RESUMO
Research on retinoid-based cancer prevention, spurred by the effects of vitamin A deficiency on gastric cancer and subsequent clinical studies on digestive tract cancer, unveils novel avenues for chemoprevention. Acyclic retinoids like 4,5-didehydrogeranylgeranoic acid (4,5-didehydroGGA) have emerged as potent agents against hepatocellular carcinoma (HCC), distinct from natural retinoids such as all-trans retinoic acid (ATRA). Mechanistic studies reveal GGA's unique induction of pyroptosis, a rapid cell death pathway, in HCC cells. GGA triggers mitochondrial superoxide hyperproduction and ER stress responses through Toll-like receptor 4 (TLR4) signaling and modulates autophagy, ultimately activating pyroptotic cell death in HCC cells. Unlike ATRA-induced apoptosis, GGA and palmitic acid (PA) induce pyroptosis, underscoring their distinct mechanisms. While all three fatty acids evoke mitochondrial dysfunction and ER stress responses, GGA and PA inhibit autophagy, leading to incomplete autophagic responses and pyroptosis, whereas ATRA promotes autophagic flux. In vivo experiments demonstrate GGA's potential as an anti-oncometabolite, inducing cell death selectively in tumor cells and thus suppressing liver cancer development. This review provides a comprehensive overview of the molecular mechanisms underlying GGA's anti-HCC effects and underscores its promising role in cancer prevention, highlighting its importance in HCC prevention.
Assuntos
Carcinoma Hepatocelular , Diterpenos , Neoplasias Hepáticas , Ácido Palmítico , Piroptose , Tretinoína , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Diterpenos/farmacologia , Ácido Palmítico/farmacologia , Piroptose/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Tretinoína/farmacologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacosRESUMO
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 13C-labeled mevalonolactone and cultured hepatoma cells, and the involvement of hepatic monoamine oxidase B in the biosynthesis of GGA was also demonstrated. The biological activity of GGA was analyzed from the retinoid (differentiation induction) and nonretinoid (cell death induction) aspects, and in particular, the nonretinoid mechanism by which GGA induces cell death in hepatoma cells was found to involve pyroptosis via ER stress responses initiated by TLR4 signaling. In addition to these effects of GGA, we also describe the in vivo effects of GGA on reproduction. In this review, based mainly on our published papers, we have shown that hepatic monoamine oxidase B is involved in the biosynthesis of GGA and that GGA induces cell death in human hepatoma-derived cell lines by noncanonical pyroptosis, one of the mechanisms of sterile inflammatory cell death.
Assuntos
Carcinoma Hepatocelular , Diterpenos , Neoplasias Hepáticas , Masculino , Humanos , Ratos , Animais , Ácidos Graxos , Ácido Mevalônico , Cromatografia Líquida , Espectrometria de Massas em Tandem , Diterpenos/farmacologia , Diterpenos/metabolismo , Neoplasias Hepáticas/patologia , Retinoides , Mamíferos/metabolismo , MonoaminoxidaseRESUMO
Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.
RESUMO
Geranylgeranoic acid (GGA) was developed as a preventative agent against second primary hepatoma, and was reported to induce cell death in human hepatoma cells via Toll-like receptor 4 (TLR4)-mediated pyroptosis. We recently reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived HuH-7 cells and that endogenous GGA is found in most rat organs including the liver. An unbiased metabolomics analysis of ice-cold 50% acetonitrile extracts from control and GGA-treated cells was performed in this study to characterize the intracellular metabolic changes in GGA-induced pyroptosis and to analyze their relationship with the mechanism of GGA-induced cell death. The total positive ion chromatograms of the cellular extracts in ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were apparently unchanged after GGA treatment, but an orthogonal partial least squares-discriminant analysis score plot clearly discriminated the intracellular metabolite profiles of GGA-treated cells from that of control cells. S-plot analysis revealed 15 potential biomarkers up-regulated by 24-h GGA treatment according to their variable importance in the projection value of more than 1, and the subsequent metabolomics analysis identified nine of these metabolites as a group of lysophospholipids containing lysophosphatidylcholine with C16:0, C20:4, or C20:3 fatty acids. The possible roles of these lysophospholipids in GGA-induced pyroptosis are discussed.
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Geranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes in hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma. We measured endogenous GGA and mRNA of monoamine oxidase (BMAOB), a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6-93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice. Hepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice; some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 24 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently; and ZAA dramatically upregulated hepatic GGA. In this study; we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.
