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Enhanced antitumor activity of vitamin C via p53 in Cancer cells
Abstract
Ascorbate is an important natural antioxidant that can selectively kill cancer cells at pharmacological concentrations. Despite its benefit, it is quite difficult to predict the antitumor effects of ascorbate, because the relative cytotoxicity of ascorbate differs between cancer cell lines. Therefore, it is essential to examine the basis for this fundamental disagreement. Because p53 is activated by DNA-damaging stress and then regulates various cellular conditions, we hypothesized that p53 can sensitize cancer cells to ascorbate. Using isogenic cancer cells, we observed that the presence of p53 can affect ascorbate cytotoxicity, and also reactivation of p53 can make cancer cells sensitive to ascorbate. p53-dependent enhancement of ascorbate cytotoxicity is caused by increased reactive oxygen species generation via a differentially regulated p53 transcriptional network. We also found that transcriptionally activated p53 was derived from MDM2 ubiquitination by ascorbate and subsequently its signaling network renders cancer cells more susceptible to oxidative stress. Similar to the p53 effect on in vitro ascorbate cytotoxicity, inhibition of tumor growth is also stronger in p53-expressing tumors than in p53-deficient ones in vivo. This is the first observation that ascorbate cytotoxicity is positively related to p53 expression, activating its transcriptional network to worsen intracellular oxidative stress and consequently enhancing its cytotoxicity. Based on our study, reactivation of p53 may help to achieve more consistent cytotoxic effects of ascorbate in cancer therapies.
... Vitamin C is an essential micronutrient and is included in the list of unorthodox therapy for cancer treatment via various metabolic pathways ( Figure 2). Vitamin C is much effective in controlling cancer despite many uncertainties [8]. Distribution of Vitamin C between intracellular and extracellular environments depends upon the absorption of Vitamin C through the transporters in membranes. ...
... Thus, Vitamin C can potentially kill cancer cells whether expressing p53 or not. Similarly, in vivo studies had also revealed preferential cytotoxicity effect of Vitamin C [8]. The ability to induce apoptosis in cancer cells is a useful strategy in cancer treatment, because apoptosis does not result in inflammation and tissue damage, as happens in necrosis [5]. ...
... From these studies, it is evident that Vitamin C regulates DNA-damaging stress in cancer cells. Cytotoxicity effect of Vitamin C is based upon generation of hydrogen peroxide (H 2 O 2 ) in affected cell which harms it anyway [8]. ...
Vitamin C is a part of cell physiology and is utilized in many metabolic reactions. Therapeutically, it was found useful in many ailments including fatal diseases such as cancer. It has ability to induce apoptotic pathway in cancer cells in order to kill them within the body at lower concentrations without affecting the non-cancerous cells. Antioxidant enzymes and reactive oxygen species are involved in the mechanism of initiation of apoptosis mediated by Vitamin C. Current review is highlighting the dose-dependent therapeutic potential of Vitamin C based upon the available literature.
... We previously showed that vitamin C stabilizes p53 by inducing MDM2 polyubiquitination/degradation. Stabilized p53 in turn aggravates intracellular oxidative stress and consequently causes cancer cells to be more sensitive to vitamin C (13). In this process, p53-dependent enhancement of vitamin C cytotoxicity is caused by increased ROS (reactive oxygen species) generation via a differentially regulated p53 transcriptional network (13). ...
... Stabilized p53 in turn aggravates intracellular oxidative stress and consequently causes cancer cells to be more sensitive to vitamin C (13). In this process, p53-dependent enhancement of vitamin C cytotoxicity is caused by increased ROS (reactive oxygen species) generation via a differentially regulated p53 transcriptional network (13). In addition, we found that the p34 SEI-1 protein level was decreased in response to vitamin C treatment. ...
... Western blotting. Western blot analysis was performed as previously described (13). The antibodies used in this study were purchased as follows: p53 (Santa Cruz Biotechnology, sc-126, USA), phospho-p53 (Ser46) (Santa Cruz Biotechnology, sc-101764), phospho-p53 (Ser15) (Cell Signaling Technology, cat. ...
Vitamin C is considered as an important anticancer therapeutic agent although this view is debatable. In this study, we introduce a physiological mechanism demonstrating how vitamin C exerts anticancer activity that induces cell cycle arrest and apoptosis. Our previous and current data reveal that p53 tumor suppressor is the prerequisite factor for stronger anticancer effects of vitamin C. In addition, vitamin C-mediated cancer cell cytotoxicity appears to be achieved at least partly through the downregulation of the p34SEI-1 oncoprotein. Our previous study showed that p34SEI-1 increases the survival of various types of cancer cells by inhibiting their apoptosis. Present data suggest that vitamin C treatment decreases the p34SEI-1 expression at the protein level and therefore alleviates its anti-apoptotic activity. Of note, SIAH1, E3 ubiquitin ligase, appears to be responsible for the p34SEI-1 polyubiquitination and its subsequent degradation, which is dependent on p53. In summary, vitamin C increases cancer cell death by inducing SIAH1-mediated polyubiquitination/degradation of the p34SEI-1 oncoprotein in a p53-dependent manner.
... It was shown later that a possible reason for the discrepancy is that intravenous Vc produces much higher plasma concentrations than those achieved by oral consumption (21,22). More recently, Chen et al. (23,24) demonstrated that megadose Vc has potent cytotoxic effects on a variety of cancer cell lines in vitro and when they were grown as xenografts, findings that have been reproduced by others (25)(26)(27). Chen et al. (23,24) also showed that megadose Vc has little or no effect on normal cells in vitro. These observations have reactivated interest in the subject and prompted further investigations, including ongoing clinical trials (28). ...
... SVCT2 is highly expressed in some breast cancers, suggesting that this type of cancer is more susceptible to megadose Vc than others. Interestingly, another group described that p53 inactivation diminishes the sensitivity of different cancer cell lines to Vc (26). The latter is important because P53 is the most frequently mutated tumor suppressor gene in human cancers (31). ...
