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OPEN Exopolysaccharides from Lactobacillus plantarum NCU116 induce c-Jun dependent Fas/Fasl- Received: 7 July 2017 Accepted: 5 October 2017 mediated apoptosis via TLR2 in Published: xx xx xxxx mouse intestinal epithelial cancer cells Xingtao Zhou 1, Tao Hong1, Qiang Yu1, Shaoping Nie1, Deming Gong1,2, Tao Xiong1 & Mingyong Xie1

Exopolysaccharides (EPS) from lactic acid bacteria (LAB) have been reported to play vital parts in the modulation of cell-cycle and apoptosis in cancer cells. However, the mechanisms by which EPS regulate the proliferation and apoptosis of cancer cells remain incompletely understood. We thus used diferent cancer cells to evaluate the anticancer ability and to investigate the underlying molecular mechanism of EPS from Lactobacillus plantarum NCU116 (EPS116). Our studies showed that EPS116 inhibited the proliferation of cancer cells in a cell type manner, and remarkably repressed the growth and survival of CT26 through induction of apoptosis. Moreover, EPS116 increased the expression of pro-apoptotic , including Fas, Fasl and c-Jun, induced the phosphorylation of c-Jun in CT26 cells. Furthermore, TLR2 (Toll like 2) was upregulated by EPS116, and the CT26 cells with TLR2 knockdown were found to be insensitive to EPS116, suggesting that the anti-cancer activity of EPS116 may be TLR2- dependent. Taken together, the suppressive efcacy of EPS116 on the proliferation of CT26 cells may be mediated via TLR2 and the activation of c-Jun dependent Fas/Fasl-mediated apoptotic pathway. Our study has, for the frst time, shown that EPS from LAB induced c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in CT26 cells.

Cancer is a class of diseases that are featured of the uncontrolled proliferation of cells with invasive and motile ability. Cancer can nearly occur anywhere in the body and has various subtypes which need diferent treatment strategies. Data released by the World Health Organization (WHO) showed that cancer is the second leading cause of death in the world and accounted for 8.8 million death in 2015 (nearly 1 in 6 of all global deaths). Colorectal carcinoma is the third most common type of cancer in human1. Despite cancer accounts for about 16% of total deaths globally, it is possible to improve the survival and cure rates of cancer patients by timely and rational treatment2. Tus, it is urgent to develop anticancer agents with high efciency and hypotoxicity, compris- ing natural products. Te success of therapy in cancer is one of the most challenging issues of modern medicine. Although we have made remarkable progress in the treatment of cancer over the past 30 years, current remedies withal mainly depend on ‘blunderbuss’ pharmaceutical therapies3. Nevertheless, it is encouraging that our comprehension of apoptosis mechanism allows us to propose the more reasonable approaches to cancer therapy. Bacteria synthesize and secrete a variety of functional and valuable polysaccharides into the surrounding envi- ronment, named exopolysaccharides (EPS), which may be composed of homopolymers or heteropolymers with

1State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, Jiangxi, 330047, China. 2New Zealand Institute of Natural Medicine Research, 8 Ha Crescent, Auckland, 2041, New Zealand. Correspondence and requests for materials should be addressed to S.N. (email: [email protected]) or M.X. (email: [email protected])

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 1 www.nature.com/scientificreports/

