Wang et al Anticancer Exopolysaccharide From B breve Iwo1 WSU-HN6 normoxia B . 80D pg/mL Time (Day) WSU-HN6 hypoxia SCC15 hy poxia 800 ugmL Time(Day) FIGURE 7 Cell proliferation inhibition of EPS from B. breve lol by CCK-8 assay(A-C)Three HNSCC cel lines were cultured in normoxia condition and treated by gradient concentration of EPs from B breve lo1(D-F)Three HNSCC cell lines were cultured in hypoxia condition. All the experiments were run in triplicate p<0.05;p<0.01 Hidalgo-Cantabrana et al, 2014). Due to the high interspecies association between the genetics and physico-chemical variability existence, and deep excavation of genome sequences characteristics (Hidalgo-Cantabrana et al., 2014). Moreover, from different database, the comparison-selected results were in this work, we did not focus on the structure of EPS used to examine the gene cluster responsible for EPS synthesis. Accordingly, future studies should focus on investigating We find complete EPs gene cluster was involved in the the genetics and physico-chemical correlation based on EPS biosynthesis of EPS and the gene cluster could be divided genome-composition-structure characters to three following groups: the gene responsible for repeated In our previous study, we already verified that the strain unit synthesis, polymerization/chain length determination and B. breve possess an anti-tumor effect on HNSCC tumor exportation. No transcriptional regulator was observed as bearing model(Wang et al, 2017). Yet, the exact mechanism in other Bifidobacterium(Ruas-Madiedo, 2014). The EPs was not fully explored. Existing studies have shown that the biosynthesis pathway was similar but not identical to that presence of bacterial molecules structure(DNA, proteins, EPS reported in B. breve UCC2003. In B. breve UCC2003, the or metabolites)could have a specific effect on the immune or EPS synthesis was bidirectional and responsible for production inflammation system(Menard et al, 2010; Hevia et al., 2014; of one or two polymers(Fanning et al., 2012). For the Hidalgo-Cantabrana et al, 2014). In the present study, we further confirmation of the exact function of each gene further examined whether the new extracted bifido-EPS may b involved in our bifdo-EPS gene cluster, mutants by deletion responsible for the anticancer bioactivity part of the EPS clusters could be used by comparing Currently, there are only few studies on the anticancer effect to their counterparts. of EPS from Bifidobacterium strains. In vitro experiments have Among the biosynthesis pathway, repeated unit synthesis shown that polysaccharide fraction extracted from B. bifidum was the key step to determine the final EPS-unit constitution BGN4 could inhibit the growth of colon cancer cell lines HT and GTF was the key enzyme involved in this part. The 29 and HCT-116, but had no effect on Caco-2 cell line. Previous amino acid identity of p-gtf among B. breve lwo1 and study has reported that the inhibition rate on HT-29 cell line other seven Bifidobacterium stains was lower. The similar was 50.5 3.6% at 80 ug/mL by Trypan blue exclusion assay sequences were observed, particularly in the carboxy terminus It is speculated that the anticancer effect may be related to the regions, due to the presence of regions involved in interaction inhibition of DNA synthesis and reduction of cell proliferation with lipid carrier (region B)and sugar specificity (region capacity(You et al, 2004). The research from Nam Joo Has group C)(Provencher et al, 2003; Yan et al, 2017). However, showed that butanol extract of B. adolescentis SPM0212 could the composition and the structure of bifido-EPS have not inhibit the growth of Caco-2, HT-29, and Sw480 cells by 70, 30, et been fully illustrated for any of the strains containing and 40%, respectively, at 200 ug/mL. The mechanism may rely on completed genomes (Hidalgo-Cantabrana et al, 2014). inducing macrophage activation and increasing the production Therefore, although the numbers and specificities of GTEs TNF-a and NO (Lee et al, 2008). In the present study, we could contribute to the different EPS-unit constitution investigated anticancer effect of bifido-EPS on HNSCC cell line, d structures, it is still not possible to fully establish the which exerted proliferation inhibition effect in dose-dependent FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044fmicb-10-01044 