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Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mSVCT2) as a central feature in breast cancer
Abstract
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.
Supplementary resource (1)
... Thus, modulation of SVCT2 or vitamin C supply may be an attractive strategy for restoring microglia homeostasis and promoting neuronal viability (172). Moreover, mitochondrial SVCT2 was previously described in U937 cells (173) and HEK293 cells (174) and it was also observed in various cancer-derived cell lines from different origin (175). This observation was also correlated with cancer pathology (175) and absent in normal cells, suggesting that mitochondrial vitamin C may be relevant for cancer cell development or survival (176). ...
... Moreover, mitochondrial SVCT2 was previously described in U937 cells (173) and HEK293 cells (174) and it was also observed in various cancer-derived cell lines from different origin (175). This observation was also correlated with cancer pathology (175) and absent in normal cells, suggesting that mitochondrial vitamin C may be relevant for cancer cell development or survival (176). ...
... Co-culture experiments in the presence of ascorbate showed that tumor cells from CNS cells were able to acquire vitamin C via SVCT2; however, compared with normal cells, the capacity for ascorbate uptake was much lower in tumor cells (189). In this regard, it is important to emphasize that the main form of vitamin C found after co-culture was ascorbate, meaning cancer cells are able to efficiently reduce DHA once inside the cell (175). Additionally, the competitive inhibition of GLUT transporters in glioblastoma cell lines did not suppress the ascorbate-induced toxicity suggesting that DHA is not the cancer cell-selective toxic species in this model (26,28,102,190). ...
For glioblastoma, the treatment with standard of care therapy comprising resection, radiation, and temozolomide results in overall survival of approximately 14-18 months after initial diagnosis. Even though several new therapy approaches are under investigation, it is difficult to achieve life prolongation and/or improvement of patient's quality of life. The aggressiveness and progression of glioblastoma is initially orchestrated by the biological complexity of its genetic phenotype and ability to respond to cancer therapy via changing its molecular patterns, thereby developing resistance. Recent clinical studies of pharmacological ascorbate have demonstrated its safety and potential efficacy in different cancer entities regarding patient's quality of life and prolongation of survival. In this review article, the actual glioblastoma treatment possibilities are summarized, the evidence for pharmacological ascorbate in glioblastoma treatment is examined and questions are posed to identify current gaps of knowledge regarding accessibility of ascorbate to the tumor area. Experiments with glioblastoma cell lines and tumor xenografts have demonstrated that high‑dose ascorbate induces cytotoxicity and oxidative stress largely selectively in malignant cells compared to normal cells suggesting ascorbate as a potential therapeutic agent. Further investigations in larger cohorts and randomized placebo‑controlled trials should be performed to confirm these findings as well as to improve delivery strategies to the brain, through the inherent barriers and ultimately to the malignant cells.
... Once inside the cells, DHA is efficiently metabolized into AA by DHA reductases. In fact, high performance liquid chromatography (HPLC) studies in immortalized cell lines have shown that independently of the vitamin C form that is incorporated by the cell, AA is the only form present inside with only traces of DHA (76). Therefore, even though DHA uptake through GLUTs is a facilitated transport, cells are able to accumulate higher intracellular concentrations of vitamin C most probably due to rapid reduction of DHA once inside the cell. ...
... Several research groups (6,23,24,70,76,87) have probed the existence of a mitochondrial AA transporter (MAT). HEK-293 cells and other human-derived cell lines express an MAT that kinetically corresponds to a low-affinity form of the SVCT2 (70,76). ...
... Several research groups (6,23,24,70,76,87) have probed the existence of a mitochondrial AA transporter (MAT). HEK-293 cells and other human-derived cell lines express an MAT that kinetically corresponds to a low-affinity form of the SVCT2 (70,76). In certain cell lines (U937 cells), a transporter also identified as SVCT2 behaves as a highaffinity transporter and its function can be inhibited by DHA (6,23,24). ...
Significance:
Vitamin C is a powerful antioxidant that has an intricate relationship with cancer, studied for more than 60 years. However, the specific mechanisms that allow malignant cells to uptake, metabolize and compartmentalize vitamin C remains unclear. In normal human cells, two different transporter systems are responsible for its acquisition: GLUTs transport the oxidized form of vitamin C (dehydroascorbic acid, DHA) and SVCTs the reduced form (ascorbic acid, AA). Here we review the mechanisms described for vitamin C uptake and metabolization in cancer. Recent Advances: Several studies performed recently in vivo and in vitro have provided the scientific community a better understanding on the differential capacities of cancer cells to acquire vitamin C: tumors from different origins do not express SVCTs in the plasma membrane, and are only able to acquire vitamin C in its oxidized form. Interestingly, cancer cells differentially express a mitochondrial form of SVCT2.
Critical issues:
Why tumors have reduced AA uptake capacity at the plasma membrane but develop the capacity of AA transport within the mitochondria remains a mystery. However, it shows that understanding vitamin C physiology in tumor survival might be key to decipher the controversies in its relation to cancer.
Future directions:
A comprehensive analysis of the mechanisms by which cancer cells acquire, compartmentalize and use vitamin C will allow the design of new therapeutic approaches in human cancer.
... On the other hand, there are several studies concerning the key role of vitamin C transport provided by GLUT in cancer cells [66,67]. A recent paper by Pena et al. suggested that the vast majority of vitamin C transferred from the extracellular space into cancer cells assumes the form of DHA [66]. ...
... On the other hand, there are several studies concerning the key role of vitamin C transport provided by GLUT in cancer cells [66,67]. A recent paper by Pena et al. suggested that the vast majority of vitamin C transferred from the extracellular space into cancer cells assumes the form of DHA [66]. The authors reported that cancer cells were able to acquire vitamin C, even if they expressed an abnormal form of SVCT2, by using GLUT transporters and converting DHA to AA inside the cells [66]. ...
... A recent paper by Pena et al. suggested that the vast majority of vitamin C transferred from the extracellular space into cancer cells assumes the form of DHA [66]. The authors reported that cancer cells were able to acquire vitamin C, even if they expressed an abnormal form of SVCT2, by using GLUT transporters and converting DHA to AA inside the cells [66]. This phenomenon is called the bystander effect and was previously thoroughly described [63]. ...
Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe2+ dependent dioxygenases (2-OGDD), which are involved in active DNA demethylation (TET proteins), the demethylation of histones, and hypoxia processes. Therefore, vitamin C may be engaged in the regulation of gene expression or in a hypoxic state. Hence, vitamin C has acquired great interest for its plausible effects on cancer treatment. Since its conceptualization, the role of vitamin C in cancer therapy has been a controversial and disputed issue. Vitamin C is transferred to the cells with sodium dependent transporters (SVCTs) and glucose transporters (GLUT). However, it is unknown whether the impaired function of these transporters may lead to carcinogenesis and tumor progression. Notably, previous studies have identified SVCTs’ polymorphisms or their altered expression in some types of cancer. This review discusses the potential effects of vitamin C and the impaired SVCT function in cancers. The variations in vitamin C transporter genes may regulate the active transport of vitamin C, and therefore have an impact on cancer risk, but further studies are needed to thoroughly elucidate their involvement in cancer biology.
... Immunoblotting experiments were performed using the protocol described in Ref. [1]. Briefly, total extracts were prepared from different cell lines, quantified, separated by SDS-PAGE and transferred to PVDF membranes. ...
... Immunofluorescence experiments were performed using the protocol described in Ref. [1]. Briefly, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, blocked with bovine serum albumin, incubated with primary antibodies and incubated with secondary antibodies, before observation by confocal microscopy. ...
... Cellular fractionation and mitochondria purification were performed using the protocol described in Ref. [1]. Briefly, mitochondria were isolated from different cell lines by differential centrifugation with all steps carried out at 4 C [3]. ...
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.
... SCVT2 was overexpressed in the mitochondria of breast cancer cells, but only marginally presented on the plasma membrane [116]. Augmented expression of mitochondrial SVCT2 appears to be a common hallmark across all human cancers and might have implications for the survival capacity of cancer cells in pro-oxidant environments [116,191]. In addition, the analysis of numerous tissue microarrays contained in the Human Protein Atlas reveals the intracellular expression of SVCT2 in different cancer tissues [116]. ...
... This effect was due to increased uptake of DHA via GLUT1 [64]. Moreover, cancer cells were able to acquire vitamin C, even if they expressed an abnormal form of SVCT2, by using GLUTs and converting DHA into ascorbate [191], a phenomenon that is called the bystander effect [68]. Recently, it was shown that the uptake of DHA largely affects the redox metabolism of human erythrocytes [208], albeit these blood cells do not express SVCTs [24]. ...
Despite the increasing number of newly diagnosed malignancies worldwide, therapeutic options for some tumor diseases are unfortunately still limited. Interestingly, preclinical but also some clinical data suggest that the administration of pharmacological ascorbate seems to respond well, especially in some aggressively growing tumor entities. The membrane transport and channel proteins are highly relevant for the use of pharmacological ascorbate in cancer therapy and are involved in the transfer of active substances such as ascorbate, hydrogen peroxide, and iron that predominantly must enter malignant cells to induce antiproliferative effects and especially ferroptosis. In this review, the relevant conveying proteins from cellular surfaces are presented as an integral part of the efficacy of pharmacological ascorbate, considering the already known genetic and functional features in tumor tissues. Accordingly, candidates for diagnostic markers and therapeutic targets are mentioned.
... For instance, adaptative up-regulation of SVCT2 has been described as a response of increased ROS levels in senescent human fibroblasts, where it translocates from an intracellular distribution to the plasma membrane [90]. Also, the exclusive expression of SVCT2 in the mitochondria of breast cancer cells has been associated to cell survival in the context of pro-oxidant environments [91]. ...
Background
Vitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited.
Results
In this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern.
Conclusions
Our results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.