diferent molecular weights4. In the development of functional food, the benefcial efects of bacteria on human health are largely due to its EPS. Anti-cancer, immunomodulatory and anti-viral activities are acknowledged bioactivities of these EPS5. Te anti-cancer activity of EPS may be exerted through the following mechanisms: (1) prevention of tumorigenesis; (2) induction of cancer cells apoptosis; (3) improvement of the immunity activity. Programmed cell death with diverse forms is indispensable throughout the life of individuals. Prominently, apoptosis plays a very important role in immunomodulation and defense of disease (e.g. colorectal cancer). Generally speaking, caspase-dependent apoptosis is triggered by external or internal factors6. Te external path- way is motivated by the involvement of death receptors (transmembrane , such as Fas and TNF-receptors) which bind with their ligands (Fas and TNF)7–9. When the ligand binds to death receptors, their cytoplasmic domains attract adaptor molecules and initiate caspases cascade. In the end, they activate Caspase-8 which successively lead to the activation of downstream caspases, like Caspase-9 or Caspase-39. Te internal path- way for apoptosis destabilizes the mitochondrial membrane and releases apoptosis associated , such as cytochrome c. Cytochrome c together with apaf-1 induces the activation of Caspase-9 which in turn activate Caspase-3, causing cell apoptosis10,11. Te mitogen activated protein kinase (MAPK) pathway play an important part in antitumor treatments. Activated MAPK transfers extracellular stimuli to modulate apoptosis, cell multiplication and growth12. Jun N-terminal kinases (JNK), a stress-activated protein kinase of the MAPK family, is initially activated in response to stress signals and engaged in numerous cellular processes, like apoptosis12,13. Te activated JNK regulates many transcriptional factors, such as ATF-2, activator protein 1 (AP-1), c-Jun and p5314,15. Upon activation, c-Jun induces apoptosis by its transcription-dependent manner16. It can be phos- phorylated and serve as a transcriptional factor to transactivate target genes, such as Fas and Fasl, initiating apoptosis17,18. TLR2, serving as receptor on the intestine epithelium cells and immune cells for bacterial products, is known to regulate signaling in response to bacterial products (such as peptidoglycan)19–21. Tese fndings imply that the activation of TLR2 on the intestinal cancer cells by EPS may have a direct role in inducing apoptosis. TLR2 forms heterodimers with TLR1 or TLR6, facilitating the interaction with diferent microbial components. To further authenticate the biological activity and molecular mechanisms of EPS116, we used diferent can- cer cells to evaluate the anticancer ability and to investigate the underlying molecular mechanism of EPS116. Our data showed that EPS116 inhibited the proliferation of cancer cells in a cell type manner, and remarkably repressed the growth and survival of mouse intestinal epithelial cancer cells CT26 through induction of apoptosis. Hence we utilized CT26 as a model to evaluate the anticancer ability and investigate the underlying molecular mechanism. We found that EPS116 induced the apoptosis of CT26 cells via TLR2 and c-Jun dependent Fas/ Fasl-mediated apoptotic pathway. Tese fndings indicate that EPS116 is a promising antitumor agent or func- tional food, and have built the foundations for the further research of EPS116. Results Homogeneity, molecular mass and monosaccharide composition of EPS116. Te homogeneity and the average molecular weight (MW) of EPS116 were analyzed by HPGPC. As shown in Fig. 1A, there was only one single narrow symmetrical peak, indicating that the purifed EPS116 was homogenous. In addition, a weak absorption peak under 280 nm wavelength was attributed to the existence of a small amount of protein. Te MW of EPS116 was estimated to be 3.84 × 105 Da, consistent with previously reported MW of EPS (from 104 to 6 × 106 Da)22. Monosaccharide composition analysis revealed that EPS116 was composed of galactosamine, glu- cosamine, glucose, mannose, and glucuronic acid with a molar ratio of 1:1.4:4:9.6:2 (Fig. 1B).

EPS116 inhibited the proliferation of CT26 cells. To assess the anti-proliferative efect of ESP116 on tumor cells, we checked the proliferation of mouse epithelial colorectal carcinoma cell line CT26, human epithe- lial colorectal carcinoma cell line Caco2, HCT116, HT29 and human cervical carcinoma cell line Hela exposed to EPS116. Interestingly, the efects of ESP116 treatment on the proliferation of CT26, Caco2, HCT116, HT29 and Hela cells were diferent (Fig. 2A). EPS116 had insignifcant efects on the viabilities of Caco2 and HCT116 cells, and had no impact on the survival of HT29 and Hela cells. However, the viabilities of CT26 cell were remarkably decreased by ESP116 treatment in a dose-dependent manner (71% and 43% of the untreated cells by 400 and 800 µg/ml of EPS116 treatments for 48 h, respectively). EPS116 induced apoptosis of CT26 cells. To evaluate whether the cytotoxic efect observed in CT26 cells upon treatment with EPS116 was due to the induction of apoptosis, treated or untreated cells were stained with Annexin V-FITC/PI or Acridine orange-Ethidium bromide (AO-EB), and then subjected to fow cytometry or fuorescence microscope. As shown in Fig. 2B and C, exposure of the cells to EPS116 at 400 and 800 µg/ml for 48 h led to remarkable increases of apoptotic cells as compared with the untreated cells.

EPS116 induced apoptosis in CT26 cells via death receptor Fas and its ligand FasL. To investi- gate which apoptotic pathway was induced by EPS116 in CT26 cells, we analyzed the changes of death receptors, Apaf1, cytochrome C, Bcl2 and Bax expression. EPS116 treatment up-regulated the expression of death receptor Fas and its ligand Fasl (Fig. 3). However, no obvious diferences were found in the expression levels of Apaf1, cytochrome C, Bcl2 and Bax (Fig. 3B). Tese data indicated that EPS116 may induce apoptosis in CT26 cells via death receptor Fas and its ligand Fasl.

EPS116 induced cleavage and activation of Caspase-3 or Caspase-8. Binding of the ligand Fasl to its receptor Fas promotes receptor clustering and formation of DISC (including pro-Caspase-8 and FADD), leading to the proteolytic maturation of Caspase-8. Activated Caspase-8 directly catalyzes Caspase-3′s matu- ration, then stimulates caspase-dependent apoptosis. Hence, we checked whether Caspase-3 and Caspase-8

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 2 www.nature.com/scientificreports/

Figure 1. Homogeneity, molecular mass and monosaccharide composition determination of the EPS116. (A) High-performance gel permeation chromatography of the EPS116; (B) Te monosaccharide composition of the EPS116.

