The butyrate derived from probiotic Clostridium butyricum exhibits an inhibitory effect on multiple myeloma through cell death induction

Abstract

Multiple myeloma (MM) is a hematological malignancy characterized by a poor prognosis. While certain probiotics have been shown to produce antitumor molecules that inhibit solid tumor progression, it remains unclear whether probiotic-derived compounds can exert similar effects on hematological tumors, such as MM. In this study, we screened the cell-free culture supernatants (CFCS) of 24 probiotic strains for antitumor effects against multiple myeloma (MM) cells and identified that the CFCS from Clostridium butyricum (C. butyricum) demonstrated the most significant reduction in MM cell viability. Further fractionation of this CFCS through reverse-phase and gel filtration chromatography revealed a high enrichment of butyrate in the antitumor fraction, as confirmed by gas chromatography-mass spectrometry. Butyrate reduced MM cell viability in a concentration-dependent manner. Butyrate was significantly more cytotoxic to RPMI-8226 cells than peripheral blood mononuclear cells (PBMCs) isolated from two non-cancerous individuals. In the xenograft model of RPMI-8226 cells, butyrate showed significant inhibition of tumor formation. Cell cycle analysis showed that butyrate induced G1 phase arrest and increased sub-G1 phase, suggesting DNA fragmentation. Western blot analysis demonstrated that butyrate treatment led to cleaved poly ADP-ribose polymerase (PARP) accumulation. Additionally, flow cytometry showed an increase in annexin V positive MM cells, indicating apoptosis. Butyrate also exhibited synergistic antitumor activity when combined with bortezomib, a proteasome inhibitor. These findings suggest that probiotic-derived molecules, including butyrate, may enhance the therapeutic effect of hematological malignancy, such as MM.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-97038-8.

Introduction

Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells, a type of cells responsible for antibody production¹. MM is characterized clonal proliferation of abnormal plasma cells in the bone marrow, leading to the production of monoclonal proteins, bone lesions, anemia, and renal dysfunction^2,3. Despite the progress in treatment, including proteasome inhibitors (PIs) and immunomodulatory drugs, and anti-CD38 monoclonal antibodies, MM remains incurable with a 5-year survival rate of approximately 55–60%^4,5. Thus, a novel therapeutic strategy has been desired.

Probiotics, defined as live microorganisms that confer health benefits to the host when administered in adequate amounts, have been highlighted because of their role in modulating the immune system and maintaining gut homeostasis⁶. Previous studies demonstrated that probiotics can influence the pathogenesis, progression, and therapeutic outcomes of solid cancers, such as gastrointestinal cancer^7,8. These anti-tumor effects of probiotics have generally been considered indirect, primarily due to their modulation of the intestinal microbiome. Meanwhile, some probiotics have been found to produce molecules that directly act on cancer cells, demonstrating anti-tumor effects. Tsai TL et al. identified anti-tumor peptides, m2163 and m2386, from the culture supernatants of Lactobacillus casei (L. casei), which directly induced cell death in colon cancer cells⁹. Additionally, Hayasaka S et al. reported that Bacillus subtilisproduces a natto peptide that inhibits the growth of HeLa cells¹⁰. We have also demonstrated that ferrichrome, produced by L. casei, exhibits anti-tumor effects against colon, pancreatic, gastric, and esophageal cancers^11–14, while heptelidic acid, produced by Aspergillus oryzae (A. oryzae), shows efficacy against pancreatic cancer and melanoma^15,16. Notably, heptelidic acid was found to cross the intestinal barrier, exerting its antitumor effects in distant organs^15,16. This provides clear evidence of a host-bacteria interaction, where probiotic-derived molecules directly act on tumor cells, exerting anti-tumor effects in the gastrointestinal tract as well as in distant tissues and organs. However, their role in hematological malignancies, including multiple myeloma (MM), remains unclear.

In this study, we aimed to evaluate the therapeutic potential of probiotic molecules for MM treatment. We investigated the effects of cell-free culture supernatants (CFCS) of 24 strains of probiotics on MM cells and their potential to inhibit MM cell proliferation. By fractionating the culture supernatant and conducting gas chromatography-mass spectrometry (GC-MS) analysis, we identified butyrate is responsible for the inhibition of MM cell growth and the induction of cell death. Butyrate showed a synergistic antitumor effect with bortezomib, a proteasome inhibitor. We have demonstrated the potential that probiotic-derived molecules may be therapeutic agents against hematological malignancies.

