Escherichia coli Nissle 1917 distinctively regulates apoptosis and cell cycling of Caco-2 cells
Schirbel A.*1, Sturm A.2
1Charité Campus Virchow Klinikum, Berlin, Germany 2DRK Klinikum Westend, Gastroenterology, Berlin, Germany
Probiotics are widely used in IBD but the mechanisms of action of the probiotic
Caco-2 cells were incubated with different concentrations of EcN conditioned medium (CM) for 24, 48 or 72h. Apoptosis was detected by FACS analysis using Annexin V/PI staining. Possibly important factors such as Fas, FasL or p27 and p53, retinoblastom (Rb) were detected by flow cytometry or immunoblotting. Mitochodrial membrane potential was measured using rhodamin123 fluorescence.
We evaluated whether apoptosis occurred caspase dependent using caspase inhibitors for caspase 3, 8 or 9.
Cell cycling was measured with flow cytometric analysis by DNA and cyclin B1 staining. CFSE staining revealed cell division after incubation with EcN.
Secretion of important pro- and anti-inflammatory cytokines after EcN-treatment were detected by cytometric bead array.
Apoptosis of Caco-2 was slightly induced compared to baseline level upon stimulation with EcN. Interestingly, mitochondrial membrane potential measured by rhodamine 123 uptake was increased upon stimulation with EcN.
While Fas expression was not affected by EcN, FasL was increased. Bid was induced dose dependently by EcN.
Whereas expression of TNF-R1 was not changed upon EcN stimulation, TNF-R2 expression was induced by stimulation with EcN (p>0.05).
The expression of the tumour suppressor proteins p27 and p53 were marginally decreased whereas the expression of Rb protein, another tumour suppressor protein, was increased upon stimulation with EcN.
Inhibition of the central executor caspase 3 and the initiator caspase 9 decreased apoptosis rate induced by EcN indicating activation of the intrinsic pathway.
CFSE labeling revealed an increased proliferation rate in EcN treated Caco-2 cells.
Cell cycling was significantly induced in a dose dependent manner measured by the regulator protein in cell cycle from S to G2/M phase Cyclin B1 and DNA staining.
Finally, CBA analysis demonstrated a distinct regulation of cytokines by EcN with upregulation of IL1β, IL8, IL6, IL17, down regulation of TNFα, IL12p70.