... Of note, we could not observe any clear correlation between the levels of endog-enous HIF1␣/HIF2␣ in normoxia in these cancer cell lines and their basal or induced (upon HIF activation) susceptibility to megadose Vc (Fig. 2E). This supports the idea that, besides HIF, other mechanisms regulate the sensitivity to Vc in cancer cells, in agreement with findings by others (26,27). Therefore, the synergistic effect of HIF in promoting the toxicity of Vc is not restricted to renal cancer cell lines and happens in cancer cell lines of different origins. ...
Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of Vc's pharmacokinetics and recent reports using cancer cell lines, have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL) defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose-transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters (SVCTs). The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient, glycolytic pathway for energy production. Our experiments thus link Vc-induced toxicity and cancer metabolism, providing a new explanation for Vc's preferential effect on cancer cells.
... One study on isogenic cancer cells reported that overexpression of p53 increased ascorbate (5-10 mM) cytotoxicity dramatically, by 43%. Additionally, p53 expression elevated ascorbate-induced H2O2 generation, whereas ascorbate also increased p53 and ROS gene mRNA levels [169]. Moreover, ascorbate protected against p53 degradation via mouse double minute 2 (MDM2) ubiquitination [169]. ...
... Additionally, p53 expression elevated ascorbate-induced H2O2 generation, whereas ascorbate also increased p53 and ROS gene mRNA levels [169]. Moreover, ascorbate protected against p53 degradation via mouse double minute 2 (MDM2) ubiquitination [169]. Similar results were also found in cervical cancer and CRC cell lines, in which case combined treatments of 10-100 µM cisplatin + 100 µg Vit-C markedly raised p53 expression and strongly induced cellular ROS generation [170,171]. ...
In recent years, the idea that Vitamin C (Vit-C) could be utilized as a form of anti-cancer therapy has generated many contradictory arguments. Recent insights into the physiological characteristics of Vit-C, its pharmacokinetics, and results from preclinical reports, however, suggest that high-dose Vit-C could be effectively utilized in the management of various tumor types. Studies have shown that the pharmacological action of Vit-C can attack various processes that cancerous cells use for their growth and development. Here, we discuss the anti-cancer functions of Vit-C, but also the potential for the use of Vit-C as an epigenetic regulator and immunotherapy enhancer. We also provide a short overview of the current state of systems for scavenging reactive oxygen species (ROS), especially in the context of their influencing high-dose Vit-C toxicity for the inhibition of cancer growth. Even though the mechanisms of Vit-C action are promising, they need to be supported with robust randomized and controlled clinical trials. Moreover, upcoming studies should focus on how to define the most suitable cancer patient populations for high-dose Vit-C treatments and develop effective strategies that combine Vit-C with various concurrent cancer treatment regimens.
... High level of oxidative stress is cytotoxic to the cell and halts proliferation by inducing apoptosis or even necrosis [142]. Additionally, Increasing oxidative stress through a differently regulated p53 transcription pathway could also increase the cytotoxicity of this vitamin [128]. ...
... vitamin C can be selectively toxic in some tumor cell types, because concentration above physiological conditions (0.1 mM), between 1 mM and 10 mM, are toxic to neoplastic cells in vitro, such as for melanoma and neuroblastoma cells, where concentrations between 10 nM to 1 mM is capable of inducing apoptosis [136]. Increasing oxidative stress through a differently regulated p53 transcription pathway have also been suggested to increase cytotoxicity of this vitamin [128]. ...
Cancer development has been directly related to oxidative stress. During chemotherapy, some cancer patients use dietary antioxidants to avoid nutritional deficiencies due to cancer treatment. Among the antioxidants consumed, there are vitamins, including retinyl palmitate (PR) and ascorbic acid (AA), which have the capacity to reduce free radicals formation, protect cellular structures and maintain the cellular homeostasis. This systematic review evaluated the antioxidant and antitumor mechanisms of retinol palmitate (a derivative of vitamin A) and/or ascorbic acid (vitamin C) in cancer-related studies. Ninety-seven (97) indexed articles in the databases PubMed and Science Direct, published between 2013 and 2017, including 23 clinical studies (5 for every single compound while 13 in interaction) and 74 non-clinical studies (37 for retinol palmitate, 36 for ascorbic acid and 1 in interaction) were considered. Antioxidant and antitumor effects, with controversies over dosage and route of administration, were observed for the test compounds in their isolated form or associated in clinical studies. Prevention of cancer risks against oxidative damage was seen in lower doses of retinol palmitate and/or vitamin C. However, at high doses, they can generate reactive oxygen species, cytotoxicity and apoptosis in test systems. Non-clinical studies using cell lines have allowed understanding the mechanisms related to antioxidants and antitumor effects of the isolated compounds, however, studies on vitamin interactions, acting as antioxidants and/or antitumor are still rare and controversial. More studies, mainly related to modulation of antineoplastic drugs are needed for understanding the risks and benefits of their use during treatment in order to achieve effectiveness in cancer therapy and patient’s quality of life.
... Accumulating evidences have reported the anticancer activity of VC on tumor cells (Chen et al. 2005;Kim et al. 2012b). Hahm et al. reported anticancer effect of VC on melanoma cell proliferation. ...
... In addition, we have examined that VC cytotoxicity is partially due to the upregulation of p53 gene as reactivation of p53 in cancer cells enhances chemotherapeutic potential of VC by enhancing ROS generation via transcriptional network of p53 (Kim et al. 2012b). Our study was in line with the published evidences. ...
Purpose:
Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells.
Methods:
We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting.
Results:
Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells.
Conclusions:
These studies provide novel approaches to combat cisplatin resistance in cervical cancer.
... Recent animal studies have investigated the effect of ascorbate and tumor growth, all using different dosing regimens and tumor models (116)(117)(118)(119)(120). However, they have consistently shown an anti-tumor effect of ascorbate supplementation in mice. ...
... However, they have consistently shown an anti-tumor effect of ascorbate supplementation in mice. Mouse Frontiers in Oncology | Cancer Molecular Targets and Therapeutics studies that used pharmacological ascorbate dosing (intravenous or intraperitoneal) showed a reduction in tumor growth rate and volume (116,(119)(120)(121). Other studies have used the Gulo−/− mouse model, in which the animals cannot synthesize ascorbate, to investigate the effect of physiological ascorbate levels (117,118), and have also shown inhibition of tumor growth in orally ascorbate-supplemented mice. ...