... Interestingly, we found higher mRNA expression of vitamin C transporters (SLC23A1 and SLC23A2) in breast cancer patients in comparison to control samples. This observation is consistent with previous studies where SVCT2 was not immunohistochemically detectable in normal breast cells in comparison to cancer ones 73 . High expression of vitamin C transporters may increase the uptake of vitamin C in plasma followed by its decreased levels. ...
... Interestingly, we found higher mRNA expression of vitamin C transporters (SLC23A1 and SLC23A2) in breast cancer patients in comparison to control samples. This observation is consistent with previous studies where SVCT2 was not immunohistochemically detectable in normal breast cells in comparison to cancer ones 73 . High expression of vitamin C transporters may increase the uptake of vitamin C in plasma followed by its decreased levels. ...
The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs’ activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5–mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.
... In the epithelial cells of the human intestine, SVCT1 is expressed in the apical membrane while SVCT2 is found in the basolateral membrane [12]. A number of studies have reported a localisation of SVCT2 to various intracellular organelles but the reason for this remains largely unknown [5, [13][14][15][16][17][18]. SVCT2 demonstrates low capacity but high affinity transport and the reported Km is in the range of 22-69 µM [10]. ...
Ascorbate plays a vital role as a co-factor for a superfamily of enzymes, the 2-oxoglutarate dependent dioxygenases (2-OGDDs), which govern numerous pathways in cancer progression, including the hypoxic response and the epigenetic regulation of gene transcription. Ascorbate uptake into most cells is through active transport by the sodium-dependent vitamin C transporter 2 (SVCT2). The aims of this study were to determine the kinetics of ascorbate uptake and retention by breast cancer cell lines under various oxygen conditions, and to investigate the role of SVCT2 in mediating ascorbate uptake and intracellular trafficking. Human MDA-MB231 cells accumulated up to 5.1 nmol ascorbate/106 cells, human MCF7 cells 4.5 nmol/106 cells, and murine EO771 cells 26.7 nmol/106 cells. Intracellular ascorbate concentrations decreased rapidly after reaching maximum levels unless further ascorbate was supplied to the medium, and there was no difference in the rate of ascorbate loss under normoxia or hypoxia. SVCT2 was localised mainly to subcellular compartments, with the nucleus apparently containing the most SVCT2 protein, followed by the mitochondria. Much less SVCT2 staining was observed on the plasma membrane. Our data showed that careful management of the doses and incubation times with ascorbate in vitro allows for an approximation of in vivo conditions. The localisation of SVCT2 suggests that the distribution of ascorbate to intracellular compartments is closely aligned to the known function of ascorbate in supporting 2-OGDD enzymatic functions in the organelles and with supporting antioxidant protection in the mitochondria.
... DHA is an oxidized form of AA that is transported into the cell via the facilitated glucose transporters GLUTs. 53 Cancer cells take in DHA and it is reduced back to AA. 54 This intracellular reduction back to AA cause a depletion of glutathione in the cell, which consequently lead to an increase in ROS, oxidative stress, energy crisis and cell death. [55][56] In another study, DHA is also reported to reacts with homocysteine thiolactone (cancer cells have high levels of homocysteine thiolactone) converting it to the toxic compound, 3-mercaptopropionaldehyde and kills the cell. ...
Introduction and aim. Over the past decades, the hypotheses that ascorbic acid (AA) can play a role as an
... The evidence on the link between VC and Warburg effect is inadequate. Since VC is easily hydroxylated into dehydroascorbic acid (DHA), which can be transported into cells via GLUTs as its structure is similar to glucose [18], whether VC would interfere with the glucose consumption and utility of cancer cells remains unknown. Also, there is need for detailed research on the relationship between VC and AMPK-mTOR pathway. ...
Malnutrition caused by insufficient nutritional supply may significantly hinder the quality of life among cancer patients. Sugar provides energy and nutritional support, but it also promotes cancer growth. Warburg effect is the reprogrammed glucose metabolic mode of cancer cells that meets the intensified ATP demand and biosynthesis. Vitamin C (VC) has anti-tumor effect. However, the relationship between cytotoxicity of VC on cancer cells and Warburg effect remains elusive, the effect of VC on glucose-induced oncogenic effect is also unclear. Based on colorectal cancer cell HCT116, our finding revealed that the discrepant oncogenic effect of different sugar is closely related to the intensification of Warburg effect, with glucose being the potent oncogenic component. Notably, as a potential Warburg effect inhibitor, VC suppressed cancer growth in a concentration-dependent manner and further reversed the glucose-induced oncogenic effect. Furthermore, VC protected tumor-bearing mice from insulin sensitivity impairment and inflammatory imbalance. These findings imply that VC might be a useful adjuvant treatment for cancer patients seeking to optimize nutritional support.
... Interestingly, we found higher mRNA expression of vitamin C transporters (SLC23A1 and SLC23A1) in breast cancer patients in comparison to control samples. This observation is consistent with previous studies where SVCT2 was not immunohistochemically detectable in normal breast cells in comparison to cancer ones [73]. Moreover, breast cancer patients exhibited negative correlation between 5-mC level and expression of SLC23A2 (p < 0.01, R = − 0.3412). ...
Background: The experimental evidence has demonstrated that active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation residues in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. TETs’ activity may be upregulated by ascorbate. Given that aberrant DNA methylation of crucial genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in active DNA demethylation process. Moreover, we tried to verify if the expression level of genes involved in active DNA demethylation and products of this process may serve as predictive markers of breast cancer.
Methods: The study included blood samples from breast cancer patients (n=74) and healthy subjects (n=71). We applied qRT-PCR with Universal Probe Library probes for measurement of expression of genes involved in active demethylation process, and two dimensional ultra performance liquid chromatography with tandem mass spectrometry detection (2D-UPLC MS/MS) for 5–mC and its derivatives assessment. Ascorbate level was determined using UPLC-MS.
Results: Breast cancer patients exhibited significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3expression was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, ascorbate level in plasma of breast cancer patients was decreased with concomitant increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2) mRNA expression level.
Conclusions: Presented study indicate particular role of TET3 in DNA demethylation in breast carcinogenesis. Furthermore, correlation between SLC23A2 and 5-mC level, may provide new path for future breast cancer research.
... Several studies indicate the critical role of vitamin C transport by GLUTs in cancer cells. Pena et al. suggested that the vast majority of vitamin C transferred from the extracellular space into cancer cells assumes the form of DHA [22]. Several cell culture studies suggest that DHA transport is an alternate or even principal pathway of vitamin C accumulation [23]. ...
Vitamin C (ascorbic acid, AA) is a weak sugar acid structurally related to glucose. All known physiological and biochemical functions of AA are due to its action as an electron donor. Ascorbate readily undergoes pH-dependent autoxidation creating hydrogen peroxide (H2O2). In vitro evidence suggests that vitamin C functions at low concentrations as an antioxidant while high concentration is pro-oxidant. Thus, both characters of AA might be translated into clinical benefits. In vitro obtained results and murine experiments consequently prove the cytotoxic effect of AA on cancer cells, but current clinical evidence for high-dose intravenous (i.v.) vitamin C’s therapeutic effect is ambiguous. The difference might be caused by the missing knowledge of AA’s actions. In the literature, there are many publications regarding vitamin C and cancer. Review papers of systematic analysis of human interventional and observational studies assessing i.v. AA for cancer patients’ use helps the overview of the extensive literature. Based on the results of four review articles and the Cancer Information Summary of the National Cancer Institute’s results, we analyzed 20 publications related to high-dose intravenous vitamin C therapy (HAAT). The analyzed results indicate that HAAT might be a useful cancer-treating tool in certain circumstances. The AA’s cytotoxic effect is hypoxia-induced factor dependent. It impacts only the anoxic cells, using the Warburg metabolism. It prevents tumor growth. Accordingly, discontinuation of treatment leads to repeated expansion of the tumor. We believe that the clinical use of HAAT in cancer treatment should be reassessed. The accumulation of more study results on HAAT is desperately needed.
... Natural products are versatile and perform important antitumor activities, as they act in different pathways, blocking tumorigenesis and controlling the progression of transformed cells [32]. A. chica protective effects involve the reduction of ROS levels and lipid peroxidation supporting the antioxidant potential of CEAC identified in the present study using the ABTS + method. In addition, this species increases of collagen content during the healing process, with analgesic properties through the inhibition of cyclooxygenases [33,[64][65][66][67] also demonstrated that the plant extract alone, or associated with vincristine, decreased serum transaminases levels, oxidative stress, and hematological toxicity. Our work is pioneer in demonstrating the role of A. chica in the modulation of hormone receptors. ...
Background
Breast Cancer (BC) is the most common cancer in women worldwide and, although 70% of patients are responsive to selective Estrogen Receptor (ER) modulators such as Tamoxifen (Tam), patients’ survival is comprised by resistance to endocrine therapy. Brazilian flora, especially the Amazon biome, is one of the richest global sources of native species with potentially bioactive compounds. Arrabidaea chica is a plant native to the Amazon that has been used in the treatment of different diseases. However, its action on BC remains unclear.
Methods
Herein the biological effects of the chloroform extract of A. chica (CEAC) were evaluated on BC cells and in in vivo model. After confirmation of CEAC antioxidant capacity, cells were treated with CEAC and Tam, alone and with CEAC+Tam. The cell viability was evaluated by MTT and hormone receptor transcripts levels were assessed ( ESR1 , ESR2 and AR ). Finally, anticarcinogenicity of CEAC was recorded in Drosophila melanogaster through Epithelial Tumor Test (ETT).
Results
The study confirmed the antioxidant activity of CEAC. CEAC was selective for MCF-7, downregulating ESR2 and AR transcripts and upregulating ESR2 expression. The modulatory effects of CEAC on ERs did not differ between cells treated with Tam and with CEAC+Tam. Interestingly, previous treatment with CEAC, followed by treatment with Tam promoted a significant decrease in cell viability. The extract also presented anticarcinogenic effect in in vivo assay.