engaged in the EPS116-induced apoptosis. CT26 cells were treated with 200, 400 and 800 µg/mL of EPS116, and caspase activities were determined. Te activities of Caspase-3 and Caspase-8 in EPS116-treated cells were increased signifcantly (Fig. 4C). In line with these results, the cleavage of Caspase-3 and Caspase-8 was evident in a dose-dependent way by comparing with the untreated cells (Fig. 4A and B). To determine whether Caspase-8 play a key role in EPS116-induced apoptosis, we added z-IETD-FMK, Caspase-8 inhibitor, to EPS116-induced CT26 cells. Our data showed that z-IETD-FMK inhibited EPS116-induced apoptosis and blocked the activation of Caspase-8 and Caspase-3 in CT26 cells (Fig. 4D and E). Tese fndings further indicated that EPS116-induced apoptosis was dependent on caspases activation.

EPS116 up-regulated expression of Rock1 and PARP2, induced cleavage of PARP1. Active Caspase-3 induces apoptotic cell death through regulation of a serial of cellular target proteins, including Actin, Acinus, GAS2, PARPs and Rock1. Hence, we investigated the expression changes of Acinus, GAS2, Lamin, PARPs and Rock1, and the cleavage of PARP1 in CT26 cells with or without EPS116. EPS116 treatment upregulated the expression of PARP2 and Rock1, and enhanced the cleavage of PARP1, as in Fig. 5. Tese data indicated that EPS116 may induce apoptosis in CT26 cells via PARPs and Rock1.

EPS116 induced c-Jun expression and activation in CT26 cells. As a transcriptional factor, c-Jun can be phosphorylated and transactivate particular genes, such as Fas and Fasl to perform apoptosis18. To investigate whether EPS116 could activate c-Jun, we examined the expression levels of c-Jun by qRT-PCR and Western blot. It was found that c-Jun mRNA and protein levels were markedly up-regulated by exposure of EPS116 (Fig. 6A and B). Te activation of c-Jun is related to its post-translational modifcation, like phosphorylation. In order to investigate the phosphorylation level of c-Jun in CT26 cells afer exposure to EPS116, Western blot using anti-phospho-c-Jun antibodies was conducted in the study. As shown in Fig. 6C and D, the phosphorylation of c-Jun at Ser63 was signifcantly increased afer EPS116 treatment. Tese results indicated that EPS116 upregu- lated c-Jun expression, specifcally enhanced c-Jun phosphorylation at Ser63 and stimulated c-Jun translocation to the nucleus in CT26 cells.

TLR2 modulated EPS116- induced apoptosis in CT26 cells. TLR2, a receptor in the intestine epithe- lium cells, is known to regulate signaling in response to bacterial products (such as peptidoglycan). As shown in Fig. 7A and B, the expression level of TLR2 were increased in CT26 cells treated with EPS116. Tese fndings indicted that the activation of TLR2 on the intestinal cancer cells by EPS116 may be involved in inducing apop- tosis. We examined the viability and apoptosis of CT26 cells whose TLR2 was dysfunctional using shRNA. Te shRNA recombinant plasmids were constructed and validated by digesting with restriction enzymes and DNA sequencing (Supplementary Figs S1 and S2), and knockdown of TLR2 in CT26 cells was verifed by qRT-PCR and Western blotting (Supplementary Figs S3 and S4). Afer that, we checked the viability and apoptosis of EPS116 on TLR2-knockdown cells. As expected, CT26 cells with TLR2 knockdown were insensitive to EPS116 treatment (Fig. 7C,D,E and F). And the expression of Fas, Fasl and c-Jun showed indistinctive change in TLR2-knockdown

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 3 www.nature.com/scientificreports/

Figure 2. Impact of EPS116 on the survivability and apoptosis of cancer cells. (A) Impact of EPS116 on the survivability of CT26, Caco2, HCT116, HT29 and Hela cells; (B,C) Impact of EPS116 on the apoptosis of CT26 cells, each sample were treated with diferent EPS116 concentration(0–800 μg/mL) for 48 h; (B) FITC Annexin /PI staining, (C) AO-EB staining (200×). Each value represents the mean of three experiments. *P < 0.05 vs control group.

cells, while it was reverse in CT26 cell with empty vector (Fig. 7G). In summary, these results implied that TLR2 regulated EPS116-induced apoptosis in CT26 cells via controlling the expression and activation of c-Jun.

