Probiotic Aspergillus oryzae produces anti-tumor mediator and exerts anti-tumor effects in pancreatic cancer through the p38 MAPK signaling pathway

Intake of probiotics or fermented food produced by some probiotic bacteria is believed to exert anti-tumor functions in various cancers, including pancreatic cancer, because several studies have demonstrated the anti-tumor effects of probiotic bacteria in vitro and in vivo in animal carcinogenesis models. However, the mechanisms underlying the anticancer effects of probiotics on pancreatic cancer have not been clarified. In this study, we assessed the anti-tumor effects of probiotic bacteria against pancreatic cancer cells. Among the known probiotic bacteria, Aspergillus oryzae exhibited a strong pancreatic tumor suppression effect. The culture supernatant of A. oryzae was separated by HPLC. Heptelidic acid was identified as an anti-tumor molecule derived from A. oryzae by LC–MS and NMR analysis. The anti-tumor effect of heptelidic acid was exhibited in vitro and in vivo in a xenograft model of pancreatic cancer cells. The anti-tumor effect of heptelidic acid was exerted by the p38 MAPK signaling pathway. Heptelidic acid traverses the intestinal mucosa and exerts anti-tumor effects on pancreatic cancer cells. This is a novel anti-tumor mechanism induced by beneficial bacteria against pancreatic cancer in which bacterial molecules pass through the intestinal tract, reach the extra-intestinal organs, and then induce apoptosis via an inducible signaling pathway.

Isolation of the tumor-suppressive molecule. The culture medium was centrifuged at 5000×g for 10 min to obtain the culture supernatant, which was then filtered through a 0.2-μm membrane. The culture supernatants were separated with a molecular weight cut-off (MWCO) spin column (GE Healthcare). The culture supernatant was separated using an AKTA Design HPLC system (GE Healthcare) with a Superdex peptide column (GE Healthcare) and eluted with distilled water at a flow rate of 1 mL/min. The fraction was applied to a Wakopak® Wakosil® C18 and C8 column (Wako Pure Chemical, Osaka, Japan) and eluted with 0.1% formic acid and 0.1% formic acid/acetonitrile in a linear gradient at a flow rate of 2.5 mL/min. The eluent was monitored by ultraviolet spectrophotometry at 210 nm.
The SRB assay. The significance of cell density by samples was determined by the SRB assay. At 24 h before stimulation, the cells were seeded on 96-well microplates at 1.0 × 10 4 cells/100 μL/well. Each sample was diluted with cell culture media and applied to the pancreatic cancer cells. At 24,48 or 72 h after stimulation, the cells were fixed in 5% trichloroacetic acid (TCA) for 1 h at 4 °C and washed 4 times in distilled water. The microplates were then dehydrated at room temperature, stained in 100 μL/well of 0.057% (wt/vol) SRB powder/distilled water, washed 4 times in 0.1% acetic acid and re-dehydrated at room temperature. The stained cells were lysed in 10 mM Tris-buffer, and the optical density (OD) was measured at 510 nm. www.nature.com/scientificreports/ tion, Kyoto, Japan) and J'sphere ODS-M80 (150 × 4.6 mml., S-4 μm, 8 nm) as a C18 column. Elusion used an acetonitrile-water gradient (5-90%; 0.4 mL/min) at 30 °C and was monitored using an ultraviolet (UV) light detector (254 nm). High-resolution MS was performed using a Waters Xevo G2 QTof. Samples were eluted by acetonitrile.

Mass spectrometry (MS).
A nuclear magnetic resonance (NMR) analysis. The 1 H NMR spectrum of the isolated compound was measured in CDCl 3 solution and reported in parts per million (δ) relative to tetramethylsilane (0.00 ppm) as the internal standard using a JEOL JMM-ECS-400 spectrometer at room temperature.
Compounds. Heptelidic   Intestinal loop study. Male BALB/c mice were purchased from Charles River Laboratories Japan, Inc.
BALB/c mice were sacrificed, and the small intestine was divided into 3 pieces, with each end ligated with silk sutures and the loops filled with DMEM supplemented with 10% (vol/vol) FBS, 2 mM l-glutamine, 50 U/ml penicillin and 50 µg/mL streptomycin containing heptelidic acid or 5-FU. Loops were incubated for 2, 6 and 18 h at 37 °C in a 5% CO 2 incubator. The outside media were collected and filtrated by a 0.45-nm membrane filter. Collected media were administered to SUIT-2 cells, and the growth changes were monitored by an SRB assay.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. At 24 h before stimulation, SUIT-2 cells were plated on a Lab-Tek® Chamber Slide™ (ThermoFisher Scientific). Heptelidic acid was diluted by culture media to 1 μg/mL and applied to the cells. At 72 h after stimulation, the slides were fixed in 4% paraformaldehyde and washed extensively with PBS. The slides were stained using an In Situ Cell Death Detection Kit and TMR red (Roche Diagnostic, Indianapolis, IN, USA) according to the manufacturer's instructions. The cells were mounted with an anti-fade mounting medium, and the TUNEL-positive cells were visualized by fluorescence microscopy (KEYENCE Corporation, Osaka, Japan).

