Abstract A81: Geranylgeraniol oxidase activity involved in oxidative formation of geranylgeranial in human hepatoma cells.
Authors: Mitake Maiko , Shidoji Yoshihiro 
Published: June 2012

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.