TLR1 facilitated TLR2 to modulate EPS116- induced apoptosis in CT26 cells. TLR2 forms heterodimers with TLR1 and TLR6, facilitating the interaction with different microbial components. Thus EPS116-induced apoptosis might depend on the functional interactions between TLR2 and TLR1 or TLR6. Tus we tried to investigate functions of these co-receptors to recognition of EPS116 by shRNA knockdown. Te shRNA recombinant plasmids were validated by digesting with restriction enzymes and DNA sequencing (Supplementary Figs S1 and S2), and knockdown of TLR1 or TLR6 in CT26 cells were verifed by qRT-PCR (Supplementary Fig. S3). Ten, we checked the apoptosis of EPS116 on TLR1 or TLR6 knockdown cells. Our data showed that EPS116 treatment had similar impacts on the apoptosis of CT26 cells with TLR6 knockdown or with empty vector, however TLR1 knockdown reversed a few EPS116-induced apoptosis (Fig. 7E and F). Tese

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 4 www.nature.com/scientificreports/

Figure 3. EPS116 induced apoptosis in CT26 cells via death receptor Fas and its ligand Fasl. (A,B) Relative expression levels of target genes; (C) Protein expression levels of Fas and Fasl; (D) Density of target genes normalized to that of β-actin. For clarity lanes were cropped from a single immunoblot. Each sample were treated with diferent EPS116 concentrations (0–800 μg/mL) for 48 h before cell lysis.

fndings implied that TLR2 might partially regulate EPS116-induced apoptosis in CT26 cells by cooperating with TLR1.

The expression of TLR2 only upregulated in CT26 cells. Our data showed that the cell viability inhibi- tion efect was only signifcantly observed in mouse colorectal carcinoma CT26 cells rather than human colorec- tal carcinoma cells Caco2, HCT116, and HT29. To understand the mechanism, we examined the expression levels of Fas, c-Jun and TLR2 in the four types of colorectal carcinoma cells by qRT-PCR. It was found that the expression of Fas, c-Jun and TLR2 was markedly up-regulated in CT26 cell by exposure of EPS116 rather than the others (Fig. 7H), which were consistent with the cell viability data. Tis fnding suggested that TLR2 might only be efectively activated in CT26 cells which promoted intracellular apoptotic pathways. Discussion Te beneft of bacteria to human health has mainly been ascribed to their EPS. Bacterial EPS exhibit great diver- sity and functions. Antitumor, antiviral and immunomodulatory activities are the partial health benefts of these EPS. In this study, EPS116 inhibited the proliferation of some but not all types of cancer cells. EPS116 at a high dose showed obvious inhibiting efect on the CT26 cells’ proliferation. Nevertheless, the similarity was not found in the other four types of cancer cells (Fig. 2A). Terefore, EPS116, as an anti-cancer agent, inhibited the cells pro- liferation in a specifc cell type manner. So we used CT26 cell as a model to investigate the molecular mechanism underlying the anti-cancer activity of EPS116. Te cytotoxic efect observed in CT26 cells upon treatment with EPS116 may be due to apoptotic, autophagic or necrotic pathway. Te further experiments revealed that EPS116 suppressed the proliferation of CT26 cells via apoptosis (Fig. 2B and C). For many antitumor drugs, apoptosis is recognized as a vital mechanism to inhibit the growth of tumor cells. To further validate the mechanism by which EPS116 induced the apoptosis of CT26 cells, we examined the changes of target genes expression in death receptors signaling pathway and mitochondrial pathway. EPS116 treatment up-regulated the expression of death receptor Fas and its ligand Fasl expression, as in Fig. 3. When the ligand Fasl binds to the , it rapidly induces the death of apoptotic cell via caspases’ activation. Upon binding, Fas form the DISC (the cytoplasmic tail of Fas, FADD and proCaspase-8) and activate Caspase-823. Activated caspase-8 initiates diverse caspases (such as Caspase-3) and result in death of apoptotic cell. Our data showed that EPS116 treatment activated Caspase-8 and Caspase-3, and z-IETD-FMK (Caspase-8 inhibi- tor) inhibited EPS116-induced apoptosis and blocked the activation of Caspase-8 and Caspase-3 in CT26 cells (Fig. 4). Tese fndings further indicated that EPS116-induced apoptosis was mainly dependent on Caspase-8 activation which sequentially led to the activation of downstream efector Caspase-3.

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 5 www.nature.com/scientificreports/

Figure 4. EPS116 induced cleavage and activation of Caspase-3 or Caspase-8. (A) Analysis of the amount of cleaved Caspase-8 or Caspase-3 by Western blot; (B) Density of target genes normalized to that of β-actin; (C) Analysis of Caspase-3 and Caspase-8 activities in CT26 cells; (D) Impact of EPS116 on the apoptosis of CT26 cells treated with 50 μm z-IETD-FMK; (E) Analysis of Caspase-3 and Caspase-8 activities in CT26 cells treated with 50 μm z-IETD-FMK. Each sample were treated with diferent EPS116 concentrations (0–800 μg/mL) for 48 h before cell lysis.