Statistical analyses.
The assay data were analyzed using Student's t-test. P values of < 0.05 were considered to indicate statistical significance.

Results
The tumor-suppressive effects of probiotic culture supernatant. To  www.nature.com/scientificreports/ B. longun. B. adolescentis]) were cultured in each media, and the culture supernatant was collected by centrifugation and membrane filtration. An SRB assay revealed a growth inhibition effect in SUIT-2 cells following treatment with the culture supernatant of A. oryzae, L. casei, L. fermentum, L. coryniformis or LGG (Fig. 1A). The culture supernatant of A. oryzae, which exhibited the strongest tumor-suppressive effect, was administered to other pancreatic cancer cell lines (MIA-PaCa-II and PANC-1 cells), and a strong tumor-suppressive effect was confirmed in these cells (Fig. 1B,C), suggesting that A. oryzae produced anti-tumor molecules against pancreatic cancer cells. These data suggest that A. oryzae-derived molecules inhibited the pancreatic cancer progression.

The fractionation of culture supernatant of A. oryzae.
To determine the size of the anti-tumor molecules derived from A. oryzae, the culture supernatant was separated by the MWCO membrane column. An SRB assay showed that the flowthrough fraction of 3 kDa maintained a tumor-suppressive effect against SUIT-2 cells ( Fig. 2A), suggesting that the size of the anti-tumor molecule derived from A. oryzae was < 3 kDa.
To separate the low-molecular-weight fraction, a gel filtration column for the separation of low-molecularweight molecules, such as peptide, was used for fractionation by high-performance liquid chromatography (HPLC). Fraction 17 (Fr17) showed the strong tumor-suppressive effect (Fig. 2B). To fractionate the collected fraction, reverse-phase chromatography using a C18 column was used. Surprisingly, the tumor-suppressive molecules derived from A. oryzae were strongly captured by the C18 column (Fr78, 79) (Fig. 2C). For the further separation of the collected fraction, a C8 column was used, and Fr68 showed a tumor-suppressive effect (Fig. 2D). Finally, the HPLC spectrum of the collected fraction was assessed, showing a single peak. This suggested that the anti-tumor molecules derived from A. oryzae were enriched in the collected fraction (Fig. 2E).  Fig. S1). LC-high-resolution mass spectrometry (HRMS) analysis was performed to determine the exact mass of the tumor-suppressive molecules (Fig. 3A), and 11 formulae were highlighted as candidates (Table 1). These 11 formulae were searched in Pubchem, and heptelidic acid was found (https:// pubch em. ncbi. nlm. nih. gov/ compo und/ Hepte lidic-acid; structure of hepte- www.nature.com/scientificreports/  www.nature.com/scientificreports/ lidic acid described in Fig. 3B). A previous investigation suggested that heptelidic acid was a metabolite of A. oryzae 24 . To determine whether or not heptelidic acid was contained in the fraction, LC and an NMR analysis were performed. LC analysis showed that the spectrum of the sample fraction was consistent with that of heptelidic acid (Fig. 3C). The NMR analysis showed that the spectrum of heptelidic acid 25 corresponded with that of the sample fraction (Fig. 3D). These data suggested that heptelidic acid was the molecule derived from A. oryzae responsible for the inhibition of pancreatic cancer cell growth.