Conclusion
The bioassays on breast tumor cells demonstrated the antiproliferative activity of the extract, which modulated the expression of hormone receptors and sensitized luminal tumor cells to Tam. These results suggest that CEAC could be a complementary treatment for BC.
... Inside the cell, it is reduced again, which consumes other antioxidants, and contributes to the development of intracellular oxidative stress [27]. On the other hand, a high expression of SVCT2 has been found in various tumors and tumor cell lines derived from breast cancer, mainly at the mitochondrial level [28,29]. Also, Lv et al. found that mega-doses of AA (0.5 mM) kill stem cells from liver cancer with a high expression of SVCT2. ...
Vitamin C is a water-soluble antioxidant associated with the prevention of the common cold and is also a cofactor of hydrolases that participate in the synthesis of collagen and catecholamines, and in the regulation of gene expression. In cancer, vitamin C is associated with prevention, progression, and treatment, due to its general properties or its role as a pro-oxidant at high concentration. This review explores the role of vitamin C in cancer clinical trials and the aspects to consider in future studies, such as plasmatic vitamin C and metabolite excretion recording, and metabolism and transport of vitamin C into cancer cells. The reviewed studies show that vitamin C intake from natural sources can prevent the development of pulmonary and breast cancer, and that vitamin C synergizes with gemcitabine and erlotinib in pancreatic cancer. In vitro assays reveal that vitamin C synergizes with DNA-methyl transferase inhibitors. However, vitamin C was not associated with cancer prevention in a Mendelian randomized study. In conclusion, the role of vitamin C in the prevention and treatment of cancer is still an ongoing area of research. It is necessary that new phase II and III clinical trials be performed to collect stronger evidence of the therapeutic role of vitamin C in cancer.
... While SVCT2 is related to sustaining ascorbate homeostasis inside the cell as well as sensitive to the fluctuations in intracellular ascorbate levels. The expression of SVCT1 in the human being is mainly limited to the epidermal cells in the kidney and duodenum [55]. ...
Aging and age-related disorders have become one of the prominent issue of world. Oxidative stress due to overproduction of reactive oxygen species is the most significant cause of aging. The aim of literature compilation was to elucidate the therapeutic effect of vitamin C against oxidative stress. Various mediators with anti-inflammatory and anti-oxidant properties might be probable competitors of vitamin C for the improvement of innovative anti-aging treatments. More attention has been paid to vitamin C due to its anti-oxidant property and potentially beneficial biological activities for inhibiting aging.Vitamin C acts as an antioxidant agent and free radical scavenger that can protect the cell from oxidative stress, disorganization of chromatin, telomere attrition, and prolong the lifetime. This review emphasizes mechanism of aging and various biomarkers that are directly related to aging and also focuses on the therapeutic aspect of vitamin C against oxidative stress and age-related disorders.
... The different vitamin C content reported in the tumor vs. normal cells might depend on the expression pattern of transporters. Indeed, in several tumors (e.g., KRAS/BRAF mutated colorectal, gastric, or breast cancer) the sensitivity to vitamin C correlated with the expression of GLUT1 and DHA adsorption [44][45][46][47]180,181]. Interestingly, the activity of vitamin C against KRAS/BRAF mutated colorectal cancer might be useful to counteract tumor resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (i.e., cetuximab, panitumumab) [182]. ...
High-dose vitamin C has been proposed as a potential therapeutic approach for patients with advanced tumors who failed previous treatment with chemotherapy. Due to vitamin C complex pharmacokinetics, only intravenous administration allows reaching sufficiently high plasma concentrations required for most of the antitumor effects observed in preclinical studies (>0.250 mM). Moreover, vitamin C entry into cells is tightly regulated by SVCT and GLUT transporters, and is cell type-dependent. Importantly, besides its well-recognized pro-oxidant effects, vitamin C modulates TET enzymes promoting DNA demethylation and acts as cofactor of HIF hydroxylases, whose activity is required for HIF-1α proteasomal degradation. Furthermore, at pharmacological concentrations lower than those required for its pro-oxidant activity (<1 mM), vitamin C in specific genetic contexts may alter the DNA damage response by increasing 5-hydroxymethylcytosine levels. These more recently described vitamin C mechanisms offer new treatment opportunities for tumors with specific molecular defects (e.g., HIF-1α over-expression or TET2, IDH1/2, and WT1 alterations). Moreover, vitamin C action at DNA levels may provide the rationale basis for combination therapies with PARP inhibitors and hypomethylating agents. This review outlines the pharmacokinetic and pharmacodynamic properties of vitamin C to be taken into account in designing clinical studies that evaluate its potential use as anticancer agent.
... SVCT2 was considered to be a potential biomarker for AA and cetuximab treatment in patients with colorectal cancer (CRC) [12] . Some studies have also shown that increased expression of mitochondrial SVCT2 intracellular form is a common feature of all human cancers [13] . Therefore, a study of the mechanisms of extracellular and intracellular transport and accumulation of AA can provide valuable insights on the therapeutic anti-cancer effects of AA. ...
L-ascorbic acid (AA) was reported to have an anti-cancer effect over 40 years. In recent years, several ongoing clinical trials are exploring the safety and efficacy of intravenous high-dose AA for cancer treatment. The lack of appropriate imaging modality limits the identification of potentially suitable patients for AA treatment. This study focuses on identifying AA-sensitive tumor cells using molecular imaging. 6-Deoxy-6-[¹⁸F] fluoro-L-ascorbic Acid (¹⁸F-DFA), a structural analog of AA, was synthesized and labeled to visualize the metabolism of AA in vivo. Colorectal cancer (CRC) cell lines with high and low expression of sodium-dependent vitamin C transporters 2 (SVCT2) were used for a series of cellular uptake tests. PET imaging was performed on xenograft tumor-bearing mice. More AA uptake was observed in CRC cells with high SVCT2 expression than in cells with low SVCT2 expression. The substrate (unlabeled AA) can competitively inhibit the ¹⁸F-DFA tracer uptake by CRC cells. The biodistribution of ¹⁸F-DFA in mice showed high radioactivity was seen in organs such as adrenal glands, kidneys, and liver that were known to have high concentrations of AA. Both PET imaging and tissue distribution showed that cancer cells with high SVCT2 expression enhanced the accumulation of ¹⁸F-DFA in mice after tumor formation. Immunohistochemistry was used to verify the corresponding results. As a radiotracer, ¹⁸F-DFA can provide powerful imaging information to identify tumor with high affinity of AA, and SVCT2 can be a potential biomarker in this process.
... Various polymorphisms of SLC23A2 have been associated with an increased risk of both colorectal adenoma and gastric cancer, while SLC23A1 polymorphisms have shown conflicting results for their link to cancer risk [227,[233][234][235]. Vitamin C has emerged as an intriguing therapeutic tool in oncology, with high concentrations of vitamin C killing colorectal cells by increasing oxidative stress in cells, inactivating GAPDH and reducing tumor size [222,236,237]. The varying localization of SLC23A2 in cells may be important for helping increase the therapeutic effect of vitamin C, as its mitochondrial localization in most cancer cell lines has allowed for mitochondria to be targeted in cancer therapeutics [238]. A recent study showed that a combinatorial therapy of doxycycline, azithromycin and vitamin C effectively eradicates cancer stem cells [239]. ...
This review aims to serve as an introduction to the solute carrier proteins (SLC) superfamily of transporter proteins and their roles in human cells. The SLC superfamily currently includes 458 transport proteins in 65 families that carry a wide variety of substances across cellular membranes. While members of this superfamily are found throughout cellular organelles, this review focuses on transporters expressed at the plasma membrane. At the cell surface, SLC proteins may be viewed as gatekeepers of the cellular milieu, dynamically responding to different metabolic states. With altered metabolism being one of the hallmarks of cancer, we also briefly review the roles that surface SLC proteins play in the development and progression of cancer through their influence on regulating metabolism and environmental conditions.
... Considering these data, at least under physiological conditions, the external source of vitamin C for cancer cells must be DHA and the transport mechanism implied is through GLUTs. Since DHA is not the most abundant form of vitamin C in vivo, Pena et al. (2019), developed an experimental system to check if AA could be oxidized locally by PMA-activated neutrophil like cells, which are producing ROS. Co-culture experiments in the presence of AA showed that cancers cells were able to acquire vitamin C in a process inhibited by cytochalasin B, a glucose transporter inhibitor, and by glucose, indicating that they were able to acquire DHA through GLUTs by bystander effect (Nualart et al., 2003). ...
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 in vitro 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 in vitro and in vivo, 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.
... To determine anticancer potential of vitamin C, it is important to determine the difference between its bioavailability in normal and cancer cells, especially that the results of studies suggest that such a difference can depend on the type of cancer [44][45][46][47]. Given that vitamin C transporters SVCT1 and SVCT2 are essential in the acquisition of this vitamin by the cell, Pena et al. showed that breast cancer samples differentially expressed a form of the SVCT2 transporter, systematically absent in normal breast tissue [48]. However, these authors observed that various cancer cell lines were not able to uptake vitamin C and acquired it by a glucose transporter by a bystander effect. ...
Vitamin C is an antioxidant that may scavenge reactive oxygen species preventing DNA damage and other effects important in cancer transformation. Dietary vitamin C from natural sources is taken with other compounds affecting its bioavailability and biological effects. High pharmacological doses of vitamin C may induce prooxidant effects, detrimental for cancer cells. An oxidized form of vitamin C, dehydroascorbate, is transported through glucose transporters, and cancer cells switch from oxidative phosphorylation to glycolysis in energy production so an excess of vitamin C may limit glucose transport and ATP production resulting in energetic crisis and cell death. Vitamin C may change the metabolomic and epigenetic profiles of cancer cells, and activation of ten-eleven translocation (TET) proteins and downregulation of pluripotency factors by the vitamin may eradicate cancer stem cells. Metastasis, the main reason of cancer-related deaths, requires breakage of anatomical barriers containing collagen, whose synthesis is promoted by vitamin C. Vitamin C induces degradation of hypoxia-inducible factor, HIF-1, essential for the survival of tumor cells in hypoxic conditions. Dietary vitamin C may stimulate the immune system through activation of NK and T cells and monocytes. Pharmacological doses of vitamin C may inhibit cancer transformation in several pathways, but further studies are needed to address both mechanistic and clinical aspects of this effect.