However, no obvious diferences were found in the expression levels of Apaf1, cytochrome C, Bcl2 and Bax afer EPS116 treatment (Fig. 3B). Tese data indicated that EPS116-induced apoptosis may be mainly related to the Fas/Fasl pathway but not mitochondrial pathway. Activation of Caspase-3 leads to apoptosis via several pathways, but which pathway might be involved in this process in unknown. To settle this problem, we checked the expression levels of Acinus, GAS2, Lamina, Rock1, PARPs and cleavage of PARP1 in CT26 cell afer EPS116 treatment. Acinus, GAS2, Lamina and Rock1 are asso- ciated with cytoskeleton stability. Lamina is thought to participate in the maintenance of the three-dimensional structure of the genome, maintain the integrity of the nuclear envelope24. Acinus is engaged in nuclear structural changes in normal cells, and essential for apoptotic chromatin condensation by caspase-325. Gas2 is a component of the microflament system26. ROCK1 (Rho-associated, coiled-coil containing protein kinase 1) is activated by caspase-3 consequently increases the level of phosphorylated myosin chain, resulting in membrane blebbing and initiating apoptosis27,28. As shown in Fig. 5, Rock1 were markedly up-regulated by treatment with 800 ug/ml EPS116, indicating that EPS116-induced apoptosis might be related with Rock1-initiated cytoskeleton instabil- ity. PARP-1 activation was investigated with regard to its role in DNA repair and had an important role in many pathways of cell death29,30. PARP1 is the substrate of Caspase-3, and its cleavage has been widely used as a bio- chemical marker of apoptosis31. Our data show that the cleavage of PARP1 was markedly increased (Fig. 5), which implied that EPS116-induced apoptosis might be through inhibition of DNA repairing. Tese results suggested that EPS116-induced activation of apoptosis might be PARPs and Rock1-dependent. JNK activation is mediated by the upstream MKK7 kinases via phosphorylation. Te activated JNK can reg- ulate a variety of transcription factors, such as c-Jun, ATF-2 and AP-1. Subsequently, a series of cellular processes are triggered, including cell proliferation, apoptosis, autophagy and DNA repair. c-Jun, a major component of

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 6 www.nature.com/scientificreports/

Figure 5. EPS116 up-regulated Rock1 and PARP2 expression, and induced PARP1 cleavage. (A) Relative expression levels of apoptosis related genes; (B) Western blot analysis of PARP1 cleavage and Rock1 expression, (C) Density of target genes normalized to that of β-actin.

Figure 6. EPS116 induced the expression and activation of c-Jun in CT26 cells. (A) Relative expression of target genes; (B) Protein expression level of c-Jun; (C) Phosphorylation level of c-Jun in nucleus; (D) Density of target genes normalized to that of β-actin or Lamin A.

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 7 www.nature.com/scientificreports/

Figure 7. TLR2 modulated EPS116-induced apoptosis in CT26 cells. (A) Relative expression levels of TLRs; (B) Protein expression of TLR2, the number indicated density of TLR2 normalized to that of β-actin; (C) Impact of EPS116 on the survivability of CT26 cells defcient in TLR2; (D) Te signifcant diference of cell viability between empty vector and TLR2-shRNA by EPS treatment, data from(C); (E) Impact of EPS116 on the apoptosis of CT26 cells defcient in TLR2, TLR1 or TLR6 (AO-EB); (F) Cells from (E) were quantifed for the number of apoptosis in a blind manner, ffy cells were counted; (G) Relative expression levels of Fas, Fasl and c-Jun in CT26 cells transfected with empty vector (pLKO.pig) or with TLR2-shRNA vector; (H) Relative expression levels of Fas, c-Jun and TLR2 in CT26, Caco2, HCT116 and HT29 cells. Each sample were treated with diferent EPS116 concentrations (0–800 μg/mL) for 48 h without special illustration.

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 8 www.nature.com/scientificreports/

