TLR4-mediated pyroptosis in human hepatoma-derived HuH-7 cells induced by a branched-chain polyunsaturated fatty acid, geranylgeranoic acid.

A branched-chain polyunsaturated fatty acid, geranylgeranoic acid (GGA; C20:4), which is an endogenous metabolite derived from the mevalonate pathway in mammals, has been reported to induce cell death in human hepatoma cells. We have previously shown that the lipid-induced unfolded protein response (UPR) is an upstream cellular process for an incomplete autophagic response that might be involved in GGA-induced cell death. Here, we found that Toll-like receptor 4 (TLR4)-mediated pyroptosis in HuH-7 cells occurred by GGA treatment. The TLR4-specific inhibitor VIPER prevented both GGA-induced cell death and UPR. Knockdown of the TLR4 gene attenuated GGA-induced cell death significantly. Upon GGA-induced UPR, caspase (CASP) 4 (CASP4) was activated immediately and gasdermin D (GSDMD) was translocated concomitantly to the plasma membrane after production of the N-terminal fragment of GSDMD. Then, cellular CASP1 activation occurred following a second gradual up-regulation of the intracellular Ca2+ concentration, suggesting that GGA activated the inflammasome. Indeed, the mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3) and interleukin-1 ß (IL1B) genes were up-regulated dramatically with translocation of cytoplasmic nuclear factor-κB (NF-κB) to nuclei after GGA treatment, indicating that GGA induced priming of the NLRP3 inflammasome through NF-κB activation. GGA-induced up-regulation of CASP1 activity was blocked by either oleic acid, VIPER, MCC950 (a selective inhibitor of the NLRP3 inflammasome), or CASP4-specific inhibitor peptide cotreatment. Pyroptotic cell death was also confirmed morphologically by bleb formation in time-series live cell imaging of GGA-treated cells. Taken together, the present results strongly indicate that GGA causes pyroptotic cell death in human hepatoma-derived HuH-7 via TLR4 signalling.

Cytoplasmic translocation of nuclear LSD1 (KDM1A) in human hepatoma cells is induced by its inhibitors.

AIM: Histone-modifiable lysine-specific demethylase-1 (LSD1/KDM1A) is an oncoprotein upregulated in cancers, including hepatoma. We previously reported that the hepatoma-preventive geranylgeranoic acid (GGA) inhibits KDM1A at the same IC50 as that of the clinically used tranylcypromine. Here, we report that these inhibitors induce the cytoplasmic translocation of nuclear KDM1A in a human hepatoma-derived cell line. METHODS & RESULTS: Immunofluorescence studies revealed that KDM1A was cytoplasmically localized in HuH-7 cells 3 h after GGA or tranylcypromine addition. However, GGA did not affect the subcellular localization of another histone lysine-specific demethylase, KDM5A. This suggests that GGA-induced translocation is KDM1A specific. CONCLUSION: These data demonstrate, for the first time, that KDM1A inhibitors specifically induce the cytoplasmic translocation of nuclear KDM1A.

The immunological function of extracellular vesicles in hepatitis B virus-infected hepatocytes.

Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.

Common SNP rs6564851 in the BCO1 Gene Affects the Circulating Levels of ß-Carotene and the Daily Intake of Carotenoids in Healthy Japanese Women.

The circulating levels of ß-carotene are modulated not only by sex, but also by autosomal gene variations and fruit intake. The aim of this study was to investigate the interactions between ß-carotene metabolism-related gene single nucleotide polymorphisms (SNPs; genetic factors) and nutrient intake (environmental factors) relating to their effects on circulating ß-carotene. The serum concentrations of ß-carotene and the habitual food intake of 92 healthy Japanese adults were examined. All subjects were genotyped for three common SNPs: rs6564851 in the ß-carotene 15,15'-oxygenase 1 (BCO1) gene, rs2278986 in the scavenger receptor class B member 1 (SCARB1) gene and rs362090 in the intestine-specific homeobox (ISX) gene. Univariate analysis revealed that the circulating ß-carotene levels were significantly higher in rs6564851 GG homozygotes (p = 0.003). Additionally, the daily intake of ß-cryptoxanthin was positively associated with the circulating ß-carotene levels in female GG homozygotes of rs6564851 (p = 0.023), and the daily intake of α- and ß-carotenes, and ß-cryptoxanthin was significantly lower in female rs6564851 T allele carries than in female GG homozygotes (p = 0.009, 0.008, 0.009, respectively). The present study apparently indicates that higher circulating ß-carotene levels in female rs6564851 GG homozygotes depend on carotenoid intake.