Geranylgeranoic acid, a 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were previously developed as synthetic “acyclic retinoids” for cancer chemoprevention. Recently, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji and Ogawa, 2004). In this present study, we present several lines of evidence to demonstrate that geranylgeranyl diphosphate taken in foods could be metabolized to GGA through geranylgeraniol and geranylgeranyl aldehyde via the following steps: 1) The conversion from geranylgeranyl diphosphate to geranylgeraniol was demonstrated to occur by the action of bovine intestinal alkaline phosphatase, with a Km of 46.1 µM. 2) Geranylgeraniol oxidase-mediated conversion of geranylgeraniol to geranylgeranyl aldehyde was revealed in rat liver homogenates, which activity was mainly localized in the mitochondrial fraction. The mitochondrial enzyme showed a Km of 92.9 µM. 3) The conversion of geranylgeranyl aldehyde to geranylgeranoic acid by geranylgeranyl aldehyde dehydrogenase in rat liver homogenates was absolutely dependent on exogenously added NAD+ or NADP+. The Km of the mitochondrial geranylgeranyl aldehyde dehydrogenase was 27.5 µM for geranylgeranyl aldehyde. Taken together, our data suggest that cancer preventive geranylgeranoic acid could be a physiological metabolite from commonly consumed foods.

Geranylgeranoic acid (GGA), a 20-carbon acyclic polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were developed as synthetic "acyclic retinoids" for cancer chemoprevention. Previously, we have shown the natural occurrence of GGA in various medicinal herbs and reported enzymatic formation of GGA from geranylgeraniol (GGOH) through geranylgeranial (GGal) by rat liver homogenates. Here, we present several lines of evidence that a putative GGOH oxidase is involved in GGA synthesis by human hepatoma cell lysates. First, conversion of GGOH to GGal did not require exogenous NAD +

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Geranylgeranoic acid (GGA) is one of the most potent cancer-preventive acyclic retinoids. GGA has been shown to induce cell death in human hepatoma-derived HuH-7 cells. We have recently reported the natural occurrence of GGA and its related compounds in several medicinal herbs such as turmeric, basil, rosehip, cinnamon and others [Shidoji and Ogawa, J. Lipid Res., 45: 1092–1103, 2004]. In the present study, we performed oral administration of turmeric tablets to healthy volunteers in order to investigate bioavailability of natural GGA. By using liquid chromatography/mass spectrometry, authentic GGA was eluted at a retention time of around 18 min as a negative ion of m/z 303.4. With healthy volunteers, plasma GGA was detected prior to the tablet intake and its concentrations were increased at 2 h after its intake and maintained at higher level until 4 h, suggesting an efficient bioavailability of preformed GGA in the turmeric tablets through oral administration. These results indicated that GGA in the turmeric tablet was absorbed as an intact form from intestinal mucosa. The present study provides a clue to conduct a research for cancer preventive roles of GGA in a number of spices.

Necdin (Ndn) transgenic (Tg) mice were generated with a bacterial artificial chromosome (BAC) clone. Droplet digital PCR (ddPCR) and inverse PCR methods revealed that the transgene consisted of four fragments with a total length of 171 kb. Two of these fragments were tandem tail-to-tail duplicates of 77 kb and 37 kb that both contained a Ndn gene. The transgene was inserted in chromosome 15qD1. Ndn is a paternally expressed imprinted gene; however, the total expression level of Ndn in hemizygous Tg mice was approximately twofold higher than that in wild-type mice. ddPCR assays with locked nucleic acid (LNA) TaqMan probes revealed that transgenic Ndn expression was almost equal to endogenous Ndn expression, despite there being two copies of the Ndn gene in the transgene, indicating an interaction between the transcriptional regulation of endogenous Ndn and the transgene. ddPCR assays with LNA TaqMan probes were also applied for imprinting analysis to confirm exclusive paternal expression in tissues with low Ndn expression. This is the first report of a Tg mouse with a tandem duplication of a Ndn transgene and Ndn overexpression, which will be useful for the in vivo study of Ndn overexpression and for rescue experiments of the neonatal lethality seen in the Ndn knockout mouse.

