Wang et al Anticancer Exopolysaccharide From B breve Iwo1 0 Hg/mL 200 Hg/mL 8O pg/mL A SCC15 SCC15 normoxia (ug/mL) 了0 MCM2 ■400ygmL aspase SCC15 PARP CL-PARP 0 CAL 2 RPS18 FIGURE 8 DAPI staining images of cell nuclear after treated with gradie concentration of eps al the cells were cultured in normoxia condition FIGURE 9 I Cell cycle and apoptosis protein expression after treating SCC15 cell line with 400 ug/mL EPS. (A)Western blot assay.( B)Relative fol magnification 4x changes by calculating relative gray value using software Quantity one."p<0.05. manner. In addition, different cells presented different sensitivity to bifido-EPS et al, 2017). Besides, rhamnose has also been reported to have As for the mechanisms of the anti-cancer property of anti-tumor activity, since it can enhance the immunogenicity EPS, except for the inhibition of DNA synthesis and immune of melanoma-associated antigen A3, which stimulates antitumor modulation reported above, EPS has anti-oxidative properties immune responses(Zhang et al, 2016). The galactose is widely and can inhibit the expression of gene involved in angiogenesis used backbone of the nanocarrier in cancer therapy, beneficial for Deepak et al., 2016) Cell proliferative activity is closely related to targeted therapy of tumors (ain et al, 2018). When conjugated the cell cycle, which is regulated by the DNA replication initiation with platinum(II), the complex has well therapeutic and target factor. MCM 2, known as DNA replication licensing factor, is effect(Wu et al, 2016). In our next study, we plan to further involved in pre-replicative complex formation during Gl phase investigate the effect of our bifido-EPS in vivo d it allows for the DNA replication initiation in the subsequent To sum up, the current work isolated one new probiotic S-Phase(Gambus et al., 2011). Downregulation of MCM2 could strain B. breve lwOl and sequenced its genome, which led to increase cell death when the cells encounter replication stress discovery of a new EPS biosynthesis cluster and prediction of during S-phase(Ibarra et al., 2008). MCM2 has also been applied its biosynthesis pathway. After extraction and purification, higl as a proliferation marker in many types of cancer(Tan et al., purity of our EPS showed to possess an anticancer activity in 2001; Yousef et al, 2017; Zhou et al., 2018). In addition, its HNSCC cell lines. In addition, the composition of bifido-EPS downregulation is considered as an attractive target in tumor showed that mannose accounted for the largest proportion. The chemotherapy(Simon and Schwacha, 2014). In this study, the preliminary mechanisms could rely on its cell cycle arrest and expression of MCM2 was downregulated, which suggested that promote cell apoptosis effect. We believe that this bifido-EPS could arrest cell cycle in Gl-S phase in HNSCC cell line. could be used to facilitate genetic and metabolic engineering and PARP is activated when cells sustained dna damage and it is the to produce tailor-made EPS for further application in functional main target of caspase 3, whose activation could mediate a latent food or drug industry specific proteolytic cleavage in apoptosis(Brauns et al., 2005; Yu et al, 2006). In our study, EPS increased the expression of caspase 3 and the cleaved-PARP proportion, while inferred cell apoptosis ETHICS STATEMENT occurred in treatment group. The chemical composition of our EPS, which included The protocol was approved Biomedical Ethics rhamnose, arabinose, galactose, glucose, and mannose, could Committee of Peking University School and Hospital of explain its anti-tumor effect to some extent. It is quite interesting Stomatology. Written informed was obtained from that mannose accounted for the largest proportion of the bifido- parents of the infant. EPS, which have been reported to have a significant anti-tumor activity. Mannose could impair tumor growth by inhibiting the proliferation while promoting the apoptosis of cancer cell. AUTHOR CONTRIBUTIONS It also revealed chemo-sensitization character. Administration of doxorubicin with mannose showed an obviously enhanced GL and CG contributed the conception and design of the anti-tumor effect(Gonzalez et al., 2018). Moreover, the level study. Lw and Kt organized the database and analysis of mannose in serum is associated with the prognosis of EPs cluster information. Yw, QL, and LX carried out esophageal adenocarcinoma patients. The patients with low level the study. Yw performed the statistical of d-mannose have higher risk of recurrence and death(Gu wrote section of the manuscript. Lw wrote FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044fmicb-10-01044 May 