Abstract
BackgroundAchlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells.MethodsTo separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing.ResultsInfection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate.ConclusionsInfection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene expression.Transcript profilingArray Express accession number E-MEXP-3496.
Highlights
Gastric cancer is among the ten most common cancers, and with a global annual death rate of approximately 700.000, it is the second most common cause of cancer related mortality [1]
Enterococci in general are thought to be of low pathogenicity, sustained overgrowth in achlorhydric mice is associated with an inflammatory response and these mice develop gastric cancer [20,32]
Using probes monitoring intracellular reactive oxygen species (ROS) production, we found that 30 minutes of infection stimulated a significant increase in intracellular ROS formation (Figure 1A)
Summary
Gastric cancer is among the ten most common cancers, and with a global annual death rate of approximately 700.000, it is the second most common cause of cancer related mortality [1]. Chronic inflammation favors an overproduction of DNA damaging reactive oxygen species (ROS), whose production can be incidental to oxidative phosphorylation (oxphos) reactions in the mitochondria (oxphosdependant) or produced from outside the mitochondria most commonly by nicotinamid adenine dinucleotide phosphate (NADPH) oxidases (oxphos-independent) (for review see [7,8,9]). Chronic production of ROS cause DNA damage, allowing the accumulation of mutations which in turn can activate oncogenes and/or inactivate tumor suppressor genes thereby increasing the risk of cancer development [3]. Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells
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