... The value of the in vitro study performed by Peña et al. 1 relates to vitamin C transport in cellular and intracellular membranes. It does not contradict the findings of thousands of other studies of vitamin C; the preponderance of evidence published over the decades based on in vitro, in vivo, and clinical studies indicates that intravenous vitamin C therapy has several mechanisms of action that are beneficial in the management of cancer. ...
A news brief circulated by the EFE News Media
Company in July 2019 claimed that scientists from
Chile had found that vitamin C immortalizes cancer
cells. This study was carried out by a group of
highly qualified scientists and published in a reputable
scientific journal. The news agency, however,
reported the study in a way that did not capture its
main findings, and the story confused the general
public and even medical professionals. It is important
to clarify that this was an in-vitro study, and the
study did not actually find that cancer cells become
immortal with vitamin C. In addition, this study
did not contradict thousands of published studies
that explain the many biological mechanisms and
beneficial effects of vitamin C use in cancer. The
purpose of the present article is to clarify the EFE
news report and put into perspective the bigger
picture of the findings in this study.
... Intracellular H 2 O 2 can be transported from the extracellular medium by aquaporins or by passive diffusion across plasma cellular membranes [46] but also generated by intracellular AA upon import through SVCTs (mainly SVCT2) [47]. Although in breast cancer cells, AA may not be imported by SVCT2, but only to mitochondria via mitochondrial SVCT2 (mitSVCT2), AA can be oxidized extracellularly to DHA and then imported through glucose transporters to be reduced by GSH-dependent mechanisms to AA intracellularly [48], consuming intracellular GSH [49,50]. AA treatment was cytotoxic toward most of breast cancer cells tested and its higher cytotoxicity compared to DHA was mainly linked to H 2 O 2 generation. ...
Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells.
Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H2O2) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation
and had less cytotoxicity. A “redoxome” approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.
Objectives
This study was conducted to evaluate the effects of vitamin C on apoptotic and proliferative genes in injured HepG2 cells.
Methods
In silico analysis was performed using molecular docking of chemical compounds with vascular endothelial growth factor (VEGF). The different computational tools used were AutoDock Vina, BIOVIA DISCOVERY studio, and PyMOL. Drug likeness and toxicity were analyzed by SWISS ADMET. Cells that were 60–70% confluent were treated with different concentrations of hydrogen peroxide (H2O2) (100–2000 μM) and ascorbic acid (30, 60, 90 μg/mL). The MTT cell proliferation assay was performed to compare the proliferative potential of HepG2 cells treated with H2O2 or ascorbic acid with untreated HepG2 cells using 96-well plates.
Results
The lowest binding energy of VEGF with vitamin C –5.2 kcal/mol and L-ascorbic acid-2 glycoside −4.7 kcal/mol was observed by in silico analysis. Vitamin C was selected because it exhibited a high interaction with VEGF and fulfilled Lipinski's rule, and had better oral viability and pharmacokinetics compared to L-ascorbic acid-2 glycoside. Cell viability assays showed that vitamin C had significant apoptotic effects (P < 0.0001). After treating HepG2 cells with ascorbic acid, reduced VEGF (angiogenesis) was observed as determined by apoptotic and proliferative gene expression. Ascorbic acid treatment of HepG2 cells led to downregulation of the proliferation markers, proliferating cell nuclear antigen, Ki67, and DNA topoisomerase II alpha. Increased apoptosis after treatment with vitamin C was observed due to upregulation of p53 and annexin V.
Conclusion
The results of this study showed that vitamin C inhibited the growth of cancer cells, thus protecting HepG2 cells from oxidative stress. Vitamin C exhibited antiproliferative activity as observed in silico and in vitro, as well as by the inhibited expression of genes involved in protein synthesis.
There is increased evidence that the excessive production of free radicals in the human body plays a major role in the pathophysiology and development of various diseases, closely associated with oxidative damage. In this frame, the consumption of antioxidant nutrients through food or dietary supplements may prevent free radicals from harming human cells. This work proposes a holistic approach consisting of distinct methodologies, suitable to evaluate the antioxidant and chemoprotective activity of three novel dietary supplements, each one containing active substances with complementary properties. In one step, this approach includes in vitro studies to evaluate the antioxidant activity of the dietary supplements by measuring the parameters of free radical scavenging capacity, of reductive power activity, as well as, their ability to protect biomolecules from oxidation. Furthermore, their antimutagenic and antigenotoxic evaluation is also presented. In another step, the specific effects of the dietary supplements were examined in three cancer cell lines (HepG2, HeLa, MKN45), by measuring redox biomarkers such as glutathione, reactive oxygen species and thiobarbituric acid reactive substances, using flow cytometry and spectrophotometry. Our results indicatthat all the dietary supplements exhibit high antioxidant, antimutagenic, antigenotoxic and lipid protective activity. The most prominent result is their capability to induce the damage caused by cancer cells due to critical downregulation of the levels of their intracellular glutathione as well as increase of ROS and lipid peroxidation levels after the administration of non-cytotoxic concentrations. We suggest that the proposed methodology could constitute a valuable tool for the characterization of dietary supplements based on their chemical and functional properties.
The Na⁺-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.
Breast cancer is the most common cancer among women and is considered a developed country disease. Moreover, is a heterogenous disease, existing different types and stages of breast cancer development, therefore, better understanding of cancer biology, helps to improve the development of therapies. The conventional treatments accessible after diagnosis, have the main goal of controlling the disease, by improving survival. In more advance stages the aim is to prolong life and symptom palliation care. Surgery, radiation therapy and chemotherapy are the main options available, which must be adapted to each person individually. However, patients are developing resistance to the conventional therapies. This resistance is due to alterations in important regulatory pathways such as PI3K/AKt/mTOR, this pathway contributes to trastuzumab resistance, a reference drug to treat breast cancer.
Therefore, is proposed the repurposing of drugs, instead of developing drugs de novo, for example, to seek new medical treatments within the drugs available, to be used in breast cancer treatment. Providing safe and tolerable treatments to patients, and new insights to efficacy and efficiency of breast cancer treatments. The economic and social burden of cancer is enormous so it must be taken measures to relieve this burden and to ensure continued access to therapies to all patients. In this review we focus on how conventional therapies against breast cancer are leading to resistance, by reviewing those mechanisms and discussing the efficacy of repurposed drugs to fight breast cancer.
Premise: Mitochondria represent critical sites for reactive oxygen species (ROS) production, which dependent on concentration is responsible for the regulation of both physiological and pathological processes.
Purpose: Antioxidants in mitochondria regulate the redox balance, prevent mitochondrial damage and dysfunction and maintain a physiological ROS-dependent signalling. The aim of the present review is to provide critical elements for addressing this issue in the context of various pharmacological approaches using antioxidants targeted or non-targeted to mitochondria. Furthermore, this review focuses on the mitochondrial antioxidant effects of ascorbic acid (AA), providing clues on the complexities associated with the cellular uptake and subcellular distribution of the vitamin.
Conclusions: Antioxidants that are not specifically targeted to mitochondria fail to accumulate in significant amounts in critical sites of mitochondrial ROS production and may eventually interfere with the ensuing physiological signaling. Mitochondria-targeted antioxidants are more effective, but are expected to interfere with the mitochondrial ROS-dependent physiologic signaling. AA promotes multiple beneficial effects in mitochondria. The complex regulation of vitamin C uptake in these organelles likely contributes to its versatile antioxidant response, thereby providing a central role to the vitamin for adequate control of mitochondrial dysfunction associated with increased mitochondrial ROS production.
Significance:
Cardiovascular disorders are the most important cause of morbidity and mortality in the Western world. Monogenic developmental disorders of the heart and vessels are highly valuable to study the physiological and pathological processes in cardiovascular system homeostasis. The arterial tortuosity syndrome (ATS) is a rare, autosomal recessive connective tissue disorder showing lengthening, tortuosity, and stenosis of the large arteries, with a propensity for aneurysm formation. In histopathology it associates with fragmentation and disorganization of the elastic fibers in several tissues, including the arterial wall. ATS is caused by pathogenic variants in SLC2A10, encoding the facilitative glucose transporter GLUT10.
Critical issues:
Although several hypotheses have been forwarded, the molecular mechanisms linking disrupted GLUT10 activity with arterial malformations are largely unknown. Recent Advances: The vascular and systemic manifestations, and natural history of ATS patients have been largely delineated. GLUT10 was identified as an intracellular transporter of dehydroascorbic acid, which contributes to collagen and elastin crosslinking in the ER, redox homeostasis in the mitochondria and global and gene-specific methylation/hydroxymethylation affecting epigenetic regulation in the nucleus. We revise here the current knowledge on ATS and the role of GLUT10 within the compartmentalization of ascorbate in physiological and diseased states.
Future directions:
Centralization of clinical, treatment and outcome data will enable better management for ATS patients. Establishment of representative animal disease models could facilitate the study of the pathomechanisms underlying ATS. This might be relevant for other forms of vascular dysplasia, such as isolated aneurysm formation, hypertensive vasculopathy and neovascularization.