Ascorbate is a specific co-factor for a large family of enzymes known as the Fe- and 2-oxoglutarate-dependent dioxygenases. These enzymes are found throughout biology and catalyze the addition of a hydroxyl group to various substrates. The proline hydroxylase that is involved in collagen maturation is well known, but in recent times many new enzymes and functions have been uncovered, including those involved in epigenetic control and hypoxia-inducible factor (HIF) regulation. These discoveries have provided crucial mechanistic insights into how ascorbate may affect tumor biology. In particular, there is growing evidence that HIF-1-dependent tumor progression may be inhibited by increasing tumor ascorbate levels. However, rigorous clinical intervention studies are lacking. This review will explore the physiological role of ascorbate as an enzyme co-factor and how this mechanism relates to cancer biology and treatment. The use of ascorbate in cancer should be informed by clinical studies based on such mechanistic hypotheses.
... VC functions as a cofactor for prolyl-hydroxylase, which is responsible for HIF-1α hydroxylation and degradation (Gan et al., 2019;Li et al., 2010;Nytko et al., 2011;Fischer and Miles, 2017). In addition, VC promotes cancer cell apoptosis by regulating p53 (Lee et al., 2015;Gong et al., 2016;Rubis et al., 2019;Kim et al., 2012). In the current study, we identified that Casitas B Cell lymphoma (CBL), as an E3 ligase of HIF-1α and p53, could balance the two genes under VC treatment. ...
Vitamin C (VC), in regard to its effectiveness against tumors, has had a controversial history in cancer treatment. However, the anticancer mechanisms of VC are not fully understood. Here, we reported that VC exerted an anticancer effect on cancer cell and xenograft models via inhibiting HIF-1α-dependent cell proliferation and promoting p53-dependent cell apoptosis. To be specific, VC modulated the competitive binding of HIF-1α and p53 to their common E3 ubiquitin ligase CBL, thereby inhibiting tumorigenesis. Moreover, VC treatment activated SIRT1, resulting in p53 deacetylation and CBL-p53 complex dissociation, which in turn facilitated CBL recruitment of HIF-1α for ubiquitination in a proteasome-dependent manner. Altogether, our results provided a mechanistic rationale for exploring the therapeutic use of VC in cancer therapy.
... The efficiency of numerous chemotherapeutic drugs such as cisplatin has been enhanced by the combination of vitamin C [259,260]. The p53 gene, responsible for inducing apoptosis is stabilized by vitamin C [261]. One of the possible reasons for the inbuilt resistance to chemotherapeutic drugs could be the altered p53 pathway. ...
Cervical cancer (CC) caused by human papillomavirus (HPV) is one of the largest causes of malignancies in women worldwide. Cisplatin is one of the widely used drugs for the treatment of CC is rendered ineffective owing to drug resistance. This review highlights the cause of resistance and the mechanism of cisplatin resistance cells in CC to develop therapeutic ventures and strategies that could be utilized to overcome the aforementioned issue. These strategies would include the application of nanocarries, miRNA, CRIPSR/Cas system, and chemotherapeutics in synergy with cisplatin to not only overcome the issues of drug resistance but also enhance its anti-cancer efficiency. Moreover, we have also discussed the signaling network of cisplatin resistance cells in CC that would provide insights to develop therapeutic target sites and inhibitors. Furthermore, we have discussed the role of CC metabolism on cisplatin resistance cells and the physical and biological factors affecting the tumor microenvironments.
... Another study has also reported that PD-L1 and p53 protein levels are correlated in patients with lung adenocarcinoma [107]. It has been shown that vitamin C treatment induces an up-regulation of p53 [108]. ...
Vitamin C also known as L-ascorbic acid is a nutrient naturally occurring in many fruits and vegetables and widely known for its potent antioxidant activity. Several studies have highlighted the importance of using high dose vitamin C as an adjuvant anti-cancer therapy. Interestingly, it has been shown that vitamin C is able to modulate the anti-cancer immune response and to help to overcome the resistance to immune checkpoints blockade (ICB) drugs such as cytotoxic T-lymphocyte antigen 4 (CLTA-4) and programmed cell death ligand 1 (PD-L1/PD-1) inhibitors. Indeed, it was reported that vitamin C regulates several mechanisms developed by cancer cells to escape T cells immune response and resist ICB. Understanding the role of vitamin C in the anti-tumor immune response will pave the way to the development of novel combination therapies that would enhance the response of cancer patients to ICB immunotherapy. In this review, we discuss the effect of vitamin C on the immune system and its potential role in empowering cancer immunotherapy through its pro-oxidant potential, its ability to modulate epigenetic factors and its capacity to regulate the expression of different cytokines involved in the immune response.
... The obvious cytotoxicity of vitamin C was observed in cancer cell lines due to extracellular H 2 O 2 generation via ascorbate radical as the electron donor (Chen et al. 2005). In addition, several other studies also confirmed the antitumor effect of vitamin C in subcutaneous tumor-bearing glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, and breast cancer models (Deubzer et al. 2010;Du et al. 2010;Ullah et al. 2011;Kim et al. 2012). However, the sensitivity and mechanism of vitamin C treatment in different tumors were discrepant according to previous reports (Seo et al. 2015). ...
Vitamin C has re-emerged as a promising anticancer agent. This study attempts to analyze the differential gene expression of profiles GSE11919 to look for some clues, and the most significant cell cycle pathway caused by vitamin C was identified by integrated bioinformatics analysis. Inspired by this, we investigated the effect of vitamin C treatment on gastric carcinoma cells by detection of cell cycle, apoptosis, and autophagy. Vitamin C significantly elevated the percentage of cells at G0/G1 phase, whereas the percentage of S phase cells was decreased. Meanwhile, vitamin C treatment resulted in downregulation of cell cycle-related protein Cyclin D1. We deduced that the downregulation of Cyclin D1 by vitamin C accompanied by significantly increased 5′AMP-activated protein kinase and induced autophagy in MKN45 cells. These results suggest that vitamin C has the antiproliferation effect on gastric carcinoma cells via the regulation of cell cycle and autophagy by Cyclin D1.
