geranylgeranic-acid and Liver-Neoplasms

Geranylgeranoic acid (GGA) was first reported in 1983 as one of the mevalonic acid metabolites, but its biological significance was not studied for a long time. Our research on the antitumor effects of retinoids led us to GGA, one of the acyclic retinoids that induce cell death in human hepatoma-derived cell lines. We were able to demonstrate the presence of endogenous GGA in various tissues of male rats, including the liver, testis, and cerebrum, by LC-MS/MS. Furthermore, the biosynthesis of GGA from mevalonic acid in mammals including humans was confirmed by isotopomer spectral analysis using

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, Liquid; Diterpenes; Fatty Acids; Humans; Liver Neoplasms; Male; Mammals; Mevalonic Acid; Monoamine Oxidase; Rats; Retinoids; Tandem Mass Spectrometry

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 β (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.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Diterpenes; Gene Knockdown Techniques; Humans; Liver Neoplasms; Pyroptosis; Toll-Like Receptor 4; Unfolded Protein Response

The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells.

Topics: Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Cell Nucleus; Diterpenes; DNA-Binding Proteins; Fatty Acids, Unsaturated; Humans; Inhibitory Concentration 50; Liver Neoplasms; Microtubule-Associated Proteins; Oleic Acid; Palmitates; Regulatory Factor X Transcription Factors; RNA Splicing; Transcription Factors; Unfolded Protein Response; X-Box Binding Protein 1

Metabolic alternation in cancer cells is one of the most common characteristics that distinguish malignant cells from normal cells. Many studies have explained the Warburg hypothesis that cancer cells obtain more energy from aerobic glycolysis than mitochondrial respiration. Here, we show that a branched-chain C-20 polyunsaturated fatty acid, geranylgeranoic acid (GGA), induces upregulation of the cellular protein levels of TP53-induced glycolysis and apoptosis regulator (TIGAR) and synthesis of cytochrome c oxidase 2 (SCO2) in human hepatoma-derived HuH-7cells harboring the mutant TP53 gene, suggesting that GGA may shift an energetic state of the tumor cells from aerobic glycolysis to mitochondrial respiration. In addition, UPLC/TOF/MS-based metabolomics analysis supported the GGA-induced energetic shift, as it revealed that GGA induced a time-dependent increase in the cellular contents of fructose 6-phosphate and decrease of fructose 1,6-diphosphate. Furthermore, metabolomics analysis revealed that GGA rapidly induced spermine accumulation with slight decrease of spermidine. Taken together, the present study strongly suggests that GGA may shift HuH-7 cells from aerobic glycolysis to mitochondrial respiration through the immediate upregulation of TIGAR and SCO2 protein levels.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Diterpenes; Fatty Acids, Unsaturated; Fructosediphosphates; Fructosephosphates; Glycolysis; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Metabolomics; Mitochondrial Proteins; Molecular Chaperones; Multivariate Analysis; Mutation; Phosphoric Monoester Hydrolases; Tumor Suppressor Protein p53; Up-Regulation

Geranylgeranoic acid (GGA) and its derivatives are currently under development as chemopreventive agents against second primary hepatoma in Japan. We aimed to evaluate chemoprevention targets of GGA and a surrogate marker of chemopreventive response to clarify the molecular mechanism of hepatoma chemoprevention with GGA. Human hepatoma-derived cell lines such as HuH-7, PLC/PRF/5, and HepG-2, were treated with GGA and its derivatives. Cellular dynamics of several cell-cycle-related proteins were assessed by either immunoblotting or immunofluorescence method. The cellular expression of cyclin D1 protein was suppressed immediately after GGA treatment. This reduction was partially blocked by pretreatment with 26S proteasome inhibitor MG-132, indicating that proteasomal degradation was involved in GGA-induced disappearance of cyclin D1. A phosphorylation of retinoblastoma protein (RB) at serine 780, a target site of cyclin D1-dependent kinase 4, was rapidly decreased in GGA-treated HuH-7 cells. Furthermore, subcellular fractionation, Western blotting, and immunofluorescence revealed GGA-induced nuclear accumulation of RB. These results strongly suggest that cyclin D1 may be a target of chemopreventive GGA in human hepatoma cells. GGA-induced rapid repression of cyclin D1, and a consequent dephosphorylation and nuclear translocation of RB, may influence cell cycle progression and may be relevant to GGA-induced cell death mechanisms.