RESUMO
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.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Diterpenos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Piroptose/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Diterpenos/uso terapêutico , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from 13C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.
Assuntos
Diterpenos/metabolismo , Fígado/enzimologia , Monoaminoxidase/metabolismo , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Humanos , Espaço Intracelular/metabolismo , Monoaminoxidase/deficiência , Monoaminoxidase/genética , OxirreduçãoRESUMO
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.
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Gene-environment (G × E) interactions involving job stress and mental health on risk factors of cardiovascular disease (CVD) are minimally explored. This study examined the association and G × E interaction effects of vascular endothelial growth factor receptor-2 (VEGFR-2) gene polymorphisms (rs1870377, rs2071559) on cardiometabolic risk in Chinese Malaysian adults. Questionnaires: Job Stress Scale (JSS); Depression, Anxiety, and Stress Scale (DASS-21); and Rhode Island Stress and Coping Inventory (RISCI) were used to measure job stress, mental health, and coping with perceived stress. Cardiometabolic risk parameters were evaluated in plasma and genotyping analysis was performed by real-time polymerase chain reaction. The subjects were 127 Chinese Malaysian adults. The allele frequencies for rs1870377 (A allele and T allele) and rs2071557 (A allele and T allele) polymorphisms were 0.48 and 0.52, and 0.37 and 0.63, respectively. Significant correlations include scores from JSS dimensions with blood glucose (BG) (p = 0.025-0.045), DASS-21 dimensions with blood pressure, BMI, and uric acid (p = 0.029-0.047), and RISCI with blood pressure and BG (p = 0.016-0.049). Significant G × E interactions were obtained for: rs1870377 with stress on total cholesterol (p = 0.035), low density lipoprotein cholesterol (p = 0.019), and apolipoprotein B100 (p = 0.004); and rs2071559 with anxiety on blood pressure (p = 0.006-0.045). The significant G × E interactions prompt actions for managing stress and anxiety for the prevention of CVD.
Assuntos
Povo Asiático/genética , Doenças Cardiovasculares/genética , Saúde Mental/estatística & dados numéricos , Polimorfismo de Nucleotídeo Único/genética , Estresse Psicológico/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Doenças Cardiovasculares/epidemiologia , Feminino , Predisposição Genética para Doença , Humanos , Malásia , Masculino , Doenças Metabólicas/epidemiologia , Doenças Metabólicas/genética , Pessoa de Meia-Idade , Fatores de Risco , Estresse Psicológico/epidemiologiaRESUMO
Geranylgeranoic acid (GGA) has been reported to induce autophagic cell death via upregulation of lipid-induced unfolded protein response in several human hepatoma-derived cell lines, and its 4,5-didehydro derivative has been developed as a preventive agent against second primary hepatoma in clinical trials. We have previously reported that GGA is a natural diterpenoid synthesized in several medicinal herbs. Here, we provide unequivocal evidence for de novo GGA biosynthesis in mammals. First, with normal male Wistar rats, the levels of GGA in liver were found to be far greater than those in other organs analyzed. Second, we demonstrated the metabolic GGA labeling from the 13C-labeled mevalonolactone in the human hepatoma-derived cell line, HuH-7. Isotopomer spectral analysis revealed that approximately 80% of the cellular GGA was newly synthesized from mevalonate (MVA) in 12 h and the acid picked up preexisting farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), suggesting that GGA is derived from FPP and GGPP through the MVA pathway. Third, zaragozic acid A, a squalene synthase inhibitor, induced dose-dependent upregulation of endogenous GGA content in HuH-7 cells and their concomitant cell death. These results strongly suggest that a cancer-preventive GGA is biosynthesized via the MVA pathway in mammals.
Assuntos
Diterpenos/metabolismo , Ácido Mevalônico/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Masculino , Ratos , Ratos Wistar , Ácidos Tricarboxílicos/farmacologiaRESUMO
The reduction pathway leading to the formation of dolichol was clarified in 2010 with the identification of SRD5A3, which is the polyprenol reductase. The finding inspired us to reanalyze the length of the major chain of polyprenol and dolichol from several plant leaves, including mangrove plants, as well as from animal and fish livers by 2D-TLC. Polyprenol- and dolichol-derived metabolites such as polyprenylacetone and epoxydolichol were found together with rubber-like prenol. This review focuses on analyses of polyprenol and its derivatives, including recently found epoxypolyprenol and polyprenylacetone. Attention has also been paid to the chromatographic behavior of rubber-like prenol on TLC.