... In addition, vitamin C counteracts cell proliferation by stabilizing transcription factor protein 53 (P53) [25]. Kim et al. claim that the presence of p53 may represent one of the reasons for differing ascorbate cytotoxicity among cancer cell lines [26]. ...
The most frequent cancer in women is breast cancer, which is a major cause of death. Currently, there are many pharmacological therapies that have made possible the cure and resolution of this tumor. However, these therapies are accompanied by numerous collateral effects that influence the quality of life (QoL) of the patients to varying degrees. For this reason, attention is turning to the use of complementary medicine to improve QoL. In particular, there are increased trials of intravenous injection of vitamin C at high doses to enhance the antitumor activity of drugs and/or decrease their side effects. This review intends to underline the anticancer mechanisms of vitamin C that could explain its efficacy for treating breast cancer, and why the use of vitamin C at high doses could help patients with breast cancer to enhance the efficacy of pharmacological therapies and/or decrease their side effects.
... Recent studies suggested that, after intravenous administration, ascorbic acid can have cytotoxic effects to cancer cells (Ma et al. 2014;Kim et al. 2012;Vuyyuri et al. 2013;). Yun et al., (2015 reported that the oxidized form of vitamin C, dehydroascorbate (DHA) plays a major role in apoptosis of colorectal cancers C cells with KRAS or BRAF mutations, when exposed to high levels of vitamin C. ...
... Recent studies suggested that, after intravenous administration, ascorbic acid can have cytotoxic effects to cancer cells (Ma et al. 2014;Kim et al. 2012;Vuyyuri et al. 2013;). Yun et al., (2015 reported that the oxidized form of vitamin C, dehydroascorbate (DHA) plays a major role in apoptosis of colorectal cancers C cells with KRAS or BRAF mutations, when exposed to high levels of vitamin C. ...
... The addition of a pharmacological concentration of ascorbate to p53 +/+ HTC116 cells (colon cancer) caused an extension of the p53 half-life by induction of MDM2 degradation. Furthermore, the authors suggested that the tumor response to ascorbate treatment may depend on p53 expression in the cell line under study and the efficacy may be enhanced by combining vitamin C therapy with another cancer drug [52]. In view of this, it appears that the combination of VC and CDDP can be very useful in increasing therapeutic efficacy against the cervical cancer cell line and suggests the benefits of vitamin C. ...
Vitamin C has been known for decades. It is common in everyday use as an element of the diet, supplementation, and a preservative. For years, research has been conducted to precisely determine the mechanism of action of ascorbate in the cell. Available results indicate its multi-directional cellular effects. Vitamin C, which belongs to antioxidants scavenging free radicals, also has a ‘second face’—as a pro-oxidative factor. However, whether is the latter nature a defect harmful to the cell, or whether a virtue that is a source of benefit? In this review, we discuss the effects of vitamin C treatment in cancer prevention and the role of ascorbate in maintaining redox balance in the central nervous system (CNS). Finally, we discuss the effect of vitamin C supplementation on biomarkers of oxidative DNA damage and review the evidence that vitamin C has radioprotective properties.
... Additionally, p53 has been associated with ROS generation and ROS-induced oxidative stress [11,51]. Both antioxidants have been shown to selectively increase p53 gene expression in tumour cells compared to the neighbouring normal cells, since tumour cells have been known to be down-regulated and deficient from p53 [52,53]. ...
Antioxidants are known to minimize oxidative stress by interacting with free radicals produced as a result of cell aerobic reactions. Oxidative stress has long been linked to many diseases, especially tumours. Therefore, antioxidants play a crucial role in the prevention or management of free radical-related diseases. However, most of these antioxidants have anticancer effects only if taken in large doses. Others show inadequate bioavailability due to their instability in the blood or having a hydrophilic nature that limits their permeation through the cell membrane. Therefore, entrapping antioxidants in liposomes may overcome these drawbacks as liposomes have the capability to accommodate both hydrophilic and hydrophobic compounds with a considerable stability. Additionally, liposomes have the capability to accumulate at the cancer tissue passively, due to their small sizes, with enhanced drug delivery. Additionally, liposomes can be engineered with targeting moieties to increase the delivery of chemotherapeutic agents to specific tumour cells with decreased accumulation in healthy tissues. Therefore, combined use of liposomes and antioxidants, with or without chemotherapeutic agents, is an attractive strategy to combat varies tumours. This mini review focuses on the liposomal delivery of selected antioxidants, namely ascorbic acid (AA) and alpha-lipoic acid (ALA). The contribution of these nanocarriers in enhancing the antioxidant effect of AA and ALA and consequently their anticancer potentials will be demonstrated.
... In fact, numerous anticancer drugs used for CRC patients are involved in oxidative stress production [67]. One explanation for the effect of high-dose ascorbic acid on CRC cell lines is that it is dependent on the expression of p53, a tumor suppressor [68]. p53 expressing CRC cell lines were shown to be sensitive to ascorbic acid-induced ROS generation and cell death, while p53-negative cells showed less response. ...
Oxidative stress is recognized as a cancer-initiating stress response in the digestive system. It is produced through mitochondrial respiration and induces DNA damage, resulting in cancer cell transformation. However, recent findings indicate that oxidative stress is also a necessary anticancer response for destroying cancer cells. The oxidative stress response has also been reported to be an important step in increasing the anticancer response of newly developed molecular targeted agents. Oxidative stress might therefore be a cancer-initiating response that should be downregulated in the precancerous stage in patients at risk of cancer but an anticancer cell response that should not be downregulated in the postcancerous stage when cancer cells are still present. Many commercial antioxidant agents are marketed as “cancer-eliminating agents” or as products to improve one’s health, so cancer patients often take these antioxidant agents. However, care should be taken to avoid harming the anticancerous oxidative stress response. In this review, we will highlight the paradoxical effects of oxidative stress and antioxidant agents in the digestive system before and after carcinogenesis.