Materials and methods

Cell culture

RPMI-8226 (RRID: CVCL_0014) and KMS-27 (RRID: CVCL_2993) cells were purchased from Japanese Collection of Research Bioresources (JCRB) and grown in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin in a humidified atmosphere containing 5% CO₂. All human cell lines have been authenticated using STR (or SNP) profiling within the last three years.

Microorganisms

Microorganisms were purchased from the American Tissue Culture Collection (ATCC) or NITE Biological Resource Center (NBRC). These bacteria were cultured in appropriate media and atmosphere for one to three days until OD₆₀₀ exceeded 1.0. The bacterial culture conditions are described in Supplemental Table 1. The CFCSs of each bacterium were obtained by filtration through a 0.2-µm membrane following centrifugation at 5000 ×g for 10 min.

Peripheral blood mononuclear cells (PBMCs)

Blood samples were collected from the peripheral veins of non-cancerous human volunteers. This study was approved by the ethics committee of Asahikawa Medical University (Approval Number: 19213), and informed consent was obtained from each volunteer. All experiments were performed in accordance with relevant guidelines and regulations. The blood samples were drawn into tubes containing 3.2% sodium citrate as an anticoagulant. The samples were then diluted with PBS at a 1:1 ratio (PBS: blood). Next, 6 mL of the diluted blood was carefully layered over 3 mL of Lymphoprep™ (Serumwerk Bernburg) in a 15 mL tube. The tubes were centrifuged at 800 × g for 20 min using a swinging rotor at 20 °C. After centrifugation, the mononuclear cell layer at the interface of Lymphoprep™ was carefully collected, suspended in PBS, and centrifuged again at 250 × g for 10 min to isolate PBMCs.

The isolation of the tumor-suppressive molecule

The CFCS of C. butyricum was separated using an AKTA Design HPLC system (GE Healthcare). The fraction was applied to a Wakopak® Wakosil® C18 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 fraction was further separated using a Superdex peptide column (GE Healthcare) and eluted with distilled water at a flow rate of 1 ml/min.

MTT assay

The cells were seeded on 96-well microplates at 2.0 × 10⁴ per well/90µL, and then 10µL/well of each CFCS or fractions were treated to the cells. Cell growth was assessed using an MTT cell proliferation kit (Roche Applied Science) according to the manufacturer’s instructions. The optical density (OD) was measured at a 590 nm test wavelength and a 650 nm reference wavelength.

Gas chromatography-mass spectrometry

The CFCS or isolated fractions were diluted 1:50 with distilled water. An SPME fiber (85 μm Carboxen/PDMS, Merck) was immersed in the sample for 1 min before injection into a GC-MS (JEOL JMS T100 GCV). The GC-MS analysis parameters were as follows: separation was achieved using a DB-WAX fused silica capillary column (30 m x 0.25 mm x 0.25 μm, Agilent Technologies, USA), with helium as the carrier gas at a flow rate of 1.5 mL/min. Injector temperatures were set to 280 °C. The oven temperature was initially set to 40 °C for 3 min, then increased to 250 °C at a rate of 16 °C/min and held for 5 min. The transfer line temperature was set to 250 °C. Mass spectrometry was conducted in electron impact (EI) mode at 70 eV with a source temperature of 250 °C, covering a mass range of 40–500 m/z. Selected ion monitoring focused on m/z 73. For the quantification of butyrate contained in the CFCS, a standard fitting curve was generated using peak area of 10, 50, 200 µg/ml of butyrate.

Compounds

Butyrate and bortezomib were purchased from Wako Pure Chemical and Tokyo Chemical Industries, respectively.

Xenograft

Severe combined immunodeficient (SCID) mice, aged 6 weeks, were purchased from The Jackson Laboratory, Japan. The back region of female mice was subjected to a subcutaneous injection of RPMI-8226 cells (3 × 10⁶ cells). 2% Butyrate or distilled water was administered by drinking water for 5 days on a 2-day off cycle. The tumor sizes were calculated from digital caliper raw data using the formula: Volume = (major tumor diameter) × (minor tumor diameter)²/2. Mice were euthanized by cervical dislocation following anesthesia with 5% isoflurane for induction.

Cell cycle analysis

The cells were collected using centrifugation (2,000 rpm, 5 min), washed with PBS, and fixed in cold ethanol. They were then treated with RNase A and stained using a propidium iodide (PI) solution (propidium iodide 50 µg/ml), and then 20,000 cells were analyzed using a SONY SH800S cell sorter.

