Status of kinases in Epstein-Barr virus and Helicobacter pylori Coinfection in gastric Cancer cells

Abstract

Background Helicobacter pylori (H. pylori) and Epstein - Barr virus (EBV) plays a significant role in aggressive gastric cancer (GC). The investigation of genes associated with these pathogens and host kinases may be essential to understand the early and dynamic progression of GC. Aim The study aimed to demonstrate the coinfection of EBV and H. pylori in the AGS cells through morphological changes, expression of the kinase and the probable apoptotic pathways. Methods Genomic DNA isolation of H. pylori and its characterization from clinical samples were performed. RT-qPCR of kinases was applied to scrutinize the gene expression of kinases in co-infected GC in a direct and indirect (separated through insert size 0.45 μm) H. pylori infection set up. Morphological changes in co-infected GC were quantified by measuring the tapering ends of gastric epithelial cells. Gene expression profiling of apoptotic genes was assessed through RT-qPCR. Results An interleukin-2-inducible T-cell kinase (ITK) showed significant upregulation with indirect H. pylori infection. Moreover, Ephrin type-B receptor six precursors (EPHB6) and Tyrosine-protein kinase Fyn (FYN) showed significant upregulation with direct coinfection. The tapering ends in AGS cells were found to be extended after 12 h. A total of 24 kinase genes were selected, out of which EPHB6, ITK, FYN, and TYK2 showed high expression as early as 12 h. These kinases may lead to rapid morphological changes in co-infected gastric cells. Likewise, apoptotic gene expression such as APAF-1 and Bcl2 family genes such as BAD, BID, BIK, BIM, BAX, AND BAK were significantly down-regulated in co-infected AGS cells. Conclusion All the experiments were performed with novel isolates of H. pylori isolated from central India, for the functional assessment of GC. The effect of coinfection with EBV was more profoundly observed on morphological changes in AGS cells at 12 h as quantified by measuring the tapering of ends. This study also identifies the kinase and apoptotic genes modulated in co-infected cells, through direct and indirect approaches. We report that ITK, EPHB6, TYK2, FYN kinase are enhanced, whereas apoptotic genes such as APAF-1, BIK, FASL, BAX are significantly down-regulated in AGS cells coinfected with EBV and H. pylori.

Helicobacter pyloriand Epstein-Barr virus coinfection stimulates the aggressiveness in gastric cancer through the regulation of gankyrin

“…About one-fifth of total human oncogenesis is associated with infectious agents. Pathogens modulate the cellular milieu by dysregulation the gene expression, fit for the potential carcinogenesis (24, 25). Studies suggested that H. pylori and EBV promote oncogenesis by reframing the expression of cellular genes at the level of transcription and translation in infected cells (2).…”

“…In this study, AGS cell lines were used which mimic the primary gastric epithelial cells except for adherent’s junction. It is well established that H. pylori and EBV infect the AGS cells and NCI-N87 in equal efficiency (2, 25). Thus, all these above reasons elicited us to investigate the synergistic effect of H. pylori exposed AGS cells for the successful infection of EBV and further how the microenvironment of EBV infected AGS cells favour the fortunate infection of H. pylori .…”

“…Various studies reported that H. pylori exposed cellular environmental conditions are suitable for the enhanced EBV infection and vice-versa (2, 25). Moreover, we also observed the enhanced EBV-GFP fluorescence in fluorolog analysis at various time intervals in all the coinfection models except in infection-I (data not shown).…”

“…HEK293T cells containing GFP-EBV (obtained as a gift from the Erle S. Robertson’s laboratory, University of Pennsylvania, USA) were cultured and used for EBV amplification. Cultured the virus and purified as described previously (2, 39, 25)…”

“…H. pylori strain (I10) was a kind gift by Dr. Asish Kumar Mukhopadhyay (National Institute of Cholera and Enteric Diseases, ICMR, Kolkata, India) H. pylori was grown according to the conditions described earlier (25).…”