... However, different nutrients exhibit different effects on these biomarkers. For example, the higher availably of vitamin C increases the activity of p53 which regulate the cell cycle and increases apoptosis rate so more cancer cell start to eliminate or die (Kim et al., 2012). Selenium-containing proteins also elevate the activity of p53 by interacting with them in the cells. ...
... However, different nutrients exhibit different effects on these biomarkers. For example, the higher availably of vitamin C increases the activity of p53 which regulate the cell cycle and increases apoptosis rate so more cancer cell start to eliminate or die (Kim et al., 2012). Selenium-containing proteins also elevate the activity of p53 by interacting with them in the cells. ...
The developments in the field of nutrigenomics and metabolomics have led to the discovery of biomarkers that are normally used for the early detection of cancer and efficacy of the cancer treatment. The statistics indicate breast cancer as the second important cause of death after cardiovascular diseases. Globally over 0.55 million deaths occur due to breast cancer where as in Pakistan one out of nine women encounter this menace at certain phase during the lifespan. The well-known risk factors for breast cancer are age, gender, family history, early menarche, overweight, smoking or exposure to smoke, night shift work, alcohol and abusive use of medicines and radiation therapy. Among these various factors diet, obesity, physical inactivity and tobacco use are modifiable. The most important one is early screening and diagnosis of cancer through biomarkers. Now-a-days the screening of the DNA and serum plasma is helpful to track cancer growth, development and reaction to therapy. Some of the most studied biomarkers and their potential to develop malignant in women are p53, HER2, BRCA1 & BRCA2, Ki-67 and Cyclin D-1. These are present on different chromosomes and alteration in them results in cancer. Influence of diet on these biomarkers also very important. The dietary interventions commonly used for management of cancer include plant-based proteins with every meal, low/moderate fat intake, minimum intake of processed and refined grain products, vitamin-mineral supplements and higher intake of fluids. In nutshell, balanced nourishment may lessen the occurrence and severity of breast cancer. In this review article, current scenario of bear cancer, risk factors, pathogenesis, diagnosis, current & emerging biomarker and influence of diet on these biomarkers has been briefly reviewed.
... In addition to the effects mentioned above, ascorbic acid has the potential to protect against cancers. High concentration of ascorbic acid induces cytotoxicity against cancer cells in vitro ( Vuyyuri et al., 2013;Tian et al., 2014) and delays tumor growth in xenograft models ( Kim et al., 2012;). The animal model study further demonstrated that feeding Apc/Kras G12D mutant mice high-dose ascorbic acid may impair tumor growth ( Yun et al., 2015). ...
Aging is the progressive loss of organ and tissue function over time. Growing older is positively linked to cognitive and biological degeneration such as physical frailty, psychological impairment, and cognitive decline. Oxidative stress is considered as an imbalance between pro- and antioxidant species, which results in molecular and
cellular damage. Oxidative stress plays a crucial role in the development of age-related diseases. Emerging research evidence has suggested that antioxidant can control the autoxidation by interrupting the propagation of free radicals or by inhibiting the formation of free radicals and subsequently reduce oxidative stress, improve immune function, and increase healthy longevity. Indeed, oxidation damage is highly dependent on the inherited or acquired defects in enzymes involved in the redox-mediated signaling pathways. Therefore, the role of molecules with antioxidant activity that promote healthy aging and counteract oxidative stress is worth to discuss further. Of particular interest in this article, we highlighted the molecular mechanisms of antioxidants involved in the prevention of age-related diseases. Taken together, a better understanding of the role of antioxidants involved in redox modulation of inflammation would provide a useful approach for potential interventions, and subsequently promoting healthy longevity.
... The discovery of ascorbic acid as APE of DEFB1 could explain better why this vitamin protects against cancer due to DEFB1 upregulation (Cruz Diaz, et al., 2015), because DEFB1 has been considered as tumour suppressor gene (Sun, et al., 2006). Known antitumour effects of vitamin C include increased ROS generation via transcriptionally activated p53 and downregulation of HIF1A transcription (Kawada, et al., 2013;Kim, et al., 2012). ...
Human β-defensin 1 (hBD-1) is a multifaceted antimicrobial peptide being a tumour suppressor and, depending on call of duty, capable of inducing self-nets and neutrophil extracellular traps (NETs) to trap and/or kill bacteria, participates in inflammatory responses in chronic diseases including hBD-3 upregulation and also capable of up/downregulation in the presence of certain species of Lactobacillus sp. thus regulating host microbiota. Alleles, genotypes and/or altered gene expression of its coding gene, DEFB1, have been associated with several human diseases/conditions ranging from metabolic/chronic (e.g. cancer), infectious (e.g. tuberculosis, HIV/AIDS), inflammatory (gastrointestinal diseases), male infertility and more recently, neurologic (e.g. depression and Alzheimer) and autoimmune diseases (e.g. vitiligo and systemic lupus erythematosus). The present update focuses on novel DEFB1/hBD-1 properties and biomarker features, its biological function and the pharmaceutical potential uses of antimicrobial peptide elicitors (APEs) or the engineered peptide in the treatment of hBD-1-related human diseases.
... Various gene mutations can influence the effectiveness of AA treatment in specific cancers. It has been shown that tumor suppressor 101F6 or p53 can sensitize cancer cells to AA-induced cell death [36,37]. Activation of the hypoxia-inducible factor (HIF) pathway has also been found to promote AA-induced toxicity in renal cancer cells [38]. ...
Cholangiocarcinoma (CC) is a devastating malignancy with late diagnosis and poor response to conventional chemotherapy. Recent studies have revealed anti-cancer effect of vitamin C (L-ascorbic acid, ascorbate) in several types of cancer. However, the effect of L-ascorbic acid (AA) in CC remains elusive. Herein, we demonstrated that AA induced cytotoxicity in CC cells by generating intracellular reactive oxygen species (ROS), and subsequently DNA damage, ATP depletion, mTOR pathway inhibition. Moreover, AA worked synergistically with chemotherapeutic agent cisplatin to impair CC cells growth both in vitro and in vivo. Intriguingly, sodium-dependent vitamin C transporter 2 (SVCT-2) expression was inversely correlated with IC50 values of AA. Knockdown of SVCT-2 dramatically alleviated DNA damage, ATP depletion, and inhibition of mTOR pathway induced by AA. Furthermore, SVCT-2 knockdown endowed CC cells with the resistance to AA treatment. Finally, the inhibitory effects of AA were further confirmed in patient-derived CC xenograft models. Thus, our results unravel therapeutic potential of AA alone or in combination with cisplatin for CC. SVCT2 expression level may serve as a positive outcome predictor for AA treatment in CC.