Topics: Cell Cycle; Cell Line, Tumor; Cyclin D1; Diterpenes; Down-Regulation; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Leupeptins; Liver Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction

GGA (geranylgeranoic acid) is a natural polyprenoic acid, derivatives of which has been shown to prevent second primary hepatoma. GGA induces mitochondria-mediated PCD (programmed cell death), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP (green fluorescent protein)-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3β-I was appreciably converted into LC3β-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles. On the other hand, LC3β-II as well as p62/SQSTM1 (sequestosome 1) continuously accumulated and co-localized in the cytoplasmic puncta after GGA treatment. Furthermore, GGA treatment of HuH-7/mRFP (monomeric red fluorescent protein)-GFP-LC3 cells showed yellow fluorescent puncta, whereas glucose deprivation of the cells gave red fluorescent puncta. These results strongly suggest that GGA induces the initial phase of autophagy, but blocks the maturation process of autolysosomes or late stages of autophagy, insomuch that GGA provides substantial accumulation of autophagosomes under serum-starvation conditions in human hepatoma cells.

Topics: Adaptor Proteins, Signal Transducing; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Diterpenes; Humans; Liver Neoplasms; Microtubule-Associated Proteins; Mitochondria; Sequestosome-1 Protein

Geranylgeranoic acid (GGA; all-trans 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenoic acid) has been shown to induce apoptosis in a human hepatoma-derived cell line, HuH-7. We aimed not only to confirm the apoptogenic properties of GGA and its derivatives, but also to search for natural GGA in medicinal herbs. GGA induced apoptosis in human hepatoma-derived cell lines, HuH-7, PLC/PRF-5, and mouse transformed hepatocyte-derived cell line, MLE-10, in a dose- and time-dependent manner, but failed to induce cell death in human hepatoblastoma-derived HepG-2 and mouse primary hepatocytes in the same condition. Besides GGA, 4,5-didehydro GGA, 14,15-dihydro GGA, and 2,3-dihydro GGA were also active to induce cell death in HuH-7 cells, while 4,5-didehydro-10,11, 14,15-tetrahydro GGA, 4,5,8,9-tetrahydro GGA, farnesoic acid, and geranylgeraniol were inert. By using liquid chromatography/mass spectrometry, we found natural GGA as a negative ion of m/z 303.4 in a Chinese herb, Schisandra chinensis, and Schisandra GGA was identified by derivatization with both mild methylation and catalytic hydrogenation. Some other GGAs hydrogenated in the different degrees, including phytanic acid (perhydro GGA), were also found in S. chinensis. GGA and phytanic acid were detected in 24 out of 25 herbs tested. The present study is the first report of natural GGA in medicinal herbs.

Topics: Animals; Cell Death; Cells, Cultured; Chromatography, Liquid; Diterpenes; Gas Chromatography-Mass Spectrometry; Humans; Liver Neoplasms; Male; Mice; Molecular Structure; Phytotherapy; Plant Extracts; Plants, Medicinal

A synthetic geranylgeranoic acid (GGA) induced apoptotic cell death in a human hepatoma cell line, HuH-7, but not in mouse primary cultured hepatocytes. Prior to chromatin condensation, GGA induced a dramatic loss of the mitochondrial membrane potential in 1 hour and in a dose dependent manner in HuH-7 cells, but not in the primary hepatocytes. Pretreatment with synthetic tetrapeptide cysteine protease inhibitor, either acetyl-Tyr-Val-Ala-Asp-chloromethylketone or acetyl-Asp-Glu-Val-Asp-aldehyde, blocked GGA-induced apoptosis without preventing a rapid loss of the mitochondrial membrane potential. alpha-Tocopherol prevented the cells from GGA-induced apoptosis as well as from a rapid loss of the membrane potential. The present study strongly suggests that GGA induces apoptosis in hepatoma cells through derangement of mitochondrial function and subsequent activation of the cysteine protease cascade.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 1; Cell Line; Cysteine Endopeptidases; Diterpenes; Fluorescent Dyes; Humans; Intracellular Membranes; Kinetics; Liver; Liver Neoplasms; Membrane Potentials; Mice; Mitochondria; Mitochondria, Liver; Rhodamine 123; Rhodamines; Time Factors; Tumor Cells, Cultured; Vitamin E