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.

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).

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).

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).

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.

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.

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).