... Ascorbate cytotoxicity seems to be more effective in cells with higher levels of p53, and because the natural body cells have lower levels of p53 than tumor cells, ascorbate does not damage these cells as much. 23 The CTX/ Water treated group of the present study showed a significant decrease in epithelial thickness, a significant increase in damage scores, and a significant decrease in PCNA immune expression. Zhao et al. (2009) 24 found the same changes in the tongue of mice, but following radiation. ...
Background and objective: Oral mucositis is currently considered to be the most severe complication of anticancer therapy such as cyclophosphamide (CTX). Ascorbic acid is a well-known antioxidant, which can protect the body from damage caused by free radicals that can be generated during normal metabolism as well as through exposure to toxins and carcinogens. This study aimed to evaluate the effectiveness of ascorbic acid as a treatment for CTX induced oral mucositis. Methods: Forty Wister-albino rats, age about 6-8 weeks and weighing 150-200 g were used. The rats were randomly divided by simple random allocation into two groups (20 animals each). The control group was intraperitoneally injected with physiological saline and the animals were grouped randomly into two groups: Saline/Water treated group which were daily received intraperitoneal injection of distilled water, while the Saline/Ascorbic acid treated group received a daily intraperitoneal injection of ascorbic acid (12mg/kg/day). For the induction of mucositis, a single dose (300 mg/kg) of CTX was administered intraperitoneally to each animal in the study group, and the animals were grouped randomly into two groups: CTX /Water treated group which was daily received intraperitoneal injection of distilled water, while the CTX /Ascorbic acid treated group were daily received intraperitoneal injection of 12 mg/kg /day of ascorbic acid. The animals were sacrificed at day four and eight (five animals each) and the tongue was dissected from the jaw for histological and immunohistochemical analysis. Results: Ascorbic acid decreased the severity of the induced CTX oral mucositis by a significant increase in epithelial thickness, significant decrease in damage score, and significant increase in PCNA immune expression at day four and eight respectively (P <0.05). Conclusion: CTX chemotherapy has a deleterious effect on the oral mucosa leading to marked morphometric and microscopic changes. Ascorbic acid can protect the oral mucosa from CTX-induced cytotoxicity, and attenuate or decrease the associated injury.
... The tumor suppressor p53 may also play a role for this activity. P53-positive cell lines were more sensitive to both AA and H 2 O 2 treatment than p53-deficient ones (Kim et al., 2012). Thus, having two compounds from one plant with diverse mechanisms of action, we were interested to evaluate, whether their combination would reveal synergistic, antagonistic, or additive interactions. ...
Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa. This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53+/+ and HCT116p53−/− colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription (TFAM, TCERG1, RGS13, C11orf31), apoptosis-regulation (CRADD, CDK7, CDK19, CD81, TOM1) signal transduction (NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27) DNA repair (TOPBP1, RPA2), mRNA metabolism (RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2), and transporter genes (ABCA1) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA.
... With this work we concluded that AA mechanisms of action depend on the cell line under study. Meyong-Sok Lee and collaborators hypothesized that an increment of oxidative stress via a differentially regulated P53 transcriptional network could enhance AA cytotoxicity (Kim et al., 2012). In our study, C2BBe1 cells (P53-null) proved that AA can induce cell death by a P53independent pathway. ...
Colorectal cancer is a major health problem worldwide with urgent need for new and effective anti-cancer approaches that allow treating, increasing survival and improving life quality of patients. At pharmacological concentrations, ascorbic acid (AA) exerts a selective cytotoxic effect, whose mechanism of cytotoxicity remains unsolved. It has been suggested that it depends on the production of extracellular hydrogen peroxide, using ascorbate radical as an intermediate. The aim of this study was to evaluate the effects induced by AA in three colon cancer cell lines, as well as, possible cell death mechanisms involved.
... Recent studies have shown that pharmacological concentrations of vitamin C selectively kill tumor cells by induction of cytotoxicity, but not normal cells, which suggests its useful and beneficial use in the treatment of cancer [22,[28][29][30]. ...
In recent years the use of natural dietary antioxidants to minimize the cytotoxicity and the damage induced in normal tissues by antitumor agents is gaining consideration. In literature, it is reported that vitamin C exhibits some degree of antineoplastic activity whereas Mitoxantrone (MTZ) is a synthetic anti-cancer drug with significant clinical effectiveness in the treatment of human malignancies but with severe side effects. Therefore, we have investigated the effect of vitamin C alone or combined with MTZ on MDA-MB231 and MCF7 human breast cancer cell lines to analyze their dose-effect on the tumor cellular growth, cellular death, cell cycle and cell signaling. Our results have evidenced that there is a dose-dependence on the inhibition of the breast carcinoma cell lines, MCF7 and MDA-MB231, treated with vitamin C and MTZ. Moreover, their combination induces: i) a cytotoxic effect by apoptotic death, ii) a mild G2/M elongation and iii) H2AX and mild PI3K activation. Hence, the formulation of vitamin C with MTZ induces a higher cytotoxicity level on tumor cells compared to a disjointed treatment. We have also found that the vitamin C enhances the MTZ effect allowing the utilization of lower chemotherapic concentrations in comparison to the single treatments.
... We therefore propose that the indirect antimicrobial effect of ascorbic acid in NHK could be at least partially dependent on DEFB1 induction which challenges the traditional view regarding AA only as being considered a player in the later activation of the adaptive immunity [20]. This also could explain why AA acts as an antitumor agent [21][22][23], since DEFB1 is considered a tumor suppressor gene [24,25]. ...
Hosts’ innate defense systems are upregulated by antimicrobial peptide elicitors (APEs). Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA), and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulation of human β-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes (NHK). The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C) and hyperthermia (39°C) are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections.