Sufficient uptake and whole body distribution of vitamin C (ascorbate) is essential for many biochemical processes, including some that are vital for tumor growth and spread. Uptake of ascorbate into cancer cells is modulated by availability, tumor blood flow, tissue diffusion parameters, and ascorbate transport proteins. Uptake into cells is mediated by two families of transport proteins, namely, the solute carrier gene family 23, consisting of sodium-dependent vitamin C transporters (SVCTs) 1 and 2, and the SLC2 family of glucose transporters (GLUTs). GLUTs transport the oxidized form of the vitamin, dehydroascorbate (DHA), which is present at negligible to low physiological levels. SVCT1 and 2 are capable of accumulating ascorbate against a concentration gradient from micromolar concentrations outside to millimolar levels inside of cells. Investigating the expression and regulation of SVCTs in cancer has only recently started to be included in studies focused on the role of ascorbate in tumor formation, progression, and response to therapy. This review gives an overview of the current, limited knowledge of ascorbate transport across membranes, as well as tissue distribution, gene expression, and the relevance of SVCTs in cancer. As tumor ascorbate accumulation may play a role in the anticancer activity of high dose ascorbate treatment, further research into ascorbate transport in cancer tissue is vital.
Paclitaxel (PTX), especially albumin-bound PTX in clinical, has displayed significant inhibition of tumor growth in patients. But the systemic distribution and poor water solubility of PTX often lead to severe side effects, consequently limiting the anti-tumor efficacy. In this study, we developed a novel PTX-loaded polymeric micelle drug delivery system. These self-assembled polymeric micelles from core to outside consisted of poly L-phenylalanine (pPhe), DTSSP linked poly L-lysine (pLys), poly ethylene glycol (PEG) and dehydroascorbic acids (DHA). pPhe formed the hydrophobic core to encapsulate PTX; DTSSPs on pLys covalently cross-linked and formed disulfide bond to stabilize PTX from loss in blood circulation; PEG improved solubility to lower toxicity of PTX for its high hydrophilicity; DHA targeted tumors by specifically recognizing GLUT1 mainly expressed on tumor cells. Thus, PTX would be precisely released into tumor cells with high dose of glutathione to break disulfide bond. Moreover, these PTX-loaded polymer micelles significantly suppressed tumor cell viability, proliferation, and migration in vitro, and also greatly inhibited tumor growth and prolonged survival in tumor-bearing mice without detectable side effects. Therefore, the new drug delivery system could reduce severe side effects and enhance anti-tumor efficacy of PTX via peripheral stabilization, low toxicity and tumor targeting.
Background:
Many cancer patients receive supplemental ascorbate (vitamin C) in the belief that it synergizes the anticancer effects of chemotherapy and reduces its toxicity.
Methods:
A systematic review was performed to evaluate the antitumor effects and toxicity of ascorbate treatment. Medline (1946 to March 2014), EMBASE (1947 to March 2014), and the Cochrane central register (1993 to March 2014) were searched for randomized and observational studies.
Results:
Of 696 identified records, 61 full-text articles were screened and 34 were included. In total, 5 randomized controlled trials (RCTs) (n = 322), 12 phase I/II trials (n = 287), 6 observational studies (n = 7,599), and 11 case reports (n = 267) were identified. Because of study heterogeneity, no meta-analyses were performed. No RCTs reported any statistically significant improvements in overall or progression-free survival or reduced toxicity with ascorbate relative to control arm. Evidence for ascorbate's antitumor effects was limited to case reports and observational and uncontrolled studies.
Conclusion:
There is no high-quality evidence to suggest that ascorbate supplementation in cancer patients either enhances the antitumor effects of chemotherapy or reduces its toxicity. Given the high financial and time costs to patients of this treatment, high-quality placebo-controlled trials are needed.
Ascorbate is a co-factor for the hydroxylases that regulate the transcription factor hypoxia-inducible factor (HIF)-1, which provides cancer cells with a metabolic and survival advantage in the hypoxic environment of solid tumors. However, whether ascorbate affects tumor development is a highly debated issue. We aimed to determine whether tumor ascorbate was associated with HIF-1 activation and patient disease-free survival. In this study we undertook a retrospective observational analysis of tissue-banked tumor and paired normal tissue from 49 colorectal cancer patients, measuring ascorbate levels, HIF-1α and its downstream gene products BNIP3 and VEGF. Patient survival was monitored for the first six years after surgery. We found that ascorbate levels were lower in tumor tissue compared to normal tissue (p< 0.001) but overall levels varied considerably. HIF-1α, VEGF and BNIP3 were elevated in tumor samples (p< 0.01). There was an inverse relationship between tumor ascorbate content and HIF-1 pathway activation (p=0.002) and tumor size (p=0.018). Higher tumor ascorbate content was associated with significantly improved disease-free survival in the first 6 years after surgery (p=0.006), with 141 - 1,094 additional disease free days. This was independent of tumor grade and stage. Survival advantage was associated with the amount of ascorbate in the tumor, but not with the amount in adjacent normal tissue. Our results demonstrate that higher tumor ascorbate content is associated decreased HIF-1 activation, most likely due to the co-factor activity of ascorbate for the regulatory HIF hydroxylases. Our findings support the need for future studies to determine whether raising tumor ascorbate is possible with clinical intervention and whether this results in modification of hydroxylase-dependent pathways in the tumor.
Ascorbate (vitamin C) was an early, unorthodox therapy for cancer, with an outstanding safety profile and anecdotal clinical benefit. Because oral ascorbate was ineffective in two cancer clinical trials, ascorbate was abandoned by conventional oncology but continued to be used in complementary and alternative medicine. Recent studies provide rationale for reexamining ascorbate treatment. Because of marked pharmacokinetic differences, intravenous, but not oral, ascorbate produces millimolar concentrations both in blood and in tissues, killing cancer cells without harming normal tissues. In the interstitial fluid surrounding tumor cells, millimolar concentrations of ascorbate exert local pro-oxidant effects by mediating hydrogen peroxide (H2O2) formation, which kills cancer cells. We investigated downstream mechanisms of ascorbate-induced cell death. Data show that millimolar ascorbate, acting as a pro-oxidant, induced DNA damage and depleted cellular adenosine triphosphate (ATP), activated the ataxia telangiectasia mutated (ATM)/adenosine monophosphate-activated protein kinase (AMPK) pathway, and resulted in mammalian target of rapamycin (mTOR) inhibition and death in ovarian cancer cells. The combination of parenteral ascorbate with the conventional chemotherapeutic agents carboplatin and paclitaxel synergistically inhibited ovarian cancer in mouse models and reduced chemotherapy-associated toxicity in patients with ovarian cancer. On the basis of its potential benefit and minimal toxicity, examination of intravenous ascorbate in combination with standard chemotherapy is justified in larger clinical trials.
Intestinal vitamin C (ascorbate) absorption was believed to be mediated by the Na+ dependent ascorbic acid transporter, SVCT1. However, ascorbate transport across intestines of SVCT1 knockout mice is normal indicating that alternative ascorbic acid transport mechanisms exist. To investigate these mechanisms, rodents were gavaged with ascorbate or its oxidized form dehydroascorbic acid (DHA), and plasma ascorbate concentrations measured. Ascorbate concentrations doubled following DHA but not ascorbate gavage. We hypothesized that the transporters responsible were facilitated glucose transporters (GLUTs). Using xenopus oocyte expression, we investigated whether facilitative glucose transporters GLUT2, and GLUT5-12 transported DHA. Only GLUT2 and GLUT8, known to be expressed in intestines, transported DHA with apparent transport affinities (Km) of 2.33 and 3.23 mM, and maximal transport rates (Vmax) of 25.9 and 10.1 pmols/mins/ oocyte, respectively. Maximal rates for DHA transport mediated by GLUT2 and GLUT8 in oocytes were lower than maximal rates for 2-DG (Vmax of 224 and 32 pmoles/min/oocyte for GLUT2 and GLUT8, respectively) and fructose (Vmax of 406 and 116 pmols/min/oocyte for GLUT2 and GLUT8, respectively). These findings may be explained by differences in the exofacial binding of substrates, as shown by inhibition studies with ethylidine glucose. DHA transport activity in GLUT2 and GLUT8 expressing oocytes were inhibited by glucose, fructose, and by the flavonoids phloretin and quercetin. These studies indicate intestinal DHA transport may be mediated by the facilitative sugar transporters, GLUT2 and GLUT8. Furthermore, dietary sugars and flavonoids in fruits and vegetables may module ascorbate bioavailability via inhibition of small intestinal GLUT2 and GLUT8.
U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin. © 2013 IUBMB Life, 2013.
Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1–5 and SGLT1. The apparentK
m of DHA transport via GLUT1 and GLUT3 was 1.1 ± 0.2 and 1.7 ± 0.3 mm, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4
transport of DHA was only 2–4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did
not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and
identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase
chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, d-glucose, and 3-O-methylglucose among other hexoses while fructose and l-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition
by the exofacial inhibitor, 4,6-O-ethylidene-α-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose
uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2–8-fold over control cells. These
studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent
accumulation of AA.
Activation of the transcription factor hypoxia-inducible factor (HIF)-1 allows solid tumors to thrive under conditions of metabolic stress. Because HIF-1 is switched off by hydroxylation reactions that require ascorbate, inadequate intracellular ascorbate levels could contribute to HIF-1 overactivation. In this study, we investigated whether the ascorbate content of human endometrial tumors [known to be driven by HIF-1 and vascular endothelial growth factor (VEGF)] influenced HIF-1 activity and tumor pathology. We measured protein levels of HIF-1alpha and three downstream gene products [glucose transporter 1 (GLUT-1), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), and VEGF], as well as the ascorbate content of tumor and patient-matched normal endometrial tissue samples. HIF-1alpha and its downstream gene products were upregulated in tumor tissue, with the highest levels being present in high-grade tumors. High-grade tumors also had reduced capacity to accumulate ascorbate compared with normal tissue; however, all grades contained tumors with low ascorbate content. Tumors with the highest HIF-1alpha protein content were ascorbate deficient. Low ascorbate levels were also associated with elevated VEGF, GLUT-1, and BNIP3 protein levels and with increased tumor size, and there was a significant association between low tissue ascorbate levels and increased activation of the HIF-1 pathway (P = 0.007). In contrast, tumors with high ascorbate levels had lesser levels of HIF-1 activation. This study shows for the first time a likely in vivo relationship between ascorbate and HIF-1, with low tumor tissue ascorbate levels being associated with high HIF-1 activation and tumor growth.