Heptelidic acid exhibited a tumor-suppressive effect against pancreatic cancer in vitro and in vivo.
To confirm the tumor-suppressive effects of heptelidic acid in pancreatic cancer cells, an SRB assay was performed. The growth of SUIT-2, MIA-PaCa-II and PANC-1 was significantly suppressed by heptelidic acid in a concentration-dependent manner ( Fig. 4A-C). To confirm whether or not heptelidic acid exerts tumorsuppressive effects in vivo, SUIT-2 cells was transplanted into nude mice, and heptelidic acid was directly injected into the transplanted tumor daily. The tumor size was significantly suppressed by the administration of heptelidic acid (Fig. 4D). These data suggest that heptelidic acid exerts a tumor-suppressive effect in vitro and in vivo.
To clarify whether or not heptelidic acid is absorbed from the intestinal tract to affect the pancreas, the small intestine of mice was resected, and heptelidic acid or 5-fluorouracil (5-FU), which are used as oral anti-tumor agents in the clinical therapy for pancreatic cancer, was enclosed in the intestinal loop. The loops were incubated in the culture media and the outside media were administered to SUIT-2 cells, and then the growth changes were investigated. The SRB assay showed the growth-suppressing effect of the medium outside of heptelidic acid or the 5-FU enclosed intestinal loop in a concentration-and treatment time-dependent manner (Fig. 4E). This suggests that heptelidic acid inhibits the tumor progression in extra-intestinal organs, including the pancreas, via the intestinal absorption of the A. oryzae-derived tumor-suppressing molecule heptelidic acid and subsequent delivery of that molecule to extra-intestinal organs.
Heptelidic acid exerted a tumor-suppressive effect through activating p38 MAPK signal transduction, thereby mediating apoptosis of the pancreatic cancer cells. To assess the mechanisms underlying the tumor-suppressive effect of heptelidic acid, the expression of the apoptosis-related molecule poly[ADP]-ribosepolymerase (PARP) and cell cycle-related molecules cyclin D1 and B1 was evaluated.
Western blotting indicated the augmentation of cleaved PARP and cyclin B1 and downregulation of cyclin D1 (Fig. 5A), suggesting that heptelidic acid exerted tumor-suppressive functions by mediating the dysregulation of the cell cycle and induction of apoptosis. Likewise, TUNEL staining revealed that heptelidic acid induced apoptotic reactions, including DNA fragmentation, in pancreatic cancer cells (Fig. 5B). To clarify the intracellular significance of heptelidic acid treatment, the phosphorylation of p38, Akt, JNK GSK3β and ERK was assessed. Western blotting indicated that levels of phosphor-p38 were significantly increased, while levels of phosphor-Akt were significantly decreased (Fig. 5C). To investigate whether or not heptelidic acid induced an tumor-suppressive effect by p38 activation, SB203580, which is an inhibitor of p38 MAPK signaling, was administered to SUIT-2 cells, and the cell growth inhibition effect of heptelidic acid was assessed. An SRB assay indicated that the growth suppression rate by heptelidic acid was significantly decreased by treatment with SB203580 (Fig. 5D), suggesting that heptelidic acid exerted its tumor-suppressive effect via the induction of apoptosis of pancreatic cancer cells by p38 MAPK signaling transduction.