TP53 is one of the tumor suppressor genes found to be highly correlated with human tumor development, this gene can sense stress or damage to cells and prevent cell division or trigger cell death, thereby preventing the proliferation of damaged cells. The P53 protein encoded by TP53 has an anti-tumor effect and is known as the “guardian of the genome”. The mutation of TP53 gene eliminates a key cell safety mechanism, making it the trigger of cancer. In this paper, we first described the relationship between TP53 gene and tumors, and discussed the application of TP53 gene in tumor prediction and treatment, then we developed a TP53 genetic testing service to evaluate the cancer suppression ability of tumors, so that to help individuals to establish a scientific and reasonable lifestyle in a timely manner, and better grasp the initiative of health. Further, we describe the interaction between TP53 gene and nutrition and its impact on the occurrence and development of cancer. Finally, based on the analysis of the genetic testing results and food frequency questionnaires, we developed a personalized nutrition service to reduce the risk of developing the diseases with high genetic risk score.
Significance:
Vitamin C (ascorbate), in regard to its effectiveness against malignancies, has had a controversial history in cancer treatment. It has been shown that in vitro and in vivo anticancer efficacy of ascorbate relies on its pro-oxidant effect mainly from an increased generation of reactive oxygen species (ROS). A growing understanding of its anticancer activities and pharmacokinetic properties has prompted scientists to reevaluate the significance of ascorbate in cancer treatment. Recent Advances: A recent resurge in ascorbate research emerged after discovering that, at high doses, ascorbate preferentially kills K-ras- and BRAF-mutant cancer cells. In addition, some of the main hallmarks of cancer cells, such as redox homeostasis and oxygen-sensing regulation (through inhibition of HIF-1α activity), are affected by vitamin C.
Critical issues:
Currently, there is no clear consensus from literature in regards to the beneficial effects of antioxidants. Results from both human and animal studies provide no clear evidence about the benefit of antioxidant treatment in preventing or suppressing cancer development. Since pro-oxidants may affect both normal and tumor cells, the extremely low toxicity of ascorbate represents a main advantage. This guarantees the safe inclusion of ascorbate in clinical protocols to treat cancer patients.
Future directions:
Current research could focus on elucidating the wide array of reactions between ascorbate and reactive species, namely ROS, reactive nitrogen species (RNS) as well as reactive sulfide species (RSS), and their intracellular molecular targets. Unraveling these mechanisms could allow researchers to assess what could be the optimal combination of ascorbate with standard treatments.
The idea to use megadoses of ascorbate (vitamin C) for cancer treatment has recently been revived. Despite clear efficacy in animal experimentation, our understanding of the cellular and molecular mechanisms of this treatment is still limited and suggests a combined oxidative and metabolic mechanism behind the selective cytotoxicity of ascorbate towards cancerous cells. To gain more insight into the cellular effects of high doses of ascorbate, we performed a detailed analysis of metabolic changes and cell survival of both luminal and basal-like breast cancer cells treated with ascorbate and revealed a distinctive metabolic shift virtually reversing the Warburg effect and triggering a severe disruption of redox homeostasis. High doses of ascorbate were cytotoxic against MCF7 and MDA-MB231 cells representing luminal and basal-like breast cancer phenotypes. Cell death was dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD⁺/NADH, associated with a multifaceted metabolic rewiring. This included a sharp disruption of glycolysis at the triose phosphate level, a rapid drop in ATP levels, and redirection of metabolites toward lipid droplet accumulation and increased metabolites and enzymatic activity in the pentose phosphate pathway (PPP). High doses of ascorbate also inhibited the TCA cycle and increased oxygen consumption. Together the severe disruptions of the intracellular metabolic homeostasis on multiple levels “redox crisis and energetic catastrophe” consequently trigger a rapid irreversible cell death.
Much attention has been put on antioxidants as potential preventive and therapeutic agents against cancer. Vitamin C, an important antioxidant with anti-inflammatory and immune system enhancement features, could provide protection against cancer. However, experimental and epidemiologic evidence on vitamin C and cancer risk are still indefinite. Substantial literature reports that cancer patients experience vitamin C deficiency associated with decreased oral intake, infection, inflammation, disease processes, and treatments such as radiation, chemotherapy, and surgery. Studies demonstrate associations between IVC and inflammation biomarkers and propose some amelioration in symptoms, with a possible advantage in quality of life (QoL) when intravenous vitamin C (IVC) alone or in combination with oral vitamin C is administered in oncologic care. While, the anticancer impact of high doses of IVC remains debatable in spite of growing evidence that high dose vitamin C shows anti-tumorigenic activity by elevating the amount of reactive oxygen species (ROS) in cancer cells without meaningful toxicities. Hence, there is an urgent requirement for rigorous and well-controlled assessments of IVC as an adjuvant therapy for cancer before clear conclusions can be drawn. Thus, more clinical trials are required to determine the additive impact of high dose vitamin C in cancer patients.
Two series of 6-(1,2,3-triazolyl)-2,3-dibenzyl-l-ascorbic acid derivatives with the hydroxyethylene (8a-8u) and ethylidene linkers (10c-10p) were synthesized and evaluated for their antiproliferative activity against seven malignant tumor cell lines and antiviral activity against a broad range of viruses. Conformationally unrestricted spacer between the lactone and 1,2,3-triazole units in 8a-8u series had a profound effect on antitumor activity. Besides, the introduction of a long side chain at C-4 of 1,2,3-triazole that led to the synthesis of decyl-substituted 2,3-dibenzyl-l-ascorbic acid 8m accounted for a selective and potent antiproliferative activity on breast cancer MCF-7 cells cells in the nM range. Further analysis showed that compound 8m strongly enhanced expression of hypoxia inducible transcription factor 1 α (HIF-1α) and to some extent decreased expression of nitric oxide synthase 2 (NOS2) suggesting its role in regulating HIF-1α signalling pathway. The p-methoxyphenyl-substituted derivative 10g displayed specific anti-cytomegalovirus (CMV) potential, whereas aliphatic-substituted derivatives 8l and 8m had the most potent, yet relatively non-specific, anti-varicella-zoster (VZV) activity.