Pharmacologic concentrations of ascorbate may be effective in cancer therapeutics. We hypothesized that ascorbate concentrations achievable with i.v. dosing would be cytotoxic in pancreatic cancer for which the 5-year survival is <3%.
Pancreatic cancer cell lines were treated with ascorbate (0, 5, or 10 mmol/L) for 1 hour, then viability and clonogenic survival were determined. Pancreatic tumor cells were delivered s.c. into the flank region of nude mice and allowed to grow at which time they were randomized to receive either ascorbate (4 g/kg) or osmotically equivalent saline (1 mol/L) i.p. for 2 weeks.
There was a time- and dose-dependent increase in measured H(2)O(2) production with increased concentrations of ascorbate. Ascorbate decreased viability in all pancreatic cancer cell lines but had no effect on an immortalized pancreatic ductal epithelial cell line. Ascorbate decreased clonogenic survival of the pancreatic cancer cell lines, which was reversed by treatment of cells with scavengers of H(2)O(2). Treatment with ascorbate induced a caspase-independent cell death that was associated with autophagy. In vivo, treatment with ascorbate inhibited tumor growth and prolonged survival.
These results show that pharmacologic doses of ascorbate, easily achievable in humans, may have potential for therapy in pancreatic cancer.
Vitamin C is a wide spectrum antioxidant essential for humans, which are unable to synthesize the vitamin and must obtain it from dietary sources. There are two biologically important forms of vitamin C, the reduced form, ascorbic acid, and the oxidized form, dehydroascorbic acid. Vitamin C exerts most of its biological functions intracellularly and is acquired by cells with the participation of specific membrane transporters. This is a central issue because even in those species capable of synthesizing vitamin C, synthesis is restricted to the liver (and pancreas) from which is distributed to the organism. Most cells express two different transporter systems for vitamin C; a transporter system with absolute specificity for ascorbic acid and a second system that shows absolute specificity for dehydroascorbic acid. The dehydroascorbic acid transporters are members of the GLUT family of facilitative glucose transporters, of which at least three isoforms, GLUT1, GLUT3 and GLUT4, are dehydroascorbic acid transporters. Ascorbic acid is transported by the SVCT family of sodium-coupled transporters, with two isoforms molecularly cloned, the transporters SVCT1 y SVCT2, that show different functional properties and differential cell and tissue expression. In humans, the maintenance of a low daily requirement of vitamin C is attained through an efficient system for the recycling of the vitamin involving the two families of vitamin C transporters.
Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of reactive oxygen species-generating antineoplastic drugs. The therapeutic efficacy of the widely used antineoplastic drugs doxorubicin, cisplatin, vincristine, methotrexate, and imatinib were compared in leukemia (K562) and lymphoma (RL) cell lines with and without pretreatment with dehydroascorbic acid, the commonly transported form of vitamin C. The effect of vitamin C on viability, clonogenicity, apoptosis, P-glycoprotein, reactive oxygen species (ROS), and mitochondrial membrane potential was determined. Pretreatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by trypan blue exclusion and colony formation after treatment with all antineoplastic agents tested. Vitamin C given before doxorubicin treatment led to a substantial reduction of therapeutic efficacy in mice with RL cell-derived xenogeneic tumors. Vitamin C treatment led to a dose-dependent decrease in apoptosis in cells treated with the antineoplastic agents that was not due to up-regulation of P-glycoprotein or vitamin C retention modulated by antineoplastics. Vitamin C had only modest effects on intracellular ROS and a more general cytoprotective profile than N-acetylcysteine, suggesting a mechanism of action that is not mediated by ROS. All antineoplastic agents tested caused mitochondrial membrane depolarization that was inhibited by vitamin C. These findings indicate that vitamin C given before mechanistically dissimilar antineoplastic agents antagonizes therapeutic efficacy in a model of human hematopoietic cancers by preserving mitochondrial membrane potential. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.
Ascorbic acid is an essential nutrient commonly regarded as an antioxidant. In this study, we showed that ascorbate at pharmacologic concentrations was a prooxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. To test this action in vivo, normal oral tight control was bypassed by parenteral ascorbate administration. Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.
We performed a detailed kinetic analysis of the uptake of dehydroascorbic acid by HL-60 cells under experimental conditions
that enabled the differentiation of dehydroascorbic acid transport from the intracellular reduction/accumulation of ascorbic
acid. Immunoblotting and immunolocalization experiments identified GLUT1 as the main glucose transporter expressed in the
HL-60 cells. Kinetic analysis allowed the identification of a single functional activity involved in the transport of dehydroascorbic
acid in the HL-60 cells. Transport was inhibited in a competitive manner by both 3-O-methyl-D-glucose and 2-deoxy-D-glucose. In turn, dehydroascorbic acid competitively inhibited the transport of both sugars.
A second functional component identified in experiments measuring the accumulation of ascorbic acid appears to be associated
with the intracellular reduction of dehydroascorbic acid to ascorbic acid and is not directly involved in the transport of
dehydroascorbic acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was independent of the presence of external
Na, whereas the intracellular accumulation of ascorbic acid was found to be a Na-sensitive process. Thus, the transport of dehydroascorbic acid via glucose transporters is a Na-independent process which is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic
acid and its subsequent intracellular accumulation.
Although vitamin C is critical to human physiology, it is not clear how it is taken up into cells. The kinetics of cell and tissue accumulation of ascorbic acid in vitro indicate that the process is mediated by specific transporters at the cell membrane. Some experimental observations have linked the transport of ascorbic acid with hexose transport systems in mammalian cells, although no clear information is available regarding the specific role(s) of these transporters, if any, in this process. Here we use the Xenopus laevis oocyte expression system to show that the mammalian facilitative hexose transporters are efficient transporters of the oxidized form of vitamin C (dehydroascorbic acid). Two transport pathways, one with low affinity and one with high affinity for dehydroascorbic acid, were found in oocytes expressing the mammalian transporters, and these oocytes accumulated vitamin C against a concentration gradient when supplied with dehydroascorbic acid. We obtained similar results in experiments using normal human neutrophils. These observations indicate that mammalian facilitative hexose transporters are a physiologically significant pathway for the uptake and accumulation of vitamin C by cells, and suggest a mechanism for the accumulation of ascorbic acid against a concentration gradient.
Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value.
Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.
Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+), and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.
Human tumors may contain high concentrations of ascorbic acid, but little is known about how they acquire the vitamin. Certain specialized cells can transport ascorbic acid directly through a sodium ascorbate cotransporter, but in most cells, vitamin C enters through the facilitative glucose transporters (GLUTs) in the form of dehydroascorbic acid, which is then reduced intracellularly and retained as ascorbic acid. Mice with established hematopoietic and epithelial cell xenografts were studied for the accumulation of injected ascorbic acid and dehydroascorbic acid. Most hematopoietic and epithelial tumor cell lines can only transport vitamin C in the oxidized form (dehydroascorbic acid) in vitro; however, when grown as xenografts in mice, they rapidly accumulated vitamin C after administration of radiolabeled ascorbic acid. The involvement of the GLUTs in vitamin C uptake by the xenografted tumors was demonstrated by competitive inhibition with D-glucose but not L-glucose. Because the malignant cells were not capable of directly transporting ascorbic acid, we reasoned that the ascorbic acid was oxidized to dehydroascorbic acid in the tumor microenvironment. Tumor accumulation of vitamin C in animals injected with ascorbic acid was inhibited by coadministration of superoxide dismutase, implying a role for superoxide anion in the oxidation of ascorbic acid. Whereas the epithelial cancer cell lines could not generate superoxide anion in culture, the minced xenograft tumors did. Our studies show the transport of dehydroascorbic acid by GLUTs is a means by which tumors acquire vitamin C and indicate the oxidation of ascorbic acid by superoxide anion produced by cells in the tumor stroma as a mechanism for generating the transportable form of the vitamin.
Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.
Human cells acquire vitamin C using two different transporter systems, the sodium-ascorbic acid co-transporters with specificity for ascorbic acid, and the facilitative glucose transporters with specificity for dehydroascorbic acid. There is no information on the mechanism of vitamin C transport across the intestinal barrier, a step that determines the bioavailability of vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with properties expected for GLUT2. Analysis by real time quantitative PCR revealed that the post-confluent differentiation of CaCo-2 cells was accompanied by a marked increase (4-fold) in the steady-state level of SVCT1 mRNA, without changes in SVCT2 mRNA levels. Functional studies revealed that the differentiated cells expressed only one functional ascorbic acid transporter having properties expected for SVCT1, and transported ascorbic acid with a V(max) that was increased at least 2-fold compared with pre-confluent cells. Moreover, post-confluent Caco-2 cells growing as monolayers in permeable filter inserts showed selective sorting of SVCT1 to the apical membrane compartment, without functional evidence for the expression of SVCT2. The identification of SVCT1 as the transporter that allows vectorial uptake of ascorbic acid in differentiated CaCo-2 cells has a direct impact on our understanding of the mechanism for vitamin C transport across the intestinal barrier.