the transcription factor AP-1, was a transcriptional activator of Fas and could stimulate its expression levels up32. c-Jun and AP-1 activities are regulated by c-Jun N-terminal phosphorylation through JNK17. JNK can phosphoryl- ated c-Jun and promote its binding to the promoters of Fas and Fasl which would enhance the expression of Fas/ Fasl18. Binding of Fasl onto Fas can activate Caspase-8, and activated Caspase-8 can motivate downstream efector Caspase-3 to initiate apoptosis. Afer EPS116 exposure, the expression of total c-Jun and Ser63 phosphorylation of c-Jun were signifcantly increased in CT26 cells, while c-Fos, another component of AP-1, almost unchanged (Fig. 6). AP-1 complex has been implicated as both positive and negative modulators of apoptosis33, and the relative expres- sion levels of c-Jun and c-Fos might determine a positive or negative efect32. Elevated levels of c-Jun has been shown to induce apoptosis in diferent cell lines34,35. EPS116 induced phosphorylation of c-Jun at Ser63 and nuclear accu- mulation of c-Jun, which was recognized as one of the activation signals of c-Jun and contributing to c-Jun transcrip- tional activity36. Collectively, our results indicated that c-Jun played an important role in EPS116-induced apoptosis. It was well documented that polysaccharides cannot penetrate cells due to their large molecular mass, so the frst step in the might be binding to cancer cell receptors. TLRs, receptors on the intestine epi- thelium cells, are known to regulate signaling in response to bacterial products21,37. When TLRs recognize their ligands, they regulate expression via receptor-specifc and ordinary signaling pathways. All TLRs, except TLR3, utilize MyD88 as an adaptor to recruit signaling proteins, resulting in activation of numerous downstream transcription factors, like Ap1, nuclear factor-kappa B and IRF family members38. Sanchez, D. et al. reported that simultaneous activation of TLR2 and MyD88 induced apoptosis of in response to mycobacterial ligands39, and Krysko, D. V. et al. found that TLR2 and TLR9 were sensors of apoptosis40. Terefore, we checked the expression of TLRs in CT26 cells treated with EPS116. Te levels of TLR2 were signifcantly increased in CT26 cells treated with EPS116 (Fig. 7A and B). Tese fndings raised the possibility that the activation of TLR2 in the intestinal cancer cells by EPS116 had a role in inducing apoptosis. We thus examined the viability and apoptosis of EPS116 in CT26 cells defcient in TLR2 function using shRNA knockdown. As expected, TLR2-defcient CT26 cells were relatively resistant to EPS116 treatment (Fig. 7C,D,E and F). Knockdown TLR-2 incompletely blocked EPS116-induced cell death and apoptosis, probably due to the fact that it didn’t thoroughly suppress the expres- sion of TLR-2(Supplementary Figs S3 and S4). Tis result was consistent with data (TLR2-knockout macrophages didn’t undergo apoptosis) from Sanchez, D. et al.’s studies39. To investigate the mechanism that TLR2 regulated apoptosis via EPS116, we analyzed the expression changes of Fas, Fasl and c-Jun in CT26 cell with empty vector or with TLR2 shRNA. Our data showed that TLR2-knockdown inhibited the upregulation of Fas, Fasl and c-Jun in CT26 cells treated with EPS116 (Fig. 7G). Tese results suggested that TLR2 regulated EPS116-induced apoptosis in CT26 cells via controlling the expression and activation of c-Jun, which transactivated Fas and Fasl to initiate apoptosis. Taken together, the shRNA-based inhibitory experiments ofered persuasively evidences that TLR2 played a vital part in CT26 cell apoptosis induced by EPS116. TLR2 forms heterodimers with TLR1 or TLR6, facilitating the interaction with diferent microbial components and possibly promoting diferent intracellular signaling pathways. Tus diferences in apoptosis induction could depend upon the functional interactions between TLR2 and TLR1 or TLR6. Tus we tried to investigate the contri- butions of these co-receptors to recognition of EPS116 by shRNA knockdown. As shown in Fig. 7E and F, EPS116 treatment had similar impacts on the apoptosis of CT26 cells with TLR6 knockdown or with empty vector, however TLR1 knockdown reversed a few EPS116-induced apoptosis of CT26 cells (less than TLR2 knockdown). Tese fnd- ings implied that TLR2 might partially regulate EPS116-induced apoptosis in CT26 cells by cooperating with TLR1. Our data showed that the cell viability inhibition efect of EPS116 was only signifcantly observed in mouse colorectal carcinoma CT26 cells rather than human colorectal carcinoma cells Caco2, HCT116, and HT29. To understand the mechanism, we examined the expression levels of Fas, c-Jun and TLR2 of the four types of colorec- tal carcinoma cells. As shown in Fig. 7H, the expression of Fas, c-Jun and TLR2 was markedly up-regulated in CT26 cell rather than the others, which were consistent with the cell viability data. Tis fnding suggested that TLR2 might only be efectively activated by EPS116 in CT26 cells which promoted intracellular apoptotic pathways. Chemotherapeutic agents, acting as important anticancer drugs, are based on the abilities of inducing apop- tosis in cancer cells. EPS116 was demonstrated as a new inducer of apoptosis that exhibited selective antitumor activities. To further develop EPS116 as an efective anti-tumor agent in clinic, we should better understand the mechanisms that EPS116 induces apoptosis. Based on the data from this work, a model of the mechanisms that EPS116 induced apoptosis of CT26 cells was presented (Fig. 8). Tis model indicated that up-regulation and activation of c-Jun (Fig. 6) via TLR2 (Fig. 7) and up-regulation of Fas/Fasl (Fig. 3) by c-Jun acted an important part in EPS116-induced apoptosis. Tus, EPS116 frstly bound to TLR2 and activated TLR2/ MyD88/TRAF6/ MKK7 pathway, then induced the activation of JNK/c-Jun. Te activated c-Jun, upregulated the transcription and translation of Fas and Fasl, resulting in Fas-mediated apoptosis signal pathway. Fas/Fasl signaling transduction involved in FADD-activating Caspase-8 and Caspase-3 (Fig. 4). Activated Caspase-3 promoted apoptosis via PARPs and Rock1 (Fig. 5). In summary, the present study has demonstrated that EPS116/TLR2/MyD88 signaling activated JNK and promoted c-Jun phosphorylation which promoted upregulation of Fas/Fasl, and then triggered apoptotic signaling. Te detailed molecular mechanism may be helpful for the development of EPS from LAB as an efcient prevention or therapy agent for intestinal cancer in humans. Materals and Methods Materials. Cell culture products were provided by Hyclone (Logan, UT). Puromycin, streptomycin and pen- icillin were provided by Life Technologies, Inc. (Gaithersburg, MD). AO-EB, Caspase-8 and Caspase-3 fuoro- metric assay kits were provided by Beyotime Biotech (Jiangsu, China). Caspase-8 inhibitor (Z-IETD-FMK) were from MedChem Epress (Princeton, NJ). RT reagent Kit and qRT-PCR kits were from Takara Bio (Dalian, China). FuGENE HD Transfection Reagent was from Promega Corporation (Madison, WI). Restriction enzymes NcoI, AgeI-HF ®and EcoRI-HF were from New England Biolabs (Ipswich, Ma). Lactobacillus plantarum NCU116, E. coli Dh5α and pLKO.pig-puro vector were stored at −80 °C.