Pancreatic cancer is among the most lethal cancers with poorly tolerated treatments. There is increasing interest in using high-dose intravenous ascorbate (IVC) in treating this disease partially because of its low toxicity. IVC bypasses bioavailability barriers of oral ingestion, provides pharmacological concentrations in tissues, and exhibits selective cytotoxic effects in cancer cells through peroxide formation. Here, we further revealed its anti-pancreatic cancer mechanisms and conducted a phase I/IIa study to investigate pharmacokinetic interaction between IVC and gemcitabine. Pharmacological ascorbate induced cell death in pancreatic cancer cells with diverse mutational backgrounds. Pharmacological ascorbate depleted cellular NAD+ preferentially in cancer cells versus normal cells, leading to depletion of ATP and robustly increased α-tubulin acetylation in cancer cells. While ATP depletion led to cell death, over-acetylated tubulin led to inhibition of motility and mitosis. Collagen was increased, and cancer cell epithelial-mesenchymal transition (EMT) was inhibited, accompanied with inhibition in metastasis. IVC was safe in patients and showed the possibility to prolong patient survival. There was no interference to gemcitabine pharmacokinetics by IVC administration. Taken together, these data revealed a multi-targeting mechanism of pharmacological ascorbate’s anti-cancer action, with minimal toxicity, and provided guidance to design larger definitive trials testing efficacy of IVC in treating advanced pancreatic cancer.
Radiotherapy is a comprehensive method, in which the main factor that determines success in clinical radiotherapy is radiation dose. It is necessary to make the most of mathematical theories to investigate the complicated mechanisms how P53-dependent regulation pathways govern cell survival and apoptosis at molecular level. In this work, we develop an integrated method for modeling Tumor Radiotherapy System (TRS) based on Kinetic Theory of Active Particle (KTAP) and Gene-Environment Network (GEN), and then explore the dynamics of integrated TRS through correlated subsystems, and inner mechanism of cell fate decision under radiotherapy. The inner mechanisms of radiotherapy are investigated at both molecular and systematic levels, including the stochastic kinetics of DNA damage generation and repair, switch-like Ataxia Telangiectasia Mutated (ATM) activation, oscillation of P53-Mouse Mouble Minute 2 homolog (MDM2) negative feedback loop, and outcome of tumor cell degradation and genome stability under radiotherapy.
More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the
oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative
stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/KrasG12D mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.
Chlorambucil is an anticancer drug with alkylating and immunosuppressive properties. The present findings show that the combination treatment of tumor-bearing mice with ascorbic acid plus chlorambucil has much better therapeutic efficacy against murine ascites Daltons' lymphoma in comparison to chlorambucil alone and this may involve a decrease in GSH in Dalton's lymphoma tumor cells. Chlorambucil-induced chromosomal aberrations, sperm abnormalities and histopathological changes in liver, kidney and testes were decreased after combination treatment. The analysis of renal function test (RFT) and liver function test (LFT) also suggest an improvement in these parameters after combination treatment. The increased GSH level, changes in GSHrelated enzymes in the tissues i.e. kidney, liver and testes after combination treatment may be involved in its protective ability against chlorambucil-induced toxicity in the host. The present studies demonstrate a protective role of ascorbic acid against chlorambucilinduced mutagenicity and tissue toxicity in the hosts.
Intravenous administration of high-dose vitamin C has recently attracted attention as a cancer therapy. High-dose vitamin C induces pro-oxidant effects and selectively kills cancer cells. However, the anticancer mechanisms of vitamin C are not fully understood. Here, we analyzed metabolic changes induced by vitamin C in MCF7 human breast adenocarcinoma and HT29 human colon cancer cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The metabolomic profiles of both cell lines were dramatically altered after exposure to cytotoxic concentrations of vitamin C. Levels of upstream metabolites in the glycolysis pathway and tricarboxylic acid (TCA) cycle were increased in both cell lines following treatment with vitamin C, while adenosine triphosphate (ATP) levels and adenylate energy charges were decreased concentration-dependently. Treatment with N-acetyl cysteine (NAC) and reduced glutathione (GSH) significantly inhibited vitamin C-induced cytotoxicity in MCF7 cells. NAC also suppressed vitamin C-dependent metabolic changes, and NAD treatment prevented vitamin C-induced cell death. Collectively, our data suggests that vitamin C inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.
We would like to express our appreciation to all the authors for their informative contributions and the reviewers for their support and constructive critiques in making this special issue possible.
Treatment with high-dose intravenous (IV) ascorbic acid (AA) is used in complementary and alternative medicine for various conditions including cancer. Cytotoxicity to cancer cell lines has been observed with millimolar concentrations of AA. Little is known about the pharmacokinetics of high dose IV AA. The purpose of the present study was to assess the basic kinetic variables in human beings over a relevant AA dosing interval for proper design of future clinical trials. Ten patients with metastatic prostate cancer were treated for four weeks with fixed AA doses of 5, 30 and 60 g. AA was measured consecutively in plasma and indicated first-order elimination kinetics throughout the dosing range with supra-physiological concentrations. The target dose of 60g AA IV produced a peak plasma AA concentration of 20.3 mM. Elimination half-life was 1.87 hr (mean, SD ± 0.40), volume of distribution 0.19 L/kg (SD ±0.05) and clearance rate 6.02 L/hr (100mL/min). No differences in pharmacokinetic parameters were observed between weeks/doses. A relatively fast first-order elimination with half-life of about 2 hr makes it impossible to maintain AA concentrations in the potential cytotoxic range after infusion stop in prostate cancer patients with normal kidney function. We propose a regimen with a bolus loading followed by a maintenance infusion based on the calculated clearance.This article is protected by copyright. All rights reserved.
Background:
Intravenous vitamin C (IVC) is a contentious adjunctive cancer therapy, widely used in naturopathic and integrative oncology settings. We conducted a systematic review of human interventional and observational studies assessing IVC for use in cancer patients.
Methods:
We searched MEDLINE, EMBASE, The Cochrane Library, CINAHL, and AMED from inception to April 2013 for human