Human cells transport dehydroascorbic acid through facilitative glucose transporters, in apparent contradiction with evidence indicating that vitamin C is present in human blood only as ascorbic acid. On the other hand, activated host defense cells undergoing the oxidative burst show increased vitamin C accumulation. We analyzed the role of the oxidative burst and the glucose transporters on vitamin C recycling in an in vitro system consisting of activated host-defense cells co-cultured with human cell lines and primary cells. We asked whether human cells can acquire vitamin C by a "bystander effect" by taking up dehydroascorbic acid generated from extracellular ascorbic acid by neighboring cells undergoing the oxidative burst. As activated cells, we used HL-60 neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized cell lines and primary cultures of human epithelial and endothelial cells. Activated cells produced superoxide anions that oxidized extracellular ascorbic acid to dehydroascorbic acid. At the same time, there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was inhibited by the glucose transporter inhibitor cytochalasin B. Only ascorbic acid was accumulated intracellularly by the bystander cells. Glucose partially blocked vitamin C uptake by the bystander cells, although it increased superoxide production by the activated cells. We conclude that the local production of superoxide anions by activated cells causes the oxidation of extracellular ascorbic acid to dehydroascorbic acid, which is then transported by neighboring cells through the glucose transporters and immediately reduced to ascorbic acid intracellularly. In addition to causing increased intracellular concentrations of ascorbic acid with likely associated enhanced antioxidant defense mechanisms, the bystander effect may allow the recycling of vitamin C in vivo, which may contribute to the low daily requirements of the vitamin in humans.
Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.
Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H(2)O(2), and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H(2)O(2) may be beneficial.
We characterized the human Na+-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that
it functions as a Na+-dependent transporter. The properties of SVCT2 are modulated by Ca2+/Mg2+ and a reciprocal functional interaction between Na+ and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na+ increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport Km without affecting the Vmax, thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected
in a bimodal and concentration-dependent manner the Na+ cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a
transport cycle characterized by a Na+:ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na+:ascorbic acid:Na+. However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca2+/Mg2+ for function, with both cations switching the transporter from an inactive into an active conformation by increasing the
transport Vmax without affecting the transport Km or the Na+ cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including
an inactive conformation in the absence of Ca2+/Mg2+. At least three active states can be envisioned, including a low affinity conformation at Na+ concentrations below 20 mm and two high affinity conformations at elevated Na+ concentrations whose Na+ cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca2+/Mg2+-dependent transporter.
Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.
Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin.
SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed.
This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates.
Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor, which is an effective clinical therapy for patients with wild-type KRAS. Numerous combinatorial therapies have been tested to overcome the resistance to cetuximab. However, no combinations have been found that can be used as effective therapeutic strategies. In this study, we demonstrate that L-ascorbic acid partners with cetuximab to induce killing effects, which are influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human colon cancer cells with a mutant KRAS. L-ascorbic acid treatment of human colon cancer cells that express a mutant KRAS differentially and synergistically induced cell death with cetuximab in a SVCT-2-dependent manner. The ectopic expression of SVCT-2 induced sensitivity to L-ascorbic acid treatment in human colon cancer cells that do not express SVCT-2, whereas the knockdown of endogenous SVCT-2 induced resistance to L-ascorbic acid treatment in SVCT-2-positive cells. Moreover, tumor regression via the administration of L-ascorbic acid and cetuximab in mice bearing tumor cell xenografts corresponded to SVCT-2 protein levels. Interestingly, cell death induced by the combination of L-ascorbic acid and cetuximab resulted in both apoptotic and necrotic cell death. These cell death mechanisms were related to a disruption of the ERK pathway and were represented by the impaired activation of RAFs and the activation of the ASK-1-p38 pathway. Taken together, these results suggest that resistance to cetuximab in human colon cancer patients with a mutant KRAS can be bypassed by L-ascorbic acid in an SVCT-2-dependent manner. Furthermore, SVCT-2 in mutant KRAS colon cancer may act as a potent marker for potentiating L-ascorbic acid co-treatment with cetuximab.
We recently reported that U937 cell mitochondria express a functional Na(+)-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na(+) increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na(+) concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca(++) and/or Mg(++). Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na(+) and independence for Ca(++) and Mg(++), displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.
Copyright © 2015. Published by Elsevier B.V.
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 the 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 0,6 mM. 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 the 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 different cells, tissues and species.
Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic landscape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH(-) an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H(2)O(2)). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H(2)O(2) to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer.
L-ascorbate (L-ascorbic acid, vitamin C) clearly has an inhibitory effect on cancer cells. However, the mechanism underlying differential sensitivity of cancer cells from same tissue to L-ascorbate is yet to be clarified. Here, we demonstrate that L-ascorbate has a selective killing effect, which is influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human breast cancer cells. Treatment of human breast cancer cells with L-ascorbate differentially induced cell death, dependent on the SVCT-2 protein level. Moreover, knockdown of endogenous SVCT-2 via RNA interference in breast cancer cells expressing high levels of the protein induced resistance to L-ascorbate treatment, whereas transfection with SVCT-2 expression plasmids led to enhanced L-ascorbate chemosensitivity. Surprisingly, tumor regression by L-ascorbate administration in mice bearing tumor cell xenograft also corresponded to the SVCT-2 protein level. Interestingly, SVCT-2 expression was absent or weak in normal tissues, but strongly detected in tumor samples obtained from breast cancer patients. In addition, enhanced chemosensitivity to L-ascorbate occurred as a result of caspase-independent autophagy, which was mediated by beclin-1 and LC3 II. In addition, treatment with N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger, suppressed the induction of beclin-1 and LC3 II, implying that the differential SVCT-2 protein-dependent L-ascorbate uptake was attributable to intracellular ROS induced by L-ascorbate, subsequently leading to autophagy. These results suggest that functional SVCT-2 sensitizes breast cancer cells to autophagic damage by increasing the L-ascorbate concentration and intracellular ROS production and furthermore, SVCT-2 in breast cancer may act as an indicator for commencing L-ascorbate treatment.Oncogene advance online publication, 4 June 2012; doi:10.1038/onc.2012.176.
Recently, it has been proposed that pharmacologic concentrations of ascorbate (vitamin C) can be reached by intravenous injection. Because high doses of ascorbate have been described to possess anticancer effects, the therapeutic potential of these concentrations has been studied, both in vitro and in vivo. By using 2-h exposures, a protocol that mimics a parenteral use, we observed that pharmacologic concentrations of ascorbate killed various cancer cell lines very efficiently (EC50 ranging from 3 to 7 mM). The mechanism of cytotoxicity is based on the production of extracellular hydrogen peroxide and involves intracellular transition metals. In agreement with what has been previously published, our in vivo results show that both intravenous and intraperitoneal administration of ascorbate induced pharmacologic concentrations (up to 20 mM) in blood. In contrast, the concentrations reached orally remained physiological. According to pharmacokinetic data, parenteral administration of ascorbate decreased the growth rate of a murine hepatoma, whereas oral administration of the same dosage did not. We also report that pharmacologic concentrations of ascorbate did not interfere with but rather reinforced the activity of five important chemotherapeutic drugs. Taken together, these results confirm that oral and parenteral administration of ascorbate are not comparable, the latter resulting in pharmacologic concentrations of ascorbate that exhibit interesting anticancer properties.
The ascorbate transporters SVCT1 and SVCT2 are crucial for maintaining intracellular ascorbate concentrations in most cell types. Although the two transporter isoforms are highly homologous, they have different physiologic functions. The SVCT1 is located primarily in epithelial cells and has its greatest effect in reabsorbing ascorbate in the renal tubules. The SVCT2 is located in most non‐epithelial tissues, with the highest expression in brain and neuroendocrine tissues. These transporters are hydrophobic membrane proteins that have a high affinity and are highly selective for ascorbate. Their ability to concentrate ascorbate inside cells is driven by the sodium gradient across the plasma membrane as generated by Na+/K+ ATPase. They can concentrate ascorbate 20 to 60‐fold over plasma ascorbate concentrations. Ascorbate transport on these proteins is regulated at the transcriptional, translational and post‐translational levels. Available studies show that transporter function is acutely regulated by protein kinases A and C, whereas transporter expression is increased by low intracellular ascorbate and associated oxidative stress. The knockout of the SVCT2 in mice is lethal on day 1 of life, and almost half of SVCT1 knockout mice do not survive to weaning. These findings confirm the importance both of cellular ascorbate and of the two transport proteins as key to maintaining intracellular ascorbate.
LINKED ARTICLES This article is part of a themed section on Transporters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2011.164.issue‐7
Mutations in glucose transporter 10 (GLUT10) alter angiogenesis and cause arterial tortuosity syndrome (ATS); however, the mechanisms by which these mutations cause disease remain unclear. It has been reported that in most cells, mitochondria are the major source of reactive oxygen species (ROS). Moreover, mitochondria are known to incorporate as well as recycle vitamin C, which plays a critical role in redox homeostasis, although the molecular mechanism(s) underlying mitochondrial vitamin C uptake are poorly understood. We report here that GLUT10 localizes predominantly to the mitochondria of smooth muscle cells and insulin-stimulated adipocytes, where GLUT10 is highly expressed. We further demonstrate that GLUT10 facilitates transport of l-dehydroascorbic acid (DHA), the oxidized form of vitamin C, into mitochondria, and also increases cellular uptake of DHA, which in turn protects cells against oxidative stress. This protection is compromised when GLUT10 expression in mitochondria is inhibited. In addition, we found that aortic smooth muscle cells from GLUT10-mutant mice have higher ROS levels than those from wild-type mice. Our results identify the physiological role of GLUT10 as the mitochondrial DHA transporter, and demonstrate that GLUT10 protects cells from oxidative injury. Furthermore, our findings provide a mechanism to explain the ascorbate in mitochondria and show how loss-of-function GLUT10 mutations may lead to arterial abnormalities in ATS. These results also reinforce the importance of vitamin C and ROS in degenerative diseases.
