yuan Yuan 
Published: August 2014

Abstract
Aim: H. pylori is responsible for about 75% of all noncardia gastric cancers and 63.4% of all stomach cancers worldwide. Despite the close association of H. pylori infection with non-cardia gastric cancer, most infected persons do not develop the disease. The clinical outcome of H. pylori infection is determined by multiple factors, including host genetic predisposition, bacterial strain difference and environmental factors such as dietary salt intake. In the previous study, using a well-established suppression subtractive hybridization (SSH) method, we have identified a gastric cancer-specific H. pylori gene, named slyD gene, which has been characterized as a virulent factor of Legionella pneumophila and Trypanosoma cruzi. In this study, by constructing the slyD prokaryotic expression vector and purifying SlyD recombinant protein, we aimed to investigate the biological activity change of AGS cell treated with rSlyD.
Methods: Helicobacter pylori (H.pylori)slyD prokaryotic expression vector was carried out in Escherichia coli (E. coli), and rSlyD was purified by immobilized metal affinity chromatography. The proliferation, apoptosis, invasion, transformation effects of rSlyD on AGS cells was detected by CCK-8, cell cycle, caspase-3 activity assay, matrigel invasion assay and double-deck soft agar colony forming efficiency. In addition, the expressions of PCNA, KI-67, caspase3 and MMP-9 were detected by western blot and immunoﬂuorescence assay, respectively.
Results: The CCK-8 assay revealed that cell proliferation was increased in a time and dose dependent manner in AGS+rSlyD group compared with the controls (P&lt;0.05). There are significant difference of PCNA and Ki67 expressions between AGS cell line with or without rSlyD (P&lt;0.05). Soft agar colony formation assay revealed the colony number (foci &gt;100 um) of AGS+rSlyD group was 26.3 ±7.09, whereas controls (AGS or AGS+PBS) were 5.6±1.15 and 5.0±1.0 respectively (P&lt; 0.01). Colorimetric enzyme assay revealed the activity of caspase-3 was decreased after treatment with rSlyD to 31.45±0.49, whereas controls (AGS or AGS+PBS) were 55.5±0.43 and 55.1±0.25 respectively (P&lt; 0.001). Similar caspase-3 results also were confirmed by Western blot. Transwell chambers assay revealed the number of invasive cells in AGS+ rSlyD group (196.66±40.41) was higher than that of AGS or AGS+PBS group (85±22.9 and 81.66±15.27) respectively (P&lt;0.001). The MMP-9 expression in AGS+rSlyD group was also higher than that of AGS or AGS+PBS group.
Conclusion: These results suggest that the H. pylori rSlyD may play an important role in disturbing cell proliferation, apoptosis, and enhancing cell transformation and invasion in the AGS cell line. SlyD might contribute to gastric pathogenicity in H.pylori associated diseases.
Citation Format: Dan Kang, Yuehua Gong, Yuan Yuan. The biological activity of H.pylori SlyD in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3661. doi:10.1158/1538-7445.AM2013-3661