Fumi Seto-Tetsuo

Chronic periodotitis is caused by the formation of biofilms. Prevotella intermedia , a gram‐negative obligate anaerobic bacterium residing in periodontal pockets is involved in the formation of biofilms and secrets a highly potent DNA‐degrading activity. Biofilm contains extracellular DNA as a structural component, suggesting that DNase activity may influence P. intermedia's own biofilm development. Neutrophil extracellular traps (NETs) have mesh‐like structures and composed of DNA, histone and antibacterial proteins. NETs play an important role in protecting against infection, but it is possible that DNase of P. intermedia disrupts NETs. The lack of established genetic manipulation has significantly delayed the analysis of DNase pathogenic factors. Recently, we have succeeded in establishing a genetic manipulation technique for P. intermedia . In this study, we created strains lacking two DNase candidate genes, nucA (PIOMA14_I_0621) and nucD (PIOMA14_II_0624), that were highly conserved among P. intermedia strains. We examined biochemical analysis of DNase activity, their effection on biofilm formation, and their evasion of NETs. Here, we showed both of them possessed DNase activities which appeared to account all of DNase activities of the bacterium. The mutant analysis further demonstrated that NucA and NucD destroyed biofilm and NETs formations. Neither one was perfectly responsible for DNase activity, but rather they take turns depending on the conditions. In conclusion, the nucA and nucD genes encode DNases that cooperatively function on biofilm formation and suppress NETs formation in P. intermedia .

We have previously reported that the differentiation-inducing factor-1 (DIF-1), a compound identified in Dictyostelium discoideum, suppresses the growth of MCF-7 breast cancer cells by inactivating p70 ribosomal protein S6 kinase (p70^S6K). Therefore, we first examined whether the same mechanism operates in other breast cancer cells, especially triple-negative breast cancer (TNBC), the most aggressive and refractory phenotype of breast cancer. We also investigated the mechanism by which DIF-1 suppresses p70^S6K by focusing on the AMPK-mTORC1 system. We found that DIF-1 induces phosphorylation of AMPK and Raptor and dephosphorylation of p70^S6K in multiple TNBC cell lines. Next, we examined whether AMPK-mediated inhibition of p70^S6K leads to the suppression of proliferation and migration/infiltration of TNBC cells. DIF-1 significantly reduced the expression levels of cyclin D1 by suppressing the translation of STAT3 and strongly suppressed the expression levels of Snail, which led to the suppression of growth and motility, respectively. Finally, we investigated whether DIF-1 exerts anticancer effects on TNBC in vivo. Intragastric administration of DIF-1 suppressed tumor growth and spontaneous lung metastasis of 4T1-Luc cells injected into the mammary fat pad of BALB/c mice. DIF-1 is expected to lead to the development of anticancer drugs, including anti-TNBC, by a novel mechanism.

We reported that differentiation-inducing factor-1 (DIF-1) inhibited the proliferation of various cancer cells including malignant melanoma and that DIF-1 prevented lung colony formation in a mouse model of metastatic melanoma. However, the mechanisms of this action remain to be elucidated. In the present study, we investigated the anti-metastatic effects of DIF-1 in human malignant melanoma A2058 cells. Activities of cell migration and invasion were measured by the wound healing assay and cell invasion assay, respectively. Activities of cell adhesion to extracellular matrix (ECM) and vascular endothelial cells were measured by using ECM-coated plate and human umbilical vein endothelial cells (HUVECs). Expression levels of signaling molecules were measured by Western blotting. DIF-1 suppressed the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and subsequently reduced a variety of genes related to cell migration and invasion such as matrix metalloproteinase-2, vimentin, N-cadherin and twist, resulting in the inhibition of cell migration and invasion. Further, DIF-1 inhibited the melanoma cell adhesion to ECM and HUVECs. These results suggested that DIF-1 suppresses the detachment of cancer cells from the primary tumor by inhibiting cell migration and invasion, and also prevents circulating cancer cells from adhering to vascular endothelial cells. Therefore, DIF-1 may have potential to be a lead chemical compound for developing a novel anti-metastatic agent against malignant melanoma.