Gut microbiota-derived propionate reduces cancer cell proliferation in the liver

Abstract

Background: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut.

Methods: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro.

Results: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines.

Conclusion: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Figure 1

Inulin-type fructans treatment decreases the hepatic infiltration of the BaF3 cells and systemic inflammation in mice. (A) Liver and spleen weights of the control mice (CT), mice transplanted with BaF3 cells (BaF3) and mice transplanted with BaF3 cells and fed ITF (BaF3-ITF). (B) Bcr-Abl mRNA levels, ND: not detected. (C, D) Plasma ℒ-lactate and lactate dehydrogenase (LDH) activity. (E–L) Plasma levels of IL-4, IL-6, IL-10, G-CSF, IL-8, MCP-1, RANTES and IFNγ. n=7–8. *P<0.05 vs CT, ^§P<0.05 vs BaF3.

Figure 2

Inulin-type fructans treatment modifies the gut microbiota composition and increases the propionate concentration in the portal vein. (A) Denaturing gradient gel electrophoresis (DGGE) profiles of the bacterial DNA isolated from the caecal content. Cross for the CT mice; closed circle for the BaF3 mice; and open circle for the BaF3-ITF mice. (B) Levels of total bacteria, Lactobacillus spp. and Bacteroides spp. CFU: colony-forming unit. (C) Caecal tissue and caecal content weights. (D–F) Serum acetate, propionate and butyrate concentrations in the portal vein. n=7–8. *P<0.05 vs CT, ^§P<0.05 vs BaF3.

Figure 3

Short-chain fatty acids decrease BaF3 cell proliferation in vitro. (A–C) BaF3 cells were incubated in the absence or presence of acetate, propionate, butyrate for 24, 48 and 72 h before performing the MTT assay. (D) BrdU incorporation after 8 or 24 h incubation in the absence or presence of propionate. (E) Manual counting of BaF3 cells after 24, 48 and 72 h in the absence or presence of 2 mℳ propionate. (F) BaF3 cells were pre-incubated in the absence or presence of the indicated drug (U73122, 2 μℳ, 1 h; dbcAMP 1 mℳ, 30 min; IBMX, 316 μℳ, 1 h) and then incubated in the absence or presence of propionate for 24 h. Data are expressed as a percentage of the control performed for each pharmacological condition in the absence of propionate. MTT assay. The graphs represent data obtained from at least three independent experiments performed in triplicate. *P<0.05 vs the control. For F, each set of data was analysed by two-way ANOVA with Bonferroni post hoc test taking into account control values.

Figure 4

Free fatty acid receptor 2 (FFA2) activation decreases the proliferation of BaF3 cells in vitro. (A) BaF3 cells were incubated in the absence or presence of CMTB for 24, 48 and 72 h before performing MTT assay. (B) BrdU incorporation after 8 or 24 h incubation in the absence or presence of CMTB. *P<0.05 vs the control. (C) Microscopic pictures of BaF3 cells incubated in the absence or presence of CMTB. Scale bar=100 μℳ. (D) Free fatty acid receptor 2 (FFA2) mRNA levels in U937 and K562 cells. *P<0.05 vs U937. n=1 in triplicate. (E) U937 and K562 cells were incubated in the absence or presence of CMTB for 24 h. MTT assay. *P<0.05 vs U937. The MTT and BrdU assays graphs represent data obtained from at least three independent experiments performed in triplicate.