Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by microbial fermentation of indigestible fiber by gut flora. histone H3 were enhanced in a GPR41-dependent manner; expression of histone deacetylases (HDAC) 3, 4, 5, 6, 8 proteins was significantly reduced; and induction of TNF- expression was significantly enhanced. These results suggest that propionate and cisplatin synergistically and significantly induce apoptosis of HepG2 cells by increasing expression of autocrine TNF- via reduction of HDACs through GPR41 signaling. From clinical and translational perspectives, our data suggest that a combination of propionate with cisplatin may have better therapeutic effects on HCC compared with conventional treatment, and that a selective GPR41 agonist may be a candidate as an adjuvant therapeutic agent for HCC. 0.01) with NaP at 0.1 to 10 mM in HepG2 cells and HuH-7 cells (Figure 2A, 2B) and with NaP at 1 to 10 mM in JHH-4 cells and HLE cells (Figure 2C, 2D). In FACS analysis to examine whether NaP (1 mM) enhanced the sensitivity of HCC cell lines to cisplatin, the apoptotic rate at 48 h was significantly higher with NaP + cisplatin than with cisplatin alone in all HCC cell lines (Figure 3AC3D). Open in a separate window Figure 2 Effects of NaP combined with cisplatin on proliferation rate of HCC cell lines(ACD) A MTS assay was used to determine the effects of NaP (1, 10 mM) FGF2 alone, cisplatin (25 M) alone, or cisplatin (25 M) + NaP (0.1, 1, 10 mM) on proliferation of HCC cell lines for 24 h. Data are shown as mean SD of % apoptosis from three independent experiments. * 0.05, ** 0.01 by one-way ANOVA with a Scheffe test. Open in a separate window Figure 3 Effects of NaP combined with cisplatin on apoptotic rate of HCC cell lines(ACD) HCC cells were treated with cisplatin (25 M), NaP (1 mM), or both agents for 48 h. Cells were then stained with annexin V and PI, followed by cytometry analysis. Data are shown as mean SD of % apoptosis from three independent experiments. * 0.05, ** 0.01 by one-way ANOVA with a Scheffe test. NaP enhances cisplatin-induced apoptosis via GPR41 in HepG2 cells Apoptosis is regulated by highly coordinated processes that involves activation of caspases, which are cysteine proteases . Caspase-3 activation is responsible for DNA fragmentation and myonuclear apoptosis [23, 24]. Therefore, we measured the cleaved, active form of caspase-3 in HepG2 cells by Western blot analysis (Figure ?(Figure4).4). NaP + cisplatin significantly increased expression at 0.1 mM NaP (Figure ?(Figure4A).4A). Rolapitant kinase inhibitor Next, we examined whether NaP + Rolapitant kinase inhibitor cisplatin enhanced expression of cleaved caspase-3 via GPR41 or GPR43. Enhancement by NaP was completely blocked by treatment with pertussis toxin (PTX), a Gi/o-type G protein inhibitor  (Figure ?(Figure4A),4A), and was blocked by Gallein, a G blocker (Figure ?(Figure4A).4A). We further investigated whether a selective agonist of GPR41 or GPR43 enhanced cisplatin-stimulated expression of cleaved caspase-3. CPC, a GPR41-selective agonist, significantly enhanced cleavage of caspase-3 at 100 M and the enhancement effect of CPC + cisplatin was blocked by treatment with PTX (Figure ?(Figure4B).4B). In contrast, CFMB, a GPR43-selective agonist, significantly reduced cleavage at 10 M (Figure ?(Figure4C).4C). These data indicate that the enhancement effect Rolapitant kinase inhibitor of CPC + cisplatin was dependent on a Gi/o signal pathway. GPR41 gene silencing in HepG2 cells using two siRNAs (siRNA-1 and siRNA-2) was performed to clarify whether NaP-mediated enhancement of cisplatin-induced apoptosis was dependent on GPR41. Significant decreases in GPR41 mRNA and protein were found in HepG2 cells treated with siRNAs against GPR41 (Figure 4D, 4E). GPR41 silencing in HepG2 cells significantly blocked NaP-induced enhancement of cisplatin-induced cleaved caspase-3 expression (Figure 4D, 4E). Taken together, these results demonstrate that NaP enhances activation of caspase-3 by cisplatin via a GPR41-mediated pathway. Open in a separate window Figure 4 Enhancement of cisplatin-induced apoptosis by NaP in a GPR41-dependent manner in HepG2 cells(A).