Since the early studies of William J. McCormick in the 1950s, vitamin C has been proposed as a candidate for the treatment of cancer. A number of reports have shown that pharmacological ...concentrations of vitamin C selectively kill cancer cells
and decrease the growth rates of a number of human tumor xenografts in immunodeficient mice. However, up to the date there is still doubt regarding this possible therapeutic role of vitamin C in cancer, mainly because high dose administration in cancer patients has not showed a clear antitumor activity. These apparent controversial findings highlight the fact that we lack information on the interactions that occurs between cancer cells and vitamin C, and if these transformed cells can uptake, metabolize and compartmentalize vitamin C like normal human cells do. The role of SVCTs and GLUTs transporters, which uptake the reduced form and the oxidized form of vitamin C, respectively, has been recently highlighted in the context of cancer showing that the relationship between vitamin C and cancer might be more complex than previously thought. In this review, we analyze the state of art of the effect of vitamin C on cancer cells
and
, and relate it to the capacity of cancer cells in acquiring, metabolize and compartmentalize this nutrient, with its implications on the potential therapeutic role of vitamin C in cancer.
Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes ...remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.
•We describe a general mechanism for the mitochondrial acquisition of ascorbic acid.•Mitochondria express a low-affinity form of the ascorbic acid transporter SVCT2.•SVCT2 transport kinetics is altered by the intracellular ionic microenvironment.•Mitochondrial SVCT2 shows an apparent transport Km of 0.6mM.•Mitochondrial SVCT2 is fully functional under normal high ascorbate concentrations.
The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in ...breast cancer”, available in Free Radical Biology and Medicine Journal 1. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.
The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the ...uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines.
We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments.
A final model is proposed identifying three potentially regulatable steps: i) The local extracellular generation of DHA, ii) DHA transport dependent on plasmamembrane glucose transporters (GLUT), and iii) Mitochondrial uptake of accumulated AA by a SVCT2 form that is overexpressed in breast cancer. This mechanism migth be implicated in mitochondrial oxidative stress reduction. Display omitted
•Breast cancer tissues exhibit high levels of SVCT2 expression, an AA transporter, that is absent in normal tissue.•However, breast cancer cells are unable to acquire vitamin C as AA and they only have the capacity to acquire it as DHA.•SVCT2 is located at the inner membrane of mitochondria of breast cancer cells, where it mediates AA transport.•Mitochondrial SVCT2 overexpression seems to be a common hallmark across all human cancers.
The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is ...synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (
p
< 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (
p
< 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (
p
< 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane–associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in
cis-
Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.
We have shown previously that rats subjected to tourniquet shock develop an acute form of remote organ injury of the liver that is both Kupffer cell (KC) and polymorphonuclear (PMN) leukocyte ...dependent. Circulating plasma xanthine oxidase (XO) has been shown to be responsible for the development of endothelial dysfunction and for remote organ injury of the lung and intestine after ischemia-reperfusion protocols. We now hypothesize that XO is released from rat hind limbs upon reperfusion and that it is responsible for KC and PMN leukocyte activation in this shock model. Our results show that about 30% of rat gastrocnemius muscle xanthine dehydrogenase (XD) is converted to XO during the 5-h tourniquet period and that it is released into the femoral vein within 10 min of reperfusion. Total muscle xanthine oxidoreductase activity (XO + XD) decreases within 30 min of reperfusion and is paralleled by a corresponding increase in femoral vein lactic dehydrogenase. In addition, liver tissue XO increases significantly within 30 min of reperfusion without a corresponding conversion of endogenous XD. Conversion of hepatic XD becomes evident 60 min after reperfusion is initiated, as does XO, and alanine aminotransferase (ALT) release into the hepatic vein, presumably from damaged hepatocytes as a consequence of oxidative stress. Tissue myeloperoxidase activity also increases significantly after the 60-min reperfusion period. That XO mediates KC and PMN activation is supported by the following observations: a) the close relationships between plasma XO and the time courses of tumor necrosis factor-alpha TNFalpha release into the hepatic vein and colloidal carbon clearance by KCs; b) that colloidal carbon clearance, TNFalpha and ALT release, loss of tissue free thiols, lipid peroxidation (TBARS), and liver infiltration by PMN neutrophils can also be induced by the administration of exogenous XO to normal rats; and c) pretreatment of rats with allopurinol inhibits KC activation and liver leukocyte infiltration. These results suggest that XO, released from the ischemic limb on reperfusion, is taken up by the liver were it mediates KC and PMN neutrophil activation and thus contributes to the development of multiple system organ failure after hind limb reperfusion.
The liver has an extraordinary regenerative capacity in response to partial hepatectomy (PHx), which develops with neither tissue inflammation response nor alterations in the whole organism. This ...process is highly coordinated and it has been associated with changes in glutathione (GSH) metabolism. However, there are no reports indicating ascorbic acid (AA) levels after partial hepatectomy. AA and GSH act integrally as an antioxidant system that protects cells and tissues from oxidative damage and imbalance observed in a variety of diseases that affect the liver. Although rat hepatocytes are able to synthesize AA and GSH, which are the providers of AA for the whole organism, they also acquire AA from extracellular sources through the sodium-coupled ascorbic acid transporter-1 (SVCT1). Here, we show that hepatocytes from rat livers subjected to PHx increase their GSH and AA levels from 1 to 7 days post hepatectomy, whose peaks precede the peak in cell proliferation observed at 3 days post-hepatectomy. The increase in both antioxidants was associated with higher expression of the enzymes involved in their synthesis, such as the modifier subunit of enzyme glutamine cysteine ligase (GCLM), glutathione synthetase (GS), gulonolactonase (GLN) and gulonolactone oxidase (GULO). Importantly, rat hepatocytes, that normally exhibit kinetic evidence indicating only SVCT1-mediated transport of AA, lost more than 90% of their capacity to transport it at day 1 after PHx without evidence of recovery at day 7. This observation was in agreement with loss of SVCT1 protein expression, which was undetectable in hepatocytes as early as 2h after PHx, with partial recovery at day 7, when the regenerated liver weight returns to normal.
We conclude that after PHx, rat hepatocytes enhance their antioxidant capacity by increasing GSH and AA levels prior to the proliferative peak. GSH and AA are increased by de novo synthesis, however paradoxically hepatocytes from rat subjected to PHx also suppress their capacity to acquire AA from extracellular sources through SVCT1.
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•Ascorbic acid and glutathione levels increased in rat hepatocytes after partial hepatectomy.•The increase of ascorbic acid and glutathione anteceded the peak in cell proliferation.•SVCT1 mediates ascorbic acid transport in rat hepatocytes.•SVCT1 protein is decreased after partial hepatectomy without changes in mRNA expression.•Liver regeneration after partial hepatectomy was associated with a loss of ascorbic acid transport by hepatocytes.
Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute ...oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.
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•Rats synthesize their own ascorbic acid (AA) in the liver.•SVCT1 is fundamental for humans to obtain AA from the diet.•Species-specific cis-regulatory elements regulate the rat and human SVCT1 genes.•SVCT1 transcription regulation differs in species that synthesize AA differently.
Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We ...show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na+-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.
► Glutathione-dependent ascorbic acid acquisition in rat hepatocytes. ► Differential regulation of ascorbic acid transporter expression in rat hepatocytes. ► Glutathione does not regulate ascorbic acid transport in human hepatoma cells. ► Absence of SVCT2 transporter expression in glutathione-depleted rat hepatoma cells.
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