Gene transfer to restore vitamin C synthesis in human cell line

Abstract

Vitamin C (ascorbate) is deficient in humans due to the lack in the final step in the biosynthesis pathway, Gulonolactone oxidase (Gulo). Ascorbate has been studied as a possible cancer therapy since the 1970’s, however results have been inconclusive. One of the reasons for this is the variability in ascorbate transport to the tumour. This study aims to design a biological tool that allows the study of ascorbate in cancer without transport being a variable. HepG2 cells were transfected with pCMV6-­‐Gulo and tested for genome incorporation by PCR, Gulo enzyme expression by western blot and functionality by HPLC-­‐ECD. One successful clone was established and a level of ascorbate comparable to previous studies was observed when the Gulo substrate (gulonolactone) was added. Transfected cells were assayed for HIF-­1α levels as a sign of tumour aggression. When ascorbate is added to the cells in 1% oxygen HIF-­1α levels are reduced. Interestingly a similar level of reduction was also observed when gulonolactone is added to the cells