Assuntos
Pentanóis/metabolismo , Animais , Cromatografia em Camada Fina , Dolicóis/metabolismo , Hemiterpenos , Fígado/metabolismo , Folhas de Planta/metabolismoRESUMO
Gene-diet interaction using a multifactorial approach is preferred to study the multiple risk factors of cardiovascular disease (CVD). This study examined the association and gene-diet interaction effects of the angiotensin II type 1 receptor (AGTR1) gene (rs5186), and type 2 receptor (AGTR2) gene (rs1403543) polymorphisms on metabolic risk factors of CVD in Malaysian adults. CVD parameters (BMI, blood pressure, glycated hemoglobin, total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), and TC/HDL-C ratio), and constructed dietary patterns "vegetables, fruits, and soy diet" (VFSD), and "rice, egg, and fish diet" (REFD) were obtained from previous studies. Genotyping analysis was performed by real-time PCR using Taqman probes. The subjects were 507 adults (151 Malays; 179 Chinese; and 177 Indians). Significant genetic associations were obtained on blood lipids for rs5186 in Malays and Chinese, and rs1403543 in Chinese females. The significant gene-diet interaction effects after adjusting for potential confounders were: rs5186 × VFSD on blood pressure in Malays (p = 0.016), and in Chinese on blood lipids for rs5186 × REFD (p = 0.009-0.023), and rs1403543 × VFSD in female subjects (p = 0.001-0.011). Malays and Chinese showed higher risk for blood pressure and/or lipids involving rs5186 and rs1403543 SNPs together with gene-diet interactions, but not Indians.
Assuntos
Doenças Cardiovasculares/etiologia , Dieta , Doenças Metabólicas/genética , Polimorfismo Genético , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/genética , Adulto , Doenças Cardiovasculares/genética , Feminino , Predisposição Genética para Doença , Humanos , Malásia , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
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.
Assuntos
Carotenoides/sangue , Frutas , beta Caroteno/sangue , Carotenoides/metabolismo , Dieta , Ingestão de Alimentos , Feminino , Frequência do Gene , Genótipo , Proteínas de Homeodomínio/genética , Humanos , Japão , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , beta Caroteno/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Diterpenos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/farmacologia , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Glicólise , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/metabolismo , Metabolômica , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Análise Multivariada , Mutação , Monoéster Fosfórico Hidrolases , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular/patologia , Diterpenos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Neoplasias Hepáticas/patologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diterpenos/química , Ácidos Graxos Insaturados/química , Humanos , Concentração Inibidora 50 , Proteínas Associadas aos Microtúbulos/metabolismo , Ácido Oleico/farmacologia , Palmitatos/farmacologia , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Lysine-specific demethylase 1 (LSD1/KDM1A), a histone-modifying enzyme, is upregulated in many cancers, especially in neuroblastoma, breast cancer and hepatoma. We have established a simple method to measure LSD1 activity using a synthetic N-terminal 21-mer peptide of histone H3, which is dimethylated at Lys-4 (H3K4me2). After the enzyme reaction, a substrate of H3K4me2 and two demethylated products, H3K4me1 and H3K4me0, were quantitatively determined by flow injection time-of-flight mass spectrometry (FI-TOF/MS). By using recombinant human LSD1, a nonlinear fitting simulation of the data obtained by FI-TOF/MS produced typical consecutive-reaction kinetics. Apparent K m and k cat values of hLSD1 for the first and second demethylation reactions were found to be in the range of reported values. Tranylcypromine was shown to inhibit LSD1 activity with an IC50 of 6.9 µM for the first demethylation reaction and 5.8 µM for the second demethylation reaction. The FI-TOF/MS assay revealed that the endogenous LSD1 activity was higher in the nuclear extracts of SH-SY5Y cells than in HeLa or PC-3 cells, and this is in accordance with the immunoblotting data using an anti-LSD1 antibody. A simple, straightforward FI-TOF/MS assay is described to efficiently measure LSD1 activity in the nuclear extracts of cultured cells.