Western blotting

Total proteins were extracted from samples using a NP-40 cell lysis buffer (ThermoFisher Scientific) containing cOmplete ™ Protease Inhibitor Cocktail (Sigma-Aldrich). 15 µg of total protein were resolved using SDS-PAGE (12.5%), blotted onto a nitrocellulose membrane, and then blocked in SuperBlock™ (PBS) Blocking Buffer (ThermoFisher Scientific). The blots were incubated overnight at 4℃ with primary antibodies. The primary antibodies of PARP (#9542) and actin (#612656) were purchased from Cell Signaling Technology and BD Transduction Laboratories, respectively. All antibodies were diluted in 1/1000 in SuperBlock™ (PBS) Blocking Buffer and incubated with blots overnight at 4℃. The blots were washed in 0.05% Tween-20 containing PBS (T-PBS), incubated with HRP conjugated secondary antibodies (R&D systems, Minneapolis, MN), washed in T-PBS, and then developed using the Super-Signal West Pico enhanced chemiluminescence system (Thermo Science). The averaged protein expression was normalized to the actin expression.

Flow cytometry of Annexin V-stained cells

The cells were collected using centrifugation (2,000 rpm, 5 min), washed with PBS, stained using APC Annexin V Apoptosis Detection Kit (BioLegend), and then 20,000 cells were analyzed using SONY SH800S cell sorter.

Synergy/antagonism score analysis

The degree of combination synergy or antagonism between drugs was assessed using Synergistic Finder software¹⁷. The highest single agent (HSA) models quantify the degree of synergy as the excess over the maximum single-drug response. Values less than − 10 suggest that the interaction between two drugs is likely to be antagonistic, values between − 10 and 10 indicate an additive interaction, and values larger than 10 suggest a likely synergistic interaction between two drugs. The raw dose-response matrix data is visualized as a heatmap. The synergy maps indicate synergistic regions in red and antagonistic regions in green.

Statistical analysis

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

Result

Clostridium butyricum reduces MM cell proliferation

To identify the probiotics with therapeutic potential against myeloma, the cell-free culture supernatants (CFCSs) of 24 probiotic strains were diluted 10-fold using culture media and treated to myeloma cell lines, RPMI-8226 and KMS-27. MTT assay showed that the CFCS of four probiotic strains, L. casei, L. fermentum, L. coryniformis, and C. butyricum, significantly reduced the cell viability of both myeloma cell lines. Among them, the CFCS of C. butyricum showed the most significant antitumor effect, reducing cell viability at 72 h to 12 ± 1% for RPMI-8226 and 31 ± 1% for KMS-27 compared to the untreated control (Fig. 1A, B).

Fig. 1.

Conditioned media derived from probiotics reduced the progression of MM cells. An MTT assay revealed that the viability of MM cells, RPMI-8226 (A) and KMS-27 (B) were significantly lower in the conditioned media from the L. casei, L. fermentum, L. coryniformis, and C. butyricum-treated groups than in the control group. The strongest cytotoxicity against MM cells was observed in the conditioned media from the C. butyricum group. The error bars show the standard deviation (S.D.) (n = 3).

The volatile molecule produced from C. butyricim reduced the cell viability of MM cells

Following fractionation of the CFCS of C. butyricum, we found that Fr54 separated using the C18 reverse-phase column exhibited the most significant inhibitory effects on cell proliferation of RPMI-8226 cells (Fig. 2A). Fr54 was further separated using a gel filtration column. Fr21 of the gel filtration column showed the most significant antitumor effect against RPMI-8226 cells (Fig. 2B). Fr21 of the gel filtration column was freeze-dried and the reconstituted fraction was treated to RPMI-8226. The antitumor effect of the freeze-dried fraction was completely diminished, indicating that the volatile molecule from C. butyricum was an antitumor mediator against RPMI-8226 cells (Fig. 2C).

Fig. 2.

The separation of the tumor-suppressive fraction from the CFCS of C. butyricum. The CFCS of C. butyricum was separated using reverse-phase chromatography and the most significant antitumor function against RPMI-8226 cells was identified in the Fr54 (A). The fractions were further separated using size-exclusion chromatography and the most significant antitumor function against RPMI-8226 cells was identified in the Fr21 (B). The antitumor activity of the freeze-dried fraction was completely diminished against RPMI-8226 cells (C). The error bars show the S.D. (n = 3).