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 9 www.nature.com/scientificreports/

Figure 8. Schematic of apoptosis induction in CT26 cells by EPS116 via TLR2 and Fas/Fasl signaling pathways.

Isolation and purifcation of EPS116. Te Lactobacillus plantarum NCU116 was cultured in a 5 L con- ical fask containing 5 L of modifed MRS broth at 37 °C. EPS116 was isolated and purifed using the method by Cerning et al.41 with slight modifcation. Briefy, afer 20 h of incubation, cells were removed by centrifuga- tion (8,000 × g, 4 °C, 15 min) and the supernatants were collected. Ten trichloroacetic acid (TCA) was added to the supernatant to a fnal concentration of 4% (w/v), mixed with agitation overnight at 4 °C, and precipi- tated proteins were separated by centrifugalization (12,000 × g, 4 °C, 15 min). Next, 4 volumes of ethanol was added to the supernatant to precipitate EPS. Afer incubation for 12 h, EPS was gathered by centrifugalization (12,000 × g, 15 min). Te precipitates were dissolved in deionized water, then dialyzed (MW cut-of 3,500 Da, Yuanye Biotechnology Co., Shanghai, China) and lyophilized. Endotoxin concentration of purifed EPS116 was tested by Toxin Sensor Chromogenic LAL Endotoxin Assay Kit (Genscript, USA) and endotoxin-free EPS116 was used for the next experiments.™

Homogeneity and molecular mass determination of EPS116. The homogeneity and average molecular weight of the purifed EPS116 were determined by high performance gel permeation chromatography (HPGPC) using the method by Zhang, L. et al.42 with minor modifcation. Standard dextrans (10, 40, 50, 70, 500, and 2000 kDa, Fluka Chemical Co., Buchs, Switzerland) were passed through a UltrahydrogelTM linear column (7.8 × 300 mm, Waters, Japan) and eluted with 0.02% (w/v) NaN3 at a fow rate of 0.6 ml/min. Te elution volumes were plotted against the logarithms of their respective molecular weights. To determine the molecular weights of EPS116, 5 mg EPS116 in 5 ml of 0.02% NaN3 was applied to the same column equilibrated and the elution volume was then plotted in the same graph.

Monosaccharide composition analysis. Te monosaccharide composition of the purifed EPS116 was analyzed using the method by Wang, J.Q. et al.43 with slight modifcations. High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) (Dionex ICS-5000 System, Dionex Corporation, CA) was applied to determine the monosaccharide composition of the EPS116. Data was analyzed by chromeleon sofware.

Cell line and cell culture. Mouse epithelial colorectal cell line CT26, human epithelial colorectal cell line Caco2 and human cervical cancer cell line Hela were provided by Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Human epithelial colorectal cell line HT-29 and HCT-116 were from KeyGEN Biotech (Nanjing China). Cells were grown in DMEM or McCoy’s 5 A medium containing 10% FBS, 100 μg /ml penicillin and l00 μg/ml streptomycin in 5% CO2 atmosphere at 37 °C.

Cell viability assay. Cell growth and survival was measured by CCK-8 assay. Cancer cells were treated with EPS116 (100–800 μg/mL) in a 96-well microplate form. Controls were incubated in the media without EPS116. 20 μL CCK-8 solution was put in each well afer 48 h incubation, then the cells were cultured about 2 h, and the absorbance at 490 nm was detected by a microplate reader (Termo Fisher Scientifc, USA).

Analysis of cell apoptosis by fow cytometry. To determine the efect of EPS116 on the apoptosis of tumor cells, CT26 cells were treated with EPS116 (200–800 μg/mL) in a 96-well microplate form for 48 h. Ten, adherent cells were trypsinized, and stained by FITC Annexin V detection with PI (BD, USA) following the

Scientific REPorTS | 7:14247 | DOI:10.1038/s41598-017-14178-2 10 www.nature.com/scientificreports/

manufacturer’s directions. Afer staining, the number of apoptotic cells was immediately measured by a FACS Calibur fow cytometer (Becton Dickinson).

