Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis

Introduction

Probiotics are associated with various health benefits, including the conditioning of the intestinal microflora, suppression of excess allergic responses and tumour-suppressive effects1,2,3. Abnormal changes have been reported in the intestinal microflora of colon cancer patients4,5, indicating that the perturbation of the intestinal microflora is closely correlated with the initiation and progression of colon cancer cells. Moreover, probiotics, including the Lactobacillus and Bifidobacteriumspecies, have been shown to have tumour-suppressive effects in colon cancer cell lines and in mouse/rat tumour models6,7. Thus, it is suggested that the administration of sufficient amounts of probiotics may have preventive effects against tumour initiation and progression and that it may offer therapeutic benefits. However, the tumour-suppressive effect of live probiotics is affected by numerous factors, including the conditions of the bacterial culture8 and differences in the populations of intestinal microbes in the host9,10.

Previous investigations concerning host–microbial interactions have shown that some of the effector molecules secreted from beneficial bacteria activate the cell survival pathways. We revealed that competence and sporulation factor (CSF) derived from Bacillus subtilisinduces the expression of the heat shock proteins (Hsps) and activates the protein kinase B (Akt) cell survival pathway through organic cation transporter 2 (OCTN2) to exert a cytoprotective effect11. We subsequently found that inorganic polyphosphate isolated from the conditioned media of Lactobacillus brevis also induces the expression of Hsps and exerts a cytoprotective effect through the integrin β1-p38MAPK signal transduction pathway12. Furthermore, Yan et al.13identified two peptides, p75 and p40, as active components that possess anti-apoptotic properties and which activate cell survival, and found that the Akt pathway is induced by Lactobacillus GG. These studies indicate that host–microbe interaction brings health benefits to host mammals through the mediation of specific molecules that are derived from commensal bacteria and probiotics. However, the anti-tumorigenic molecules produced by the commensal bacteria and probiotics have not been identified.

We sought to identify the tumour-suppressive molecules from the culture supernatants of the Lactobacillus species and successfully identified a tumour-suppressive molecule, ferrichrome (a siderophore produced by Lactobacillus casei ATCC334). The tumour-suppressive effect of ferrichrome on colon cancer cells was greater than or equal to that of existing anticancer drugs. In contrast, ferrichrome showed little or no growth inhibition effect on non-cancerous intestinal cells. Furthermore, we found that ferrichrome induces apoptosis through a process that is mediated by the JNK-associated induction of DNA damage-inducible transcript 3 (DDIT3) in colon cancer cells. This is the first study to identify a probiotic-derived anti-tumour molecule.

Results

Cancer cell growth suppression by L. casei supernatant

Colon cancer cells, including Caco2/bbe, SKCO-1 and SW620 cells, were incubated with the culture supernatants of Lactobacillus GGATCC53103, L. casei ATCC334, Lactobacillus coryniformis ATCC25600 and Lactobacillus fermentis ATCC23271 to clarify their tumour-suppressive effects. All bacterial bodies and debris in the culture supernatants were removed by centrifugation and filtration using a 0.22-μm membrane. A sulforhodamine B (SRB) assay indicated that the culture supernatant of L. coryniformis ATCC25600 suppressed the cell growth of SKCO-1 cells, but not Caco2/bbe and SW620 cells. The bacterial culture supernatants of Lactobacillus GG ATCC53103, L. caseiATCC334 and L. fermentis ATCC23271 (especially the L. casei ATCC334 culture supernatant), suppressed the cell growth of Caco2/bbe, SKCO-1 and SW620 cells (Fig. 1a–c). These data indicate that the secreted molecule, but not the bacterial body and debris, exhibited the growth inhibition effect.

Figure 1: Conditioned media derived from the Lactobacillus spp. reduced the progression of colon cancer cells.

An SRB assay revealed that the numbers of colon cancer cells, Caco2/bbe (a), SKCO-1 (b) and SW620 cells (c), were significantly lower in the conditioned media from theLactobacillus GG ATCC53103-, L. casei ATCC334-, L. coryniformis ATCC25600- and L. fermentisATCC23271-treated groups than in the control group. The strongest tumour-suppressive effect against colon cancer cells was observed in the conditioned media from the L. caseiATCC334 group. The error bars show the s.d. (n=5).