Imprinted genes are differentially expressed in a parent-of-origin-specific manner. Parental origin of the alleles is discriminated by intragenic DNA polymorphisms. Comparisons of parental allelic expression have been analysed by semiquantitative RT-PCR. Here, we developed a novel quantitative method for allelic expression of the imprinted gene Ube3a, which inactivation and mutations cause Angelman syndrome and predominantly expressed by the maternal allele in neuronal tissues. In this method, cDNA was amplified by droplet digital PCR (ddPCR) coupled with allelespecific locked nucleic acid (LNA) TaqMan probes, which labelled by FAM and HEX were designed to detect the SNPs in the target regions. ddPCR assay demonstrated that the sense transcript of Ube3a was equally expressed from both parental alleles in adult tissues except neuronal tissues, where Ube3a expression from the paternal allele was about 10 to 14% of total Ube3a expression in adult brain, and 20% in spinal cord. The antisense transcript of Ube3a was expressed at 60% to 70% of the sense transcript of Ube3a in adult brain. Changes in the Ube3a transcripts during postnatal brain development were also evaluated by ddPCR. The ddPCR method is far more reliable and simpler to use than semiquantitative PCR to analyse skewed or faint allelic expression of imprinted genes.

Background: Geranylgeranoic acid (GGA), a 20-carbon polyprenoic acid (all-trans 3, 7, 11, 15-tetramethyl-2, 4, 6, 10, 14-hexadecatetraenoic acid) and its derivatives were developed as synthetic “acyclic retinoids” for cancer chemoprevention. The efficacy of 4,5-didhehydroGGA in preventing second primary hepatoma was proven in a placebo-controlled double-blinded and randomized phase II clinical trial with postoperative hepatoma patients with few side effects (Muto et al., 1996), and later, it was revealed that the polyprenoic acid significantly increased a 5-year survival rate after a radical therapy of primary hepatoma in these patients (Muto et al., 1999). Previously, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji & Ogawa, 2004) and recently, we found enzymatic formation of GGA from geranylgeraniol (GGOH) through granylgeranial (GGal) by rat liver homogenates (Muraguchi et al., 2011). Objective: To demonstrate that GGOH oxidase is involved in GGA synthesis by human hepatoma cell lysate, enzymatic conversion from GGOH to GGal that is accompanied by oxygen consumption was studied. Experimental procedure: GGOH was incubated under several conditions at 37°C with a human hepatoma-derived cell line, HuH-7 cell lysate. The reaction was stopped with ethanol and extracted with n-hexane. The hexane solution was analyzed by LC/MS Waters Xevo QTOF MS (ACQUITY UPLC BEH C18 column) for determination of GGal or GGA. Oxygen consumption rate was measured by MitoXpress (Luxcel Bioscience), a photoluminescent oxygen-sensitive probe measured on the Roche LightCycler 1.5 (Zitova et al., 2009). Subcellular fractionation was conducted differential centrifugation to obtain the mitochondrial (precipitated at 13,400 × g, 16 min), microsomal (precipitated at 105,000 × g, 90 min) and cytosolic (supernatant at 105,000 × g, 90 min). Authentic GGal was prepared by reaction of GGOH with MnO2 and purified by LiChroprep RB-18 column. Results: 1) Endogenous GGA was detected in HuH-7 cells. GGA contents were approximately 17.7 ng/g wet wt, when the cells were grown in standard condition. 2) The conversion from GGOH to GGal did not require exogenous NAD+, whereas the conversion from GGOH to GGA absolutely required 5 mM NAD+ with HuH-7 cell lysates. 3) GGOH-dependent consumption of oxygen was found with HuH-7 lysates. 4) GGOH-dependent GGal synthesis activity in the human hepatoma cell lysates was proteinase K-resistant and even enhanced by proteinase K treatment. Specific activity of GGOH oxidase in the control cell lysate was 0.36 AU/min/mg and it increased to 1.16 AU/min/mg when HuH-7 cell lysate was pre-incubated with proteinase K at 37°C for 10 min. 5) GGOH-dependent GGal synthetic activity was the highest in mitochondria (0.074 AU/min/mg) and negligible in the cytosolic fraction (0.005 AU/min/mg). Conclusion: These data suggest that a putative mitochondrial GGOH oxidase could be involved in the initial step of GGA synthesis from GGOH. GGOH oxidase may be a target enzyme for cancer chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A81.