AO-EB staining assay. To investigate the efect of EPS116 on the apoptosis of tumor cells, the cell morphol- ogy were analyzed by AO-EB staining. Briefy, CT26 cells were treated with EPS116 (200–800 μg/mL) in a 12-well microplate form for 48 h. Ten AO-EB was added to the live cells at room temperature in the dark and measured by a fuorescence microscope (Nikon TE2000, Nikon Corporation, Japan). In the AO-EB staining assay, four cell morphologies are observed under the fuorescence microscope. “Live cells: the cells nuclear chromatin is green has a normal structure. Early apoptotic cells: the cells nuclear chroma- tin appears green and show shrinkage-like or fragment-like morphologies. Late apoptotic cells: the cells nuclear chromatin appear orange-red and showed shrinkage-like or fragment-like structures. Non-apoptotic dead cells: the cells nuclear chromatin appears red and shows a normal structure”44.

Preparation of RNA and quantitative real-time PCR (qRT-PCR). Total RNA was isolated with TRIzol Reagent (Termo Fisher Scientifc, USA). RNA integrity was determined by RNA formaldehyde dena- turing ®gel electrophoresis. RNA concentration and purity was measured by Nanodrop2000 (Termo Fisher Scientifc, America), traces of DNA were removed and reverse transcription PCR was executed by RT reagent Kit with genomic DNA Eraser (Takara Bio, China). Te mRNA expression levels of target genes were measured by qRT-PCR with the SYBR Premix Ex Taq II (Takara Bio, China) on a Termo Fisher Scientifc Q7 system following the manufacturer’s directions. Te™ qRT-PCR primers specifc for the target genes can be found as Supplementary Table S1. All primers’ specifcity was assessed by melting curve analysis. Te qRT-PCR was performed following the next procedure: 95 °C for 2 min and then 40 cycles at 95 °C for 15 s, 59 °C for 20 s and 72 °C for 20 s. Changes in mRNA levels of target genes were expressed as the fold of induction relative to the negative control.

Western blot analysis. Total proteins (15 μg) were extracted with lysis bufer and separated by SDS-PAGE, then transferred to PVDF membrane. The specific antibodies for western blots as following: anti-Rock1, anti-PARP1 (Cell Signaling, Danvers, MA), anti-Fas, anti-c-Jun, anti-phospho-c-Jun (Ser63), anti-TLR2 (Abcam, Cambridge, MA), anti-β-actin (ZSGB-Bio, Beijing, China), anti-Fasl, anti-cleaved Caspase-3, anti-cleaved Caspase-8, and anti-lamin A (Boster-Bio, Wuhan, China). Te immunoreactive protein bands were detected by Gel Doc XR+ system (Bio-Rad Laboratories, USA) and quantifed by the ImageJ sofware.

Measurement of Caspase-3 and Caspase-8 activities. Activities of Caspase-3 and Caspase-8 were studied with caspase fuorometric assay kits (Beyotime Biotech, Jiangsu, China) following the manufacturer’s directions. Briefy, CT26 cells were collected and lysed afer EPS116 treatment. Ten the mixture of caspase sub- strate (DEVD-AFC for Caspase-3, LEHD-AFC for Caspase-8) and reaction bufer were put into 96-well plates. Activities of caspases were measured by an ELISA micro-plate reader at 405 nm.

Construction of TLRs-shRNA Vector. After double digested by restriction enzymes AgeI-HF and EcoRI-HF, the Lentiviral plasmid vector (pLKO.pig) was ligated with TLRs-targeting shRNA fragment to generate the shRNA delivery plasmid. shRNA primers specifc for TLR1, TLR2 and TLR6 can be found as Supplementary Table S2. Te recombinant plasmids were verifed by digesting with restriction enzymes (EcoRI, NcoI) and DNA sequencing.

shRNA. To generate CT26 cells that were stably defcient in TLRs, we used the method by Schaniel, C. et al.45 with minor modifcation. In brief, lentiviral plasmid vector (pLKO.pig) encoding TLRs-targeting shRNA was transfected into cells with FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s directions. Stable integration of lentivirus® was obtained by selection of cells using media containing 5 mg/mL puromycin for 24 h. A TLRs-specifc shRNA construct with the very low expression level of the TLRs mRNA was obtained. Te knockdown of TLRs was validated by qRT-PCR or Western blot analysis.

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Acknowledgements Tis study was supported by the National Natural Science Foundation of China for Excellent Young Scholars (31422042), the National Key Technology R & D Program of China (2012BAD33B06), the Outstanding Science and Technology Innovation Team Project in Jiangxi Province (2016RCYTB0030), and the Project of Science and Technology of Jiangxi Provincial Education Department (Grant 150188).

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Author Contributions Conceived the work: Xingtao Zhou, Shaoping Nie, Mingyong Xie; Performed the experiments: Xingtao Zhou, Tao Hong, Qiang Yu, Tao Xiong; Wrote the manuscript: Xingtao Zhou, Deming Gong. All authors reviewed and approved the fnal manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-14178-2. Competing Interests: Te authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© Te Author(s) 2017

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