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Polyisoprenoid alcohols from the livers of temperate sea fish (skipjack tuna, chub mackerel, red sea bream and rainbow trout) were analyzed by using 2D-TLC, electrospray ionization (ESI) mass spectrometry and NMR methods. Dolichols (Dols) were detected in all the fish livers, and they were composed of 19-22 isoprene units with Dol-20 as the predominant prenolog. In addition, Dol-like family compounds were found by using 2D-TLC on skipjack tuna samples. These compounds were found to have a larger molecular mass than the Dol family by 16 mass units. NMR analysis indicated that the Dol-like compounds were consistent with the terminal epoxide structure of Dols (the ω-oxirane derivatives of Dols). ESI analysis also revealed the occurrence of dehydro molecules in both Dols and epoxy Dols (Dol-like) fractions. The occurrence of epoxy Dols in fish is discussed in context with the biosynthesis of Dols, which is responsible for forming Dol phosphate, which lead to Dol-PP-oligosaccharide.
Assuntos
Dolicóis/análogos & derivados , Fígado/metabolismo , Atum/metabolismo , Animais , Cromatografia em Camada Fina , Dolicóis/biossíntese , Dolicóis/química , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Redes e Vias Metabólicas , Oncorhynchus mykiss/metabolismo , Perciformes/metabolismo , Dourada/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Mutant p53 proteins in human hepatoma cell lines such as HuH-7 (Y220C) and PLC/PRF/5 (R249S) accumulate in the cytoplasm, and lose their transcriptional function. Geranylgeranoic acid (GGA) is a naturally occurring acyclic diterpenoid that induces cell death in both cell lines, but not in HepG2 cells harboring wild-type p53. Here, we demonstrate that micromolar concentrations of GGA induce a rapid nuclear translocation of cytoplasmic p53 in both p53-mutant cell lines and p53 knockdown attenuates GGA-induced cell death in HuH-7 cells. Cell-free experiments demonstrate that GGA is able to release 670-kD p53-containing complexes from putative huge macromolecular aggregates in post-mitochondrial fractions as revealed on blue-native gradient PAGE. Among several p53-target genes tested, GGA upregulates PUMA gene expression, and ivermectin, an inhibitor for importin α/ß, blocks GGA-induced nuclear translocation of cytoplasmic p53 and suppresses GGA-induced upregulation of PUMA mRNA levels in HuH-7 cells. Taken together, these data suggest that GGA treatment stimulates a nuclear translocation of mutant p53 through its dissociation from cytoplasmic aggregates, which may be essential for GGA-induced cell death.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diterpenos/farmacologia , Regulação Neoplásica da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Citosol/efeitos dos fármacos , Citosol/ultraestrutura , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Ivermectina/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Lysine-specific demethylase 1 (LSD1) is upregulated in many cancers, especially neuroblastoma. We set out to explore whether geranylgeranoic acid (GGA) inhibits LSD1 activity by using recombinant human LSD1. GGA inhibited LSD1 activity with IC50 similar to that of the clinically used drug tranylcypromine. In human neuroblastoma SH-SY5Y cells, GGA induced NTRK2 gene expression alongside upregulation of histone H3 with dimethylated lysine-4 in the regulatory regions of the NTRK2 gene. Dihydrogenation of GGA reinforced the LSD1-inhibitory effect in a position-dependent manner. The inhibitory effects of dihydro-derivatives of GGA on recombinant LSD1 strongly correlated with the induction of NTRK2 gene expression in SH-SY5Y cells. These data demonstrate for the first time the efficient LSD1-inhibitor activity of GGA and its derivatives, providing a novel prospect of preventing cancer onset by using GGA to regulate epigenetic modification.
Assuntos
Diterpenos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Anticarcinógenos/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Diterpenos/química , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Farneseno Álcool/farmacologia , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Cinética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas , Receptor trkB/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Tranilcipromina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Development of an effective low-cost anti-acquired immunodeficiency syndrome (AIDS) drugs is needed for treatment of AIDS patients in developing countries. Host cell lipid raft microdomains, which are enriched with cholesterol, glycolipids, ceramide, and gangliosides, are important for human immunodeficiency virus type 1 (HIV-1) entry. Retinoid analogs have been shown to modulate ceramide levels in the cell membrane, while cholera toxin B subunit (CT-B) specifically binds to the ganglioside GM1. In this study, we found that the acyclic retinoid analogs geranylgeranoic acid (GGA) and NIK-333 as well as CT-B efficiently attenuate CXCR4-tropic, but not CCR5-tropic, HIV-1 vector infection. We also found that GGA and NIK-333 suppress CXCR4-tropic HIV-1 infection by attenuating CXCR4 expression. CT-B also attenuated CXCR4-tropic HIV-1 infection, but did not suppress CXCR4 expression. These results suggest a distinct role for lipid raft microdomains in CXCR4- and CCR5-tropic HIV-1 infections and illuminate novel agents for the development of AIDS therapy.