Butyrate produced from C. butyricum is an antitumor mediator against MM cells

To identify the antitumor molecule from C. butyricum, a gas chromatography-mass spectrometry (GC-MS) analysis was performed. GC-MS analysis of the active fraction showed that butyrate, a class of short-chain fatty acid (SCFA), was highly enriched in the fraction (Fig. 3A). Subsequently, the concentration of butyrate in the CFCS of C. butyricum was evaluated. 0.1% of butyrate was contained in CFCS of C. butyricum (Fig. 3B, C), indicating 0.01% of butyrate was treated to MM cells in Fig. 1A and B. 0.01% of butyrate exerted comparable cytotoxicity with CFCS of C. butyricum diluted in1/10 against RPMI-8226 and KMS-27 cells (Fig. 3D, E). Notably, the cytotoxicity of butyrate was selectively exerted in MM cells compared to PBMCs (Fig. 3F). These findings suggest that butyrate is the key antitumor mediator produced by C. butyricum, selectively targeting MM cells while sparing normal immune cells.

Fig. 3.

Butyrate, contained in the CFCS of C. butyricum, was an antitumor mediator against MM cells. The gas chromatography-mass spectrometry spectrum of the tumor-suppressive fraction is shown (A). The butyrate concentration of culture supernatant was calculated using a fitting curve (B, C). An MTT assay revealed that the viability of RPMI-8226 (D) and KMS-27 (E) were significantly reduced by butyrate treatment in a concentration-dependent manner. Butyrate inhibited proliferation of PBMC less than RPMI-8226 cells (F). The error bars show the S.D. (n = 3).

Butylate exerts antitumor effect in vivo RPMI-8226 xenograft model

To assess the antitumor effects of butyrate in vivo, RPMI-8226 cells were engrafted to the back of SCID mice, and then butyrate was administered in the drinking water. The tumor formation was significantly inhibited in the butyrate treatment group compared to the control group (Fig. 4).

Fig. 4.

Butyrate suppressed tumor formation of RPMI-8226 transplanted mice. In the xenograft model, the enlargement of the tumors in the butyrate-treated group was suppressed, while the tumors in the control group became enlarged (Control: n = 5, Butylate: n = 6). The error bars show the standard deviation (S.D.). *p < 0.05 by Student’s t-test.

Butyrate induces apoptosis in MM cells

Cell cycle analysis was performed to assess the mechanism of action of butyrate that mediates the cytotoxicity against MM cells. 0.01% of butyrate induced the G1 arrest and accumulation of MM cells in sub-G1 (Fig. 5A), indicating that butyrate induced the programmed cell death (PCD) and DNA fragmentation of MM cells. Western blotting analysis indicated that cleaved PARP, a landmark of PCD, was significantly induced by the treatment of 0.01% of butyrate in RPMI-8226 cells (Fig. 5B, unprocessed images were presented in Supplemental Fig. 1). Flow cytometry analysis indicated the annexin V-positive cells were increased by 0.01% of butyrate in RPMI-8226 cells (Fig. 5C). These indicate that butyrate induced apoptosis, exerting antitumor effects on MM cells.

Fig. 5.

Butyrate induced apoptosis in MM cells. Flow cytometry showed that the accumulated RPMI-8226 cells were in the G1 of the cell cycle and sub-G1 after 24 h of 0.01% of butyrate treatment (Left: Control, Right: Butyrate) (A). Western blotting revealed that the cleavage of PARP was significantly increased after 24 h of 0.01% of butyrate treatment (B) (n = 3). Annexin V staining indicated that apoptosis was induced by the treatment of 0.01% butyrate in RPMI-8226 cells (Left: Control, Right: Butyrate) (C). The error bars show the S.D. (n = 3). *p < 0.05 by Student’s t-test.

Butyrate is synergistic with bortezomib

To determine whether butyrate could contribute to the drug responsiveness of MM treatment, we examined the additive and synergistic effects of bortezomib and butyrate. Bortezomib and butyrate exerted cytotoxicity against RPMI-8226 cells in a concentration-dependent manner (Fig. 6A), and low doses of butyrate and bortezomib treatment were found to act synergistically (Fig. 6B).

Fig. 6.

Butyrate exerted synergistic therapeutic efficacy with bortezomib. An MTT assay revealed the concentration-dependent cytotoxicity of butyrate and bortezomib against RPMI-8226 (A). The synergy map indicated that butyrate and bortezomib exerted synergistic cytotoxicity against RPMI-8226 cells (B).

