Sodium butyrate has anti-proliferative, pro-differentiating, and immunomodulatory effects in osteosarcoma cells and counteracts the TNFα-induced low-grade inflammation

Abstract

Butyrate, an essential factor for colonocytes and regulator in the development of colon cancer, is partially absorbed by the gut. It influences the proliferation and differentiation of several cell types including osteoblasts. We evaluated the effects of different doses of butyrate on differentiation and functionality of osteosarcoma cells in vitro and the expression of a pro-inflammatory phenotype in a normal or inflammatory environment. SaOS-2 osteosarcoma cells were induced to differentiate and contemporarily treated for 24 h, 48 h, or 7 days with sodium butyrate 10^-4, 5 × 10^-4, or 10^-3 M in the presence or absence of tumor necrosis factor alpha (TNFα) 1 ng/mL, a pro-inflammatory stimulus. Despite the mild effects on proliferation and alkaline phosphatase activity, butyrate dose- and time-dependently induced the expression of a differentiated phenotype (RUNX2, COL1A1 gene expression, and osteopontin gene and protein expression). This was associated with a partial inhibition of nuclear factor kappa B (NF-κB) activation and the induction of histone deacetylase 1 expression. The net effect was the expression of an anti-inflammatory phenotype and the increase in the osteoprotegerin-to-receptor activator of nuclear factor kappa-B ligand (RANKL) ratio. Moreover, butyrate, especially at the highest dose, counteracted the effects of the pro-inflammatory stimulus of TNFα 1 ng/mL. Butyrate affects osteosarcoma cell metabolism by anticipating the expression of a differentiated phenotype and by inducing the expression of anti-inflammatory mediators.

Keywords: NF-κB activation; cytokines; histone deacetylases; osteosarcoma cells; sodium butyrate.

Figure 1.

Proliferation and cell viability. Effects of treatments on total DNA content of cultures (a, b) by CyQuant^® test, and cell viability, normalized to DNA content (c, d), by alamarBlue^® assay, at 24 h, 48 h, and 7 days of culture. (a) and (c) Proliferation index and cell viability, respectively, of untreated control (CTRL) and NaBu-treated cultures; (b) and (d) Proliferation index and cell viability, respectively, of CTRL, TNFα-, and TNFα + NaBu-treated cultures. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 2.

Specific activity of alkaline phosphatase (ALP). Effects of treatment on specific activity of ALP at 24 h, 48 h, and 7 days of culture. (a) ALP specific activity of untreated control (CTRL) and NaBu-treated cultures; (b) ALP specific activity of CTRL, TNFα-, and TNFα + NaBu-treated cultures. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 3.

Expression of key genes in osteoblastic differentiation. Effects of treatment on the expression of key genes in osteoblastic differentiation ((a, b): RUNX2; (c, d): COL1A1; (e, f): OPN) at 24 h, 48 h, and 7 days of culture. (a, c, and e) Changes in gene expression in untreated control (CTRL) and NaBu-treated cultures. (b, d, and f) Changes in gene expression in CTRL, TNFα-, and TNFα + NaBu-treated cultures. Relative gene expression is expressed as fold increase as compared to untreated CTRL at 24 h (placed at 1, dotted line). Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). Differences versus the untreated CTRL at 24 h are indicated by the symbol “#.” The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 4.

OPN protein expression in media and cell lysates. (a) Explicative image of PVDF membrane electroblotted from a PAGE and stained with rabbit anti-human and mouse anti-human GAPDH monoclonal antibody. Upper half: time points (24 h, 48 h, and day 7); lower half: combination of treatments (untreated CTRL, NaBu 10⁻³ M, TNFα, TNFα + NaBu 10⁻³ M); left half: molecular weight, based on the molecular marker loaded within the gel; right half: OPN forms corresponding to the different bands resolved by PAGE. (b) Densitometric analysis of the bands corresponding to the 66-kDa monomeric OPN, normalized to GAPDH protein expression. Relative gene expression is expressed as fold increase as compared to untreated CTRL at 24 h. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 5.

Expression of RELA subunit of NF-κB. Effects of treatment on gene expression of the RELA subunit of NF-κB at 24 h, 48 h, and 7 days of culture. (a) Changes in RELA gene expression in untreated control (CTRL) and NaBu-treated cultures. (b) Changes in RELA gene expression in CTRL, TNFα-, and TNFα + NaBu-treated cultures. Relative gene expression is expressed as fold increase as compared to untreated CTRL at 24 h (placed at 1, dotted line). Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). Differences versus the untreated CTRL at 24 h are indicated by the symbol “#.” The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 6.

NF-κB activation. Effects of treatment on NF-κB activation (phospho-RelA/NF-κB p65) at 24 h, 48 h, and 7 days of culture. (a) NF-κB activation status of untreated control (CTRL) and NaBu-treated cultures. (b) NF-κB activation status of CTRL, TNFα-, and TNFα + NaBu-treated cultures. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 7.

Cytokine concentrations in culture media. Effects of treatment on cytokine concentrations in culture media ((a, b): IL-6; (c, d): IL-10; (e, f): OPG; (g, h): RANKL) at 24 h, 48 h, and 7 days of culture. (a, c, e, and g) Cytokine concentrations in media from untreated control (CTRL) and NaBu-treated cultures. (b, d, f, and h) Cytokine concentrations in media from CTRL, TNFα-, and TNFα + NaBu-treated cultures. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).

Figure 8.

OPG-to-RANKL ratio in culture media. Effects of treatment on OPG-to-RANKL ratio concentrations in culture media at 24 h, 48 h, and 7 days of culture. (a) OPG-to-RANKL ratio in media from untreated control (CTRL) and NaBu-treated cultures. (b) OPG-to-RANKL ratio concentrations in media from CTRL, TNFα-, TNFα + NaBu-treated cultures. Repeated-measures ANOVA was used to evaluate inter-time changes for each condition ($: vs 24 h, §: vs 48 h); Student’s t-test was used to evaluate inter-group differences at each time point (†). The number of symbols indicates the significance level (P < 0.05, P < 0.01, P < 0.001).