Discussion
The present study revealed that conditioned media of A. oryzae exerted a strong tumor-suppressive effect against pancreatic cancer cells, suggesting that A. oryzae released tumor-suppressive molecules into the conditioned media. After the separation of the conditioned media by several columns, heptelidic acid was enriched in the tumor-suppressive fraction. The tumor-suppressive effects of heptelidic acid were confirmed in pancreatic cancer cells in vitro and in an in vivo xenograft model, and the mechanisms underlying the tumor-suppressive functions were shown to be apoptosis mediation followed by the activation of the p38 MAPK signaling pathway. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Through our study, we demonstrated the mechanism underlying the host-microbe communication that brings about health benefits, including anti-tumor effects in organs separate from the gastrointestinal tract, such as the pancreas, through mediation by probiotic-derived molecules (Fig. 6).
A. oryzae has been used to produce fermented food, such as soy sauce, and fermented liquor and is known to exert anti-tumor effect for intestinal cancer cells. However, the mechanism underlying this bacterial anti-tumor effect has been unclear. We identified several anti-tumor molecules produced by bacteria, so the effect A. oryzae was thought to be mediated by bacterial bioactive molecules. After the separation of A. oryzae culture supernatant by HPLC, heptelidic acid was identified in the isolated tumor-suppressive fraction by LC-MS and an NMR analysis. Our xenograft study showed the strong tumor-suppressive effect of heptelidic acid in vivo. TUNEL staining and Western blotting revealed that heptelidic acid induced the apoptosis of pancreatic cancer cells. In addition, the cell cycle-related molecules cyclin D1 and B1 were dysregulated by treatment with heptelidic acid, suggesting that heptelidic acid induced an abnormal cell cycle, resulting in the accumulation of cell cycle-arrested cells and the subsequent apoptosis of pancreatic cancer cells.
Heptelidic acid was first identified as a sesquiterpene antibiotic derived from three strains of fungi: Gliocladium virens, Chaetomium globosum and Trichoderma viride 26 . It was also isolated from the bacterial culture of Figure 5. Heptelidic acid exerted anti-tumor effects mediating the p38 MAPK signaling pathway in pancreatic cancer cells. A Western blotting analysis indicated that the expression of cleaved PARP and cyclin B1 was increased, and that of cyclin D1 was decreased in SUIT-2 cells by treatment with 1 μg/mL of heptelidic acid (A). TUNEL staining showed that the DNA fragmentation of SUIT-2 cells was induced by treatment with 1 μg/mL of heptelidic acid (B). Western blotting showed that the phosphorylation of p38 MAPK was augmented while that of Akt was attenuated by treatment with 1 μg/mL heptelidic acid (C). The SRB assay showed that the growth inhibition rate of heptelidic acid was decreased by treatment with SB203580 (D). The error bars show the standard deviation (S.D.) (A; p-p38; n = 6, p-Akt, p-JNK, p-GSK3β, p-ERK; n = 3) (C,D; n = 3) (B; n = 4).  30 . Likewise, p38 MAPK signaling is known to be a key regulatory signaling pathway of glycolysis [31][32][33] in pancreatic cancer cells and our findings showed that heptelidic acid exerted an anti-tumor effect which was mediated by p38 signal transduction. These findings suggest that anticancer therapy, especially pancreatic cancer treatment, utilizing heptelidic acid may prove effective. Our SRB assay showed the anti-tumor effect when heptelidic acid was directly administered to pancreatic cancer cells. Of note, to exert its anti-tumor effect in distant organs, such as pancreatic cancer, from the intestinal tract, heptelidic acid must transit the intestinal tract and reach the pancreatic tissue. A previous investigation showed that microbial metabolites, such as short-chain fatty acids (SCFAs), but not the bacterial body, were able to pass through the intracellular space of the intestinal tract and modulate the cellular functions of distant organs, including the pancreas 34 . An MS analysis showed that the molecular size of heptelidic acid was only 280.13. Furthermore, heptelidic acid was considered to have high lipophilicity because the heptelidic acid was enriched when separated by a reverse-phase column, which was selected based on the degree of lipophilicity. These suggests that heptelidic acid can pass through the cell membrane, escape the intestinal tract, enter the blood stream and reach cancer cells in extra-intestinal organs, including the pancreas.
Western blotting showed that the cell cycle regulator cyclin B1 was significantly augmented by heptelidic acid treatment. The expression of cyclin B1 is known to increase in the G2 phase, where it forms a complex with Cdk1. The formation of Cyclin 1-CDK1 complex is an indispensable step for the cell cycle to transform into the M phase 35,36 . Heptelidic acid also irreversibly inhibits GAPDH 27 , which promotes the production of ATP mediating glycolysis, thereby accelerating the cell cycle by modulating the cyclin B1-cdk1 activity 37 . These findings indicate that cell cycle arrest, especially in the G2/M phase, was induced by heptelidic acid, resulting in an increase in cyclin B1.
It was previously reported that metabolic syndrome, including diabetes, is associated with the development of pancreatic cancer 16 . Interestingly, microbiota transplantation from obese mice to germ-free mice reportedly resulted in a significant increase in total body fat 38 , and fecal transplantation from pancreatic cancer patients with a short-term survival (STS) and long-term survival differentially influenced the immune response and natural history of the disease 21 . Likewise, soybean foodstuffs fermented by A. oryzae exhibited a preventative effect on obesity by modulating the insulinotropic action in diabetic rats 39 . These findings suggest that the probiotic A. oryzae produces anti-obese molecules as well as anti-tumor molecules, such as heptelidic acid, helping prevent diabetic diseases and inhibit cancer growth.
In conclusion, we showed that heptelidic acid released by A. oryzae exerted anti-tumor functions by inducing apoptosis mediated by p38 signaling activation in pancreatic cancer cells. Furthermore, heptelidic acid exerted