One solution found in evolution to increase the number of cellular functions, without increasing the number of genes, is distribution of single gene products to more than one cellular compartment. It is well documented that in eukaryotic cells, molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized proteins are coined in this review "echoforms" indicating repetitious forms of the same protein (echo in Greek denotes repetition) distinctly placed in the cell. This term replaces the term to "isoproteins" or "isoenzymes" which are reserved for proteins with the same activity but different amino acid sequences. Echoforms are identical or nearly identical, even though, as referred to in this review may, in some cases, surprisingly have a totally different function in the different compartments. With regard to mitochondria, our operational definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a mitochondrial membrane. In this review we ask how, when and why mitochondrial proteins are dual localized in the cell. We describe mechanisms of dual targeting of proteins between mitochondria and other compartments of the eukaryotic cell. In particular, we have paid attention to situations in which dual localization is regulated in time, location or function. In addition, we have attempted to provide a broader view concerning the phenomenon of dual localization of proteins by looking at mechanisms that are beyond our simple definition of dual targeting. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
In 2008, we celebrate the 80th anniversary of the discovery of vitamin C. Since then, we know that vitamin C possesses few pharmacological actions although it is still perceived by the public as a "miracle-pill" capable to heal a variety of illnesses. Cancer is one of the most common diseases for which a beneficial role of vitamin C has been claimed. Thus, its dietary use has been proposed in cancer prevention for several years. Apart from this nutritional aspect, an extensive and often confusing literature exists about the use of vitamin C in cancer that has considerably discredited its use. Nevertheless, recent pharmacokinetic data suggest that pharmacologic concentrations of vitamin C can be achieved by intravenous injections. Since these concentrations exhibit anticancer activities in vitro, this raises the controversial question of the re-evaluation of vitamin C in cancer treatment. Therefore, the purpose of this commentary is to make a critical review of our current knowledge of vitamin C, focusing on the rationale that could support its use in cancer therapy.
Ascorbic acid, cysteine, glutathione and uric acid were determined by reverse-phase high-pressure liquid chromatography (HPLC) in 46 breast tissue samples [neoplastic (C) and non-neoplastic (N) from the same patient]. Cholesterol, alpha-tocopherol and gamma-tocopherol were quantified in 64 similar samples by extraction into heptane followed by direct-phase HPLC. DNA was measured in all samples and the percentages of epithelium, fat and connective tissue were estimated in sections adjacent to the sample. Results confirm previous findings that ascorbic acid and glutathione, expressed as mumol/g DNA, were greatly increased in the epithelium of neoplastic tissue. Similar increases in cysteine could be accounted for by the presence of inflammatory cells. Although values of alpha- and gamma-tocopherol correlated with the percentage of fat in both types of tissue, these compounds were also present in the epithelium. Because of the varying amounts of fat in the samples, no significant difference could be found between N and C values. Cholesterol correlated with fat in N and epithelium in C. Consideration of 10 cases with equal amounts of fat in C and N tissue suggests that cholesterol is reduced in C in the epithelial cells.
The water-soluble scavengers ascorbic acid (Asc), cysteine (Cys), glutathione (GSH) and uric acid (UA) as well as DNA content were determined in 40 breast tissue samples (neoplastic and non-neoplastic tissues from 20 patients). To allow proper homogenization to take place, a fixed number of sections was cut from a tissue cylinder of known diameter. Adjacent sections were stained with hematoxylin-eosin and the fractions of epithelium, fat and connective tissue were estimated as a percentage of the section area. Protein-free extracts were injected into a reverse-phase high-pressure liquid chromatography system and scavengers quantified with 2 electrochemical detectors (gold and glassy carbon). DNA and all scavengers, except UA, were greatly increased in cancer tissues in nearly all cases. Amounts of Asc and GSH in neoplastic tissue correlated closely with DNA values and percentage of epithelium, those of Cys not so closely and those of UA not at all. We assume that Asc and GSH were located mainly in the epithelium, UA mainly in the extracellular space and Cys in both spaces. When values were expressed as mumol/g DNA, a parameter related to content per cell, values were higher in neoplastic than in non-neoplastic tissue for Asc (18/20 cases), GSH (17/20) and Cys (14/20) and lower in neoplastic tissue for UA (19/20). It is known that increased GSH protects cells against certain drugs in tissue cultures. For in vivo treatment the presence of increased Asc (and to a lesser extent Cys) in addition to GSH could be of importance.
Two sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2, were cloned from a human kidney cDNA library. hSVCT1 had a 1797 bp open reading frame encoding a 598 amino acid polypeptide. The 1953 bp open reading frame of hSVCT2 encoded a 650 amino acid polypeptide. Using a Xenopus laevis oocyte expression system, both transporters were functionally expressed. By Eadie-Hofstee transformation the apparent K(m) of hSVCT1 for ascorbate was 252.0 microM and of hSVCT2 for ascorbate was 21.3 microM. Both transporters were sodium-dependent and did not transport dehydroascorbic acid. Incubation of oocytes expressing either transporter with phorbol 12-myristate 13-acetate (PMA) inhibited ascorbate transport activity. Availability of the human transporter clones may facilitate new strategies for determining vitamin C intake.
Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.
Ascorbic acid is necessary for optimal insulin secretion from pancreatic islets. We evaluated ascorbate recycling and whether it is impaired by increased glucose metabolism in the rat beta-cell line INS-1. INS-1 cells, engineered with the potential for overexpression of glucokinase under the control of a tetracycline-inducible gene expression system, took up and reduced dehydroascorbic acid to ascorbate in a concentration-dependent manner that was optimal in the presence of physiologic D-glucose concentrations. Ascorbate uptake did not affect intracellular GSH concentrations. Whereas depletion of GSH in culture to levels about 25% of normal also did not affect the ability of the cells to reduce dehydroascorbic acid, more severe acute GSH depletion to less than 10% of normal levels did impair dehydroascorbic acid reduction. Culture of inducible cells in 11.8 mM D-glucose and doxycycline for 48 h enhanced glucokinase activity, increased glucose utilization, abolished D-glucose-dependent insulin secretion, and increased generation of reactive oxygen species. The latter may have contributed to subsequent decreases in the ability of the cells both to maintain intracellular ascorbate and to recycle it from dehydroascorbic acid. Cultured beta cells have a high capacity to recycle ascorbate, but this is sensitive to oxidant stress generated by increased glucose metabolism due to culture in high glucose concentrations and increased glucokinase expression. Impaired ascorbate recycling as a result of increased glucose metabolism may have implications for the role of ascorbate in insulin secretion in diabetes mellitus and may partially explain glucose toxicity in beta cells.
In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.
Ascorbic acid and dehydroascorbic acid (DHAA, oxidized vitamin C) are dietary sources of vitamin C in humans. Both nutrients are absorbed from the lumen of the intestine and renal tubules by, respectively, enterocytes and renal epithelial cells. Subsequently vitamin C circulates in the blood and enters all of the other cells of the body. Concerning flux across the plasma membrane, simple diffusion of ascorbic acid plays only a small or negligible role. More important are specific mechanisms of transport and metabolism that concentrate vitamin C intracellularly to enhance its function as an enzyme cofactor and antioxidant. The known transport mechanisms are facilitated diffusion of DHAA through glucose-sensitive and -insensitive transporters, facilitated diffusion of ascorbate through channels, exocytosis of ascorbate in secretory vesicles, and secondary active transport of ascorbate through the sodium-dependent vitamin C transporters SVCT1 and SVCT2 proteins that are encoded by the genes Slc23a1 and Slc23a2, respectively. Evidence is reviewed indicating that these transport pathways are regulated under physiological conditions and altered by aging and disease.
Reactive oxygen species (ROS)-induced mitochondrial abnormalities may have important consequences in the pathogenesis of degenerative diseases and cancer. Vitamin C is an important antioxidant known to quench ROS, but its mitochondrial transport and functions are poorly understood. We found that the oxidized form of vitamin C, dehydroascorbic acid (DHA), enters mitochondria via facilitative glucose transporter 1 (Glut1) and accumulates mitochondrially as ascorbic acid (mtAA). The stereo-selective mitochondrial uptake of D-glucose, with its ability to inhibit mitochondrial DHA uptake, indicated the presence of mitochondrial Glut. Computational analysis of N-termini of human Glut isoforms indicated that Glut1 had the highest probability of mitochondrial localization, which was experimentally verified via mitochondrial expression of Glut1-EGFP. In vitro mitochondrial import of Glut1, immunoblot analysis of mitochondrial proteins, and cellular immunolocalization studies indicated that Glut1 localizes to mitochondria. Loading mitochondria with AA quenched mitochondrial ROS and inhibited oxidative mitochondrial DNA damage. mtAA inhibited oxidative stress resulting from rotenone-induced disruption of the mitochondrial respiratory chain and prevented mitochondrial membrane depolarization in response to a protonophore, CCCP. Our results show that analogous to the cellular uptake, vitamin C enters mitochondria in its oxidized form via Glut1 and protects mitochondria from oxidative injury. Since mitochondria contribute significantly to intracellular ROS, protection of the mitochondrial genome and membrane may have pharmacological implications against a variety of ROS-mediated disorders.
PS-341 (bortezomib, Velcade), the first proteasome inhibitor approved by the Food and Drug Administration for the treatment of patients with relapsed multiple myeloma, induces apoptosis in human cancer cell lines. Vitamin C (ascorbic acid) is an essential water-soluble vitamin required for many normal physiologic functions and has to be obtained through diet or supplemental tablets in humans. Here we studied the potential effect of vitamin C on the anticancer activity of PS-341 in human cancer cell lines.
The effects of vitamin C on apoptosis induction by PS-341 alone and by PS-341 combined with tumor necrosis factor-related apoptosis-inducing ligand were studied. In addition, the effects of vitamin C and other antioxidants on PS-341-mediated proteasome inhibition were also examined. Finally, the direct chemical interaction between vitamin C and PS-341 was determined.
Vitamin C abrogated the ability of PS-341 to induce apoptosis in various human cancer cell lines, to induce G(2)-M arrest, and to augment apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. Moreover, vitamin C suppressed PS-341-mediated inhibition of proteasome activity. PS-341 itself did not induce generation of intracellular reactive oxygen species whereas other antioxidants failed to abrogate its biological activity. Importantly, we detected a direct chemical interaction between vitamin C and PS-341.
Vitamin C directly binds to PS-431, thus inactivating PS-341 independent of its antioxidant activity. Our findings suggest that vitamin C may have a negative effect on PS-341-mediated anticancer activity.
Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.