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Biomedicines 2014, 2(1), 98-109; doi:10.3390/biomedicines2010098
Article

Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1

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Received: 20 December 2013; in revised form: 26 February 2014 / Accepted: 26 February 2014 / Published: 5 March 2014
(This article belongs to the Special Issue Gene Therapy Used in Cancer Treatment)
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Abstract: Humans are unable to synthesise ascorbate (Vitamin C) due to the lack of a functional gulonolactone oxidase (Gulo), the enzyme that catalyses the final step in the biosynthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including as a cofactor for metalloenzymes that regulate the transcription factor hypoxia-inducible factor-1 (HIF-1), which governs cell survival under hypoxia. In most animals, ascorbate is made in liver cells. This study aimed to restore ascorbate synthesis to human hepatocellular carcinoma HepG2 cells and determine the effect of internally produced ascorbate on HIF-1 activation. HepG2 cells were gene-modified with a plasmid encoding the mouse Gulo cDNA, tested for genomic incorporation by PCR and ascorbate synthesis by high performance liquid chromatography. Levels of HIF-1 protein were measured using Western blotting. Gulo-modified HepG2 cells showed increased adherence compared to control HepG2 cells. A PCR-positive clone synthesised ascorbate when the Gulo substrate, l-gulono-1,4-lactone, was supplied. Intracellular ascorbate concentrations reached 5% of saturation levels (6 nmol/106 cells). Addition of ascorbate or gulonolactone reduced HIF-1 accumulation in the Gulo clone, but also in parental HepG2 cells. Our data confirm the requirement for a number of factors in addition to Gulo in the ascorbate biosynthesis pathway in human cells.
Keywords: vitamin C; HIF-1; gene therapy vitamin C; HIF-1; gene therapy
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Flett, T.; Campbell, E.J.; Phillips, E.; Vissers, M.C.M.; Dachs, G.U. Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1. Biomedicines 2014, 2, 98-109.

AMA Style

Flett T, Campbell EJ, Phillips E, Vissers MCM, Dachs GU. Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1. Biomedicines. 2014; 2(1):98-109.

Chicago/Turabian Style

Flett, Teresa; Campbell, Elizabeth J.; Phillips, Elisabeth; Vissers, Margreet C.M.; Dachs, Gabi U. 2014. "Gulonolactone Addition to Human Hepatocellular Carcinoma Cells with Gene Transfer of Gulonolactone Oxidase Restores Ascorbate Biosynthesis and Reduces Hypoxia Inducible Factor 1." Biomedicines 2, no. 1: 98-109.


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