Abstract
Activation of inflammasome leads to the formation of an inflammatory microenvironment which plays an important role in the process of cancer development. Beta-hydroxybutyrate (BHB) is a ketone body that has recently been reported to exert anti-inflammatory effects via inhibition of NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. Here, we investigated the potential influence of BHB on the in vitro migration of C6 glioma cells and the activation of NLRP3 inflammasome. Our results indicated that administration of BHB suppressed C6 cells migration and NLRP3 inflammasome activation, reducing the levels of activated cysteinyl aspartate-specific proteinase 1 (caspase-1) and mature Interleukin 1β (IL-1β). Fully activation of NLRP3 inflammasome was induced by lipopolysaccharide (LPS) prime plus adenosine triphosphate (ATP) stimulation in C6 cells, which promoted in vitro migration of C6 cell. BHB also counteracted the LPS/ATP-promoted cell migration by suppressing the activation of caspase-1 and the maturation of IL-1β. The enhancement of phospho-signal transducer and activator of transcription 3 (p-STAT3), degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) as well as the overexpression of fibroblast growth factor 2 (FGF2) resulting from LPS/ATP treatment, and subsequent IL-1β maturation could also be compensated by BHB. Our results suggested that BHB inhibits the activation of NLRP3 inflammasome in C6 glioma cells and consequently suppressed the C6 cell migration. These findings also implicated that by inhibiting NLRP3 inflammasome, BHB reduced the inflammatory microenvironment which provided ancillary therapeutic benefits for the intervention of glioma.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Nos. 81371288, 81172170).
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Conceived and designed the experiments: LX, LH, and SS. Performed the experiments: SS and WL. Analyzed the data: SS and ZY. All authors read and approved the manuscript.
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10571_2018_617_MOESM1_ESM.tif
Supplementary Figure 1 BHB inhibits in-vitro cell migration of LN229 and U2251 cell lines. A: U251 cells; B: LN229 cells. Cells were treated with BHB for 24h, cell migration status was analyzed using Transwell chamber migration assay. The representative images were captured by phase-contrast microscope. The number of U251 and LN229 cells migrated into the down chamber were obtained from counting of five microscopic fields (up, down, left, right and middle). ∗P < 0.05, ∗∗P < 0.01, compared with the control group. (TIF 34525 KB)
10571_2018_617_MOESM2_ESM.tif
Supplementary Figure 2 BHB inhibits the activity of NLRP3 inflammasome in LN229 and U251 cells. A: U251 cells; B: LN229 cells. LN229 and U251 cells were treated with BHB for 24h, respectively. The protein levels of Pro-caspase-1, Pro-IL-1β and their mature form of caspase-1 and IL-1β were detected by Western blotting. β-actin was served as an internal control. ∗P < 0.05, ∗∗P < 0.01, compared with the control group. (TIF 88324 KB)
10571_2018_617_MOESM3_ESM.tif
Supplementary Figure 3 BHB inhibited the LPS/ATP induced cell migration in LN229 cell line. LN229 cells were pre-treated with LPS for 4 h, and then treated with BHB for 2 h, followed by 5 mM ATP simulation for 30 min. 24 h later, cell migration status was analyzed by Transwell chamber migration assay. A: The representative images were captured by phase-contrast microscope. B: The number of LN229 cells migrated into the down chamber were obtained from counting of five microscopic fields (up, down, left, right and middle). ∗P < 0.05, ∗∗P < 0.01, compared with the control group. # P <0.05, ## P < 0.01, compared with the LPS/ATP group. (TIF 23066 KB)
10571_2018_617_MOESM4_ESM.tif
Supplementary Figure 4 BHB inhibited the LPS/ATP induced NLRP3 inflammasome activation in LN229 cell line. LN229 cells were pre-treated with LPS for 4 h, and then treated with BHB for 2 h, followed by 5 mM ATP simulation for 30 min. a The protein levels of Pro-caspase-1, Pro-IL-1β and their mature form of caspase-1 and IL-1β were detected by Western blotting. β-actin was served as an internal control. b, c, d, e Densitometry of immoblots shown in a. ∗P < 0.05, ∗∗P < 0.01, compared with the control group. # P <0.05, ## P < 0.01, compared with the LPS/ATP group (TIF 41897 KB)
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Shang, S., Wang, L., Zhang, Y. et al. The Beta-Hydroxybutyrate Suppresses the Migration of Glioma Cells by Inhibition of NLRP3 Inflammasome. Cell Mol Neurobiol 38, 1479–1489 (2018). https://doi.org/10.1007/s10571-018-0617-2
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DOI: https://doi.org/10.1007/s10571-018-0617-2