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. 2013 Jul 23;8(7):e69102.
doi: 10.1371/journal.pone.0069102. Print 2013.

Lactobacillus gasseri SF1183 affects intestinal epithelial cell survival and growth

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Lactobacillus gasseri SF1183 affects intestinal epithelial cell survival and growth

Blanda Di Luccia et al. PLoS One. .

Retraction in

Abstract

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host's intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HCT116 cell response to L. gasseri CM with or without TNFα treatment.
Western blot with anti-PARP-1 antibody of whole cell extracts from HCT116 cells incubated in (A) complete cell culture medium supplemented or not with TNFα (1 nM) for 2, 8 or 24 hours; (B) complete cell culture medium supplemented or not with CM (20%v/v) for 16 hours before treatment with 1 nM TNFα for 8 hours; (C) complete cell culture medium supplemented or not with TNFα (1 nM) for 8 hours and MDM (20%v/v) or MDM+lactic acid pH4 (20%v/v). After the treatments cells were collected, lysed and protein concentration determined. Equal amount of cell lysates were fractionated on SDS-PAGE and analyzed by western blotting with antibodies against PARP-1. Actin was used as a loading control.
Figure 2
Figure 2. L. gasseri secretes thermostable, bioactive molecule(s) of proteinaceous nature during the stationary phase of growth.
HCT116 cells were incubated in complete cell culture medium supplemented or not with TNFα (1 nM) for 8 hours and with A) CM fractionated with a cut-off of 3 kDa, or B) CM treated with different enzymes [Trypsin, Proteinase K, DNAse I, RNAse A], or C) CM treated at 100°C for 30 minutes, or D) CM of cultures at the indicated phases of growth. After the treatments, cells were collected, lysed and total cell extracts were analyzed by western blotting with antibodies against PARP-1. Actin was used as a loading control. PARP-1 band intensity was evaluated by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition. Values expressed as ratio with the corresponding actin values and normalised to the reference point (PARP-1 cleavage in medium). Percentage of increase (+) or decrease (–) with respect to the intensity of the reference point are indicated.
Figure 3
Figure 3. The CM of L. gasseri SF1183 affects HCT116 cell number but not cell viability.
Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v). After 24 hours (A) controls (NT) and CM-treated (CM) cells were collected and counted in a Burker chamber; or (B) incubated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium as a substrate and the absorbance of converted formazan measured at 490 nm.
Figure 4
Figure 4. The CM of L. gasseri SF1183 affects cell proliferation of HCT116 cells.
Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v) and/or TNFα (1 nM). After the treatments, cells were collected and treated for flow-cytometric analysis (A and Fig. S3 in File S1) or western blot (B) with the indicated antibodies.
Figure 5
Figure 5. The anti-apoptotic effect of L. gasseri is not specific for TNFα-induced apoptosis.
Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v) and/or cisplatin (30 µM). After the treatments, cells were collected and treated for flow-cytometric analysis (A and Fig. S4 in File S1) or western blot (B) with the indicated antibodies.

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This work was supported by the FARO project (terza tornata) of the University of Naples and Istituto San Paolo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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