Discussion

In this study, we showed that probiotics can suppress the progression of MM cells. Probiotic-derived butyrate, a type of short-chain fatty acid (SCFA), exerts a direct antitumor effect on MM cells and acts synergistically with bortezomib, which is used for standard of care in MM treatment. This synergistic interaction suggests that probiotic molecules could enhance the therapeutic effect of refractory hematological malignancy, such as relapsed MM.

The mammalian intestinal tract is the largest immune organ in the body and comprises cells from non-hemopoietic (epithelia, Paneth cells, goblet cells) and hemopoietic (macrophages, dendritic cells, T cells) origin, and is also a dwelling for trillions of microbes collectively known as the microbiota¹⁸. While complex cellular interactions occur, there are three possible mechanisms by which bacteria-derived molecules can inhibit cancer progression in the host: activation of the host immune system, increased production of secondary messengers in host cells, and direct actions on tumor cells. Recent studies highlight the ability of bacteria-derived molecules to exert anticancer effects directly. We demonstrated that ferrichrome, a molecule produced by L. casei, directly interacts with colon cancer cells, activates the JNK signaling pathway, and induces apoptosis, leading to an anti-cancer effect¹¹. Similarly, it has been shown that heptelidic acid derived from A. oryzaecan pass the gastrointestinal tract and reach distant organs when administered orally^15,16. These findings suggest that bacterial-derived molecules can circulate throughout the body, reach various organs, and exert therapeutic effects by directly acting on the cells of the targeted tissues. On the other hand, SCFA, a bacterial metabolite, was thought to suppress the progression of tumors indirectly by influencing intestinal immunity¹⁹. However, Park et al. found that propionate, a type of SCFA, directly reduced the proliferative potential of breast cancer cells in vitro. Moreover, they showed that oral administration of propionate to mice with breast cancer suppressed tumor growth²⁰. These suggest that SCFA has dual antitumor functions by activating the immune systems of the host and directly suppressing the tumor cell growth. The anti-tumor effect of butyrate observed in this study is thought to result from its direct action on MM cells. Our analysis indicated that butyrate directly induced apoptosis for its antitumor functions against MM cells. Additionally, our xenograft study also shows that oral administration of butyrate to mice exerts an anti-myeloma effect, which is expected to inhibit tumor growth systemically. Interestingly, blood levels of butyrate were higher in MM patients in remission²¹, further supporting its potential role in therapy. These emphasize the broader therapeutic application of probiotic molecules that directly act on tumor cells in oncology, including MM.

We have demonstrated probiotic molecules can attenuate intestinal inflammation by modulating the production of secondary messengers from host cells. Long-chain polyphosphate produced by Lactobacillus brevisacts on intestinal epithelial cells, macrophages, and fibroblasts, modulating the production of cytokines and Transforming Growth Factor-β (TGF-β)²². Similarly, it induces the release of growth factors encapsulated within platelets to support epithelial cell repair²³. These findings suggest that certain probiotics may produce molecules that exert anti-tumor effects by regulating secondary messenger expression in host cells. In co-culture systems with MM cells and other cell types of the host, such as epithelial or fibroblast cells, probiotic CFCS could potentially exhibit anti-tumor effects, even though a direct anti-tumor effect was not confirmed in this study.

Our MTT assay demonstrated that butyrate inhibited the growth of MM cells while sparing on PBMCs, indicating that MM cells are more sensitive to butyrate than non-cancerous immune cells. Additionally, the administration of 2% butyrate showed antitumor effects in MM xenograft mice. Several studies have already confirmed the safety of butyrate in vivo. For example, Chen et al. reported that a high dose of butyrate (500 mg/kg) did not significantly reduce body weight in xenograft mice bearing A549 lung cancer cells²⁴. Similarly, Beisner et al. demonstrated that free access to drinking water containing 5% butyrate alleviated obesity-induced intestinal barrier dysfunction²⁵. These findings suggest that butyrate can be a safe and effective treatment option for MM.

MM patients, with a median age at diagnosis of 69 years, often experience relapses, highlighting the need for combination therapies to improve their outcomes^26,27. Our study has demonstrated that probiotic-derived butyrate induces apoptosis in MM cells, acting synergistically with the proteasome inhibitor, bortezomib. Thus, the combination of butyrate and bortezomib showed potential as a treatment option for patients with relapsed and refractory MM. These offer the clinical potential of probiotic molecules to enhance outcomes in older MM patients.