May 8, 2019 Time: 14:36 # 9 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 7 | Cell proliferation inhibition of EPS from B. breve lw01 by CCK-8 assay. (A–C) Three HNSCC cell lines were cultured in normoxia condition and treated by gradient concentration of EPS from B. breve lw01. (D–F) Three HNSCC cell lines were cultured in hypoxia condition. All the experiments were run in triplicate. ∗p < 0.05; ∗p < 0.01. Hidalgo-Cantabrana et al., 2014). Due to the high interspecies variability existence, and deep excavation of genome sequences from different database, the comparison-selected results were used to examine the gene cluster responsible for EPS synthesis. We find complete EPS gene cluster was involved in the biosynthesis of EPS and the gene cluster could be divided into three following groups: the gene responsible for repeated unit synthesis, polymerization/chain length determination and exportation. No transcriptional regulator was observed as in other Bifidobacterium (Ruas-Madiedo, 2014). The EPS biosynthesis pathway was similar but not identical to that reported in B. breve UCC2003. In B. breve UCC2003, the EPS synthesis was bidirectional and responsible for production of one or two polymers (Fanning et al., 2012). For the further confirmation of the exact function of each gene involved in our bifdo-EPS gene cluster, mutants by deletion part of the EPS clusters could be used by comparing to their counterparts. Among the biosynthesis pathway, repeated unit synthesis was the key step to determine the final EPS-unit constitution and GTF was the key enzyme involved in this part. The amino acid identity of p-gtf among B. breve lw01 and other seven Bifidobacterium stains was lower. The similar sequences were observed, particularly in the carboxy terminus regions, due to the presence of regions involved in interaction with lipid carrier (region B) and sugar specificity (region C) (Provencher et al., 2003; Yan et al., 2017). However, the composition and the structure of bifido-EPS have not yet been fully illustrated for any of the strains containing completed genomes (Hidalgo-Cantabrana et al., 2014). Therefore, although the numbers and specificities of GTFs could contribute to the different EPS-unit constitution and structures, it is still not possible to fully establish the association between the genetics and physico-chemical characteristics (Hidalgo-Cantabrana et al., 2014). Moreover, in this work, we did not focus on the structure of EPS. Accordingly, future studies should focus on investigating the genetics and physico-chemical correlation based on EPS genome-composition-structure characters. In our previous study, we already verified that the strain B. breve possess an anti-tumor effect on HNSCC tumor￾bearing model (Wang et al., 2017). Yet, the exact mechanism was not fully explored. Existing studies have shown that the presence of bacterial molecules structure (DNA, proteins, EPS or metabolites) could have a specific effect on the immune or inflammation system (Menard et al., 2010; Hevia et al., 2014; Hidalgo-Cantabrana et al., 2014). In the present study, we further examined whether the new extracted bifido-EPS may be responsible for the anticancer bioactivity. Currently, there are only few studies on the anticancer effect of EPS from Bifidobacterium strains. In vitro experiments have shown that polysaccharide fraction extracted from B. bifidum BGN4 could inhibit the growth of colon cancer cell lines HT- 29 and HCT-116, but had no effect on Caco-2 cell line. Previous study has reported that the inhibition rate on HT-29 cell line was 50.5 ± 3.6% at 80 µg/mL by Trypan blue exclusion assay. It is speculated that the anticancer effect may be related to the inhibition of DNA synthesis and reduction of cell proliferation capacity (You et al., 2004). The research from Nam Joo Ha’s group showed that butanol extract of B. adolescentis SPM0212 could inhibit the growth of Caco-2, HT-29, and SW480 cells by 70, 30, and 40%, respectively, at 200 µg/mL. The mechanism may rely on inducing macrophage activation and increasing the production TNF-α and NO (Lee et al., 2008). In the present study, we investigated anticancer effect of bifido-EPS on HNSCC cell line, which exerted proliferation inhibition effect in dose-dependent Frontiers in Microbiology | www.frontiersin.org 9 May 2019 | Volume 10 | Article 1044