Original article
Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice
Graphical abstract
Introduction
Ascorbate has long been proposed to have anti-cancer activity [1], [2], [3], [4], [5], [6], and is frequently administered by complementary medicine practitioners as an adjunct to cancer treatments, often as high-dose infusions [7]. In the past decade, improved understanding of the pharmacokinetics of ascorbate uptake and transport has led to renewed interest in its potential activity in cancer. Recent investigations have shown that high-dose ascorbate can affect tumour growth in mouse models [8], [9], [10], [11], and there are currently a number of human clinical trials aiming to determine efficacy [12], [13], [14], [15], [16]. However, the use of ascorbate in cancer remains an area of controversy, due to a lack of robust clinical data and of a proven mechanism of action.
Ascorbate could exert a number of possible anti-tumour activities: at high local concentrations, achievable by intravenous administration, autoxidation results in significant generation of H2O2 that is cytotoxic to tumour cells in vitro [17], [8], [18]. High concentrations of dehydroascorbate (DHA), the oxidized form of ascorbate, are proposed to lead to an energetic crisis in glycolytic cancer cells, causing cell death [11]. Ascorbate regulates the activity of the transcription factor hypoxia-inducible factor (HIF)-1 [19], [20], [21] and decreased intracellular levels up-regulate the hypoxic response [22], [23], [24]. HIF-1 drives tumour angiogenesis, modifies the extracellular pH-environment, and affects cell death and survival pathways, and its activation culminates in an aggressive cancer phenotype [25], [26], [27].
HIF-1 is controlled by proline hydroxylases (PHDs 1, 2, 3) and an asparagine hydroxylase (factor-inhibiting HIF, FIH), collectively known as the HIF hydroxylases, that together regulate the stability of the alpha subunit and transcriptional activity [28], [29], [30]. The HIF hydroxylases are iron-containing 2-oxoglutarate-dependent dioxygenases that require molecular oxygen and 2-oxoglutarate as substrates [20], [31]. They also require ascorbate as a cofactor, to retain optimum activity and prevent irreversible oxidation of the active site iron [20], [21]. We, and others, have shown that intracellular ascorbate availability is a major determinant in the regulation of HIF-1 activity in response to mild hypoxia, 2-oxoglutarate deprivation or metal poisoning with Co2+ or Ni2+ [22], [19].
How intravenous or high-dose administration of ascorbate affects tumour growth is unknown, with the mechanism of anti-tumour activity by ascorbate not having been monitored in an in vivo setting. This information is required to inform human clinical study protocols. Pharmacokinetic studies in humans have demonstrated that the uptake of ascorbate following oral ingestion is tightly regulated [32], [33], and that the bioavailability is increased by 70-fold when administered intravenously [7], [33], [14]. We have previously proposed that these supra-physiological plasma concentrations could allow ascorbate to overcome the diffusion barrier of poorly-perfused solid tumours [34], thereby boosting intracellular levels. To date, however, no study has monitored the pharmacokinetics of ascorbate uptake into tumour tissue following high dose ascorbate (high-dose vitamin C, HDVC) administration.
Uptake of ascorbate into cells varies between tissues, and is mediated by the sodium-dependent vitamin C transporter that exists as two isoforms, SVCT1 and SVCT2 [35]. The SVCTs show distinct tissue distribution, and together ensure the effective uptake and regulation of plasma and cellular ascorbate concentrations [36], [35]. SVCT1 is responsible for uptake through the gut and re-absorption in the kidneys, and is thought to largely regulate plasma levels, whereas SVCT2 is concentrated in more metabolically active cells throughout the body, ensuring an adequate intracellular supply to support crucial intracellular functions [36], [35]. Both SVCT1 and SVCT2 have been found in lung tissue, with SVCT1 being identified solely in the blood vessels in one study [37], and on the apical surfaces of rat lung columnar epithelial cells in another study [52], both reporting widespread distribution of SVCT2. Transporter status has not been studied in tumour tissue, and this, together with ascorbate availability and tumour vessel patency, is likely to play an important role in the anti-cancer activity of HDVC.
The present study was designed to investigate whether HDVC could affect ascorbate levels in pre-existing tumours and could impact on tumour growth, and whether increased tumour ascorbate levels affected HIF-1 activity. We have utilized the Gulo-/- mouse which, like humans, carries a mutation in the gulonolactone oxidase (Gulo) gene that encodes the terminal enzyme in the ascorbate synthesis pathway, and thus relies entirely on dietary ascorbate [38]. Using this model, we have previously reported that variable dietary ascorbate intake affects tumour ascorbate levels, HIF-1 expression and tumour growth rate in three different syngeneic tumour models, namely Lewis Lung carcinoma (LL/2), B16-F10 melanoma and CMT93 colorectal cancer [39], [40]. All three tumour models showed a significant inverse association between tumour ascorbate content and HIF-1 levels. In the current study we have utilized the LL/2 model to specifically investigate the pharmacokinetics of ascorbate uptake into established tumours following HDVC, its ability to boost ascorbate levels in the tumour and in non-tumour tissue, and the effect on HIF-1 activity and subsequent tumour growth.
Section snippets
Materials
Mouse Lewis lung carcinoma (LL/2), CMT-93 colorectal carcinoma and B16-F10 melanoma cells were from American Type Culture Collection, Cryosite Distribution, Australia; the control mouse mammary carcinoma cell line EO771 was kindly provided by Dr Andreas Moeller, QIMR Berghofer, Australia. Sodium L-ascorbate was from Sigma-Aldrich, St Louis, MO, USA.Pimonidazole was obtained from Hypoxyprobe™-1, Burlington, Massachusetts, USA. Antibodies against mouse HIF-1α, carbonic-anhydrase IX (CA-IX), and
Ascorbate transporter status of murine cell lines in vitro
In our previous studies with three different tumour cell lines, LL/2, CMT-93 and B16-F10, we have shown that intracellular ascorbate levels affect HIF-1 activation both in vitro, and in vivo following dietary intervention in the Gulo-/- mouse [39], [40]. All three cell lines are able to accumulate ascorbate in vitro [40], and were shown to express the SVCT2 transporter, the isoform mostly responsible for uptake by metabolically active cells [35] (Fig. 1). LL/2 cells were also assayed for SVCT1
Discussion
We investigated the pharmacokinetic parameters of high dose ascorbate administration in plasma, liver and tumour tissue in the Gulo-/- mouse and have monitored the effect of tumour ascorbate uptake on the activation of HIF-1 and on tumour growth. This is the first study to monitor these processes together in established tumours following HDVC. We have shown that in response to a single dose of HDVC, there is a prolonged presence of ascorbate in the tumour, compared to plasma and liver. We also
Conclusion
This study has provided novel tumour-specific pharmacokinetic information on ascorbate uptake and accumulation. We have shown that ascorbate elimination from solid tumours is markedly slower than from plasma and liver. Furthermore, daily administration of HDVC was required to maintain optimal levels of ascorbate in tumours, and this led to reduced angiogenesis and reduced tumour hypoxia, resulting in a tumour growth delay. This mechanistic information has been lacking to date and will inform
Conflicts of interest
None.
Acknowledgements
This study was funded by the Health Research Council of New Zealand (11/460, MCMV, GUD), the Mackenzie Charitable Foundation (GUD) and the University of Otago (Ph.D Scholarships for EJC and CW).
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