Butyrate has been suggested to inhibit proteasome activity by blocking histone deacetylase activity in human colon cells²⁸. Bortezomib, on the other hand, inhibits proteasome function by reversibly binding to the β5 subunit with chymotrypsin-like activity and the β1 subunit with caspase-like activity, leading to the accumulation of ubiquitinated proteins and subsequent cellular stress, which induces apoptosis in tumor cells. When butyrate and bortezomib are administered simultaneously, their distinct mechanisms of proteasome inhibition are thought to act synergistically to enhance antitumor effects. Furthermore, butyrate has been widely reported to induce autophagy²⁹, which can promote programmed cell death by triggering apoptosis³⁰. The activation of apoptosis through multiple pathways may explain the observed synergistic effect.

Butyrate has been reported to exert cytotoxic effects on various cancer cells, including colorectal³¹ and breast cancer³². In our study, butyrate demonstrated a strong anti-myeloma effect by inducing apoptosis in MM cells while exhibiting lower cytotoxicity toward PBMCs from non-cancerous individuals. This finding implies that butyrate could be utilized as part of combination therapy to improve patient outcomes. Although further studies are necessary to compare antitumor effects of butyrate across different cancer types comprehensively, its strong anti-tumor activity highlights its potential as a promising therapeutic agent for various cancers.

This study demonstrated that probiotic-derived butyrate has a therapeutic effect on MM, a refractory hematologic malignancy. Proteasome inhibitors such as bortezomib and carfilzomib have been shown to be effective in treating diffuse large B-cell lymphoma (DLBCL)³³. Since butyrate exhibits proteasome-inhibitory activity and has shown a synergistic effect with bortezomib, as mentioned above, it may also have therapeutic potential for DLBCL and other hematologic malignancies beyond MM.

Our MTT assays have demonstrated that several CFCSs as well as CFCS of C. butyricum exhibit cytotoxic effects on two MM cell lines. L. casei has been shown to mediate antitumor activity in gastrointestinal cancers through three specific molecules, m2163, m2386, and ferrichrome, suggesting that these may exert therapeutic effects against MM. The CFCS of L. fermentumhas displayed antitumor effects against colorectal cancer³⁴, while L. coryniformis has been reported to regulate intestinal function and stimulate an immune response^35,36, though its antitumor potential in MM remains unknown. Future analysis may reveal how these bacteria exert their anticancer effects on MM.

Conclusion

We demonstrated the cytotoxicity of butyrate produced by probiotic C. butyricum against MM cells. Butyrate induced the cell cycle arrest in G1 phase and induced apoptosis of MM cells. Butyrate enhanced the antitumor effect of bortezomib. These findings indicate that butyrate may be a promising chemotherapeutic for treating MM.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Hiroaki Konishi: Conceptualization; Funding acquisition; Investigation; Supervision; Writing—original draft. Takeshi Saito: Conceptualization; Writing—review & editing. Shuichiro Takahashi: Writing—review & editing. Hiroki Tanaka: Methodology; Validation. Katsuhiro Okuda: Investigation; Methodology. Hiroaki Akutsu: Investigation; Methodology. Tatsuya Dokoshi: Writing—review & editing. Aki Sakatani: Writing—review & editing. Keitaro Takahashi: Writing—review & editing. Katsuyoshi Ando: Writing—review & editing. Nobuhiro Ueno: Writing—review & editing. Shin Kashima: Writing—review & editing. Kentaro Moriichi: Funding acquisition; Investigation; Writing—review & editing. Naoki Ogawa: Resources; Writing—review & editing. Mikihiro Fujiya: Conceptualization; Funding acquisition; Project administration; Resources; Supervision, Writing—review & editing.

Funding

This paper was supported by Grants-in-Aid for Scientific Research, No. 21 KK0291 (H. Konishi), 22 K15363 (H. Konishi), 21 K07929 (M. Fujiya) and 22 K08047 (K. Moriichi), Intractable Disease Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare (M. Fujiya), and Hirose Foundation (H. Konishi).

Data availability

The data that support the findings of this study are available from the corresponding author, H.K., upon request.

Declarations

Competing interests

Hiroaki Konishi and Mikihiro Fujiya report that Kamui Pharma Inc. provides financial support. They are part of a joint research department funded by Kamui Pharma Inc. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics statement

All experiments were performed according to the Guidelines of the Public Health Service Policy on the Humane Use and Care of Laboratory Animals. The study received ethical approval for using an opt-out methodology from the Medical Ethics Committee of Asahikawa Medical University (Approval No. R6-005). All animal experiments were reported in accordance with the ARRIVE guidelines.