8, 2019 Time: 14:36 # 10 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 FIGURE 8 | DAPI staining images of cell nuclear after treated with gradient concentration of EPS. All the cells were cultured in normoxia condition. magnification 4×. manner. In addition, different cells presented different sensitivity to bifido-EPS. As for the mechanisms of the anti-cancer property of EPS, except for the inhibition of DNA synthesis and immune modulation reported above, EPS has anti-oxidative properties and can inhibit the expression of gene involved in angiogenesis (Deepak et al., 2016). Cell proliferative activity is closely related to the cell cycle, which is regulated by the DNA replication initiation factor. MCM 2, known as DNA replication licensing factor, is involved in pre-replicative complex formation during G1 phase and it allows for the DNA replication initiation in the subsequent S-phase (Gambus et al., 2011). Downregulation of MCM2 could increase cell death when the cells encounter replication stress during S-phase (Ibarra et al., 2008). MCM2 has also been applied as a proliferation marker in many types of cancer (Tan et al., 2001; Yousef et al., 2017; Zhou et al., 2018). In addition, its downregulation is considered as an attractive target in tumor chemotherapy (Simon and Schwacha, 2014). In this study, the expression of MCM2 was downregulated, which suggested that EPS could arrest cell cycle in G1-S phase in HNSCC cell line. PARP is activated when cells sustained DNA damage and it is the main target of caspase 3, whose activation could mediate a latent specific proteolytic cleavage in apoptosis (Brauns et al., 2005; Yu et al., 2006). In our study, EPS increased the expression of caspase 3 and the cleaved-PARP proportion, while inferred cell apoptosis occurred in treatment group. The chemical composition of our EPS, which included rhamnose, arabinose, galactose, glucose, and mannose, could explain its anti-tumor effect to some extent. It is quite interesting that mannose accounted for the largest proportion of the bifido￾EPS, which have been reported to have a significant anti-tumor activity. Mannose could impair tumor growth by inhibiting the proliferation while promoting the apoptosis of cancer cell. It also revealed chemo-sensitization character. Administration of doxorubicin with mannose showed an obviously enhanced anti-tumor effect (Gonzalez et al., 2018). Moreover, the level of mannose in serum is associated with the prognosis of esophageal adenocarcinoma patients. The patients with low level of d-mannose have higher risk of recurrence and death (Gu FIGURE 9 | Cell cycle and apoptosis protein expression after treating SCC15 cell line with 400 µg/mL EPS. (A) Western blot assay. (B) Relative fold changes by calculating relative gray value using software Quantity One. ∗p < 0.05. et al., 2017). Besides, rhamnose has also been reported to have anti-tumor activity, since it can enhance the immunogenicity of melanoma-associated antigen A3, which stimulates antitumor immune responses (Zhang et al., 2016). The galactose is widely used backbone of the nanocarrier in cancer therapy, beneficial for targeted therapy of tumors (Jain et al., 2018). When conjugated with platinum (II), the complex has well therapeutic and target effect (Wu et al., 2016). In our next study, we plan to further investigate the effect of our bifido-EPS in vivo. To sum up, the current work isolated one new probiotic strain B. breve lw01 and sequenced its genome, which led to discovery of a new EPS biosynthesis cluster and prediction of its biosynthesis pathway. After extraction and purification, high purity of our EPS showed to possess an anticancer activity in HNSCC cell lines. In addition, the composition of bifido-EPS showed that mannose accounted for the largest proportion. The preliminary mechanisms could rely on its cell cycle arrest and promote cell apoptosis effect. We believe that this bifido-EPS could be used to facilitate genetic and metabolic engineering and to produce tailor-made EPS for further application in functional food or drug industry. ETHICS STATEMENT The protocol was approved by the Biomedical Ethics Committee of Peking University School and Hospital of Stomatology. Written informed consent was obtained from parents of the infant. AUTHOR CONTRIBUTIONS GL and CG contributed the conception and design of the study. LW and KT organized the database and analysis EPS cluster information. YW, QL, and LX carried out the study. YW performed the statistical analysis and wrote section of the manuscript. LW wrote the first draft Frontiers in Microbiology | www.frontiersin.org 10 May 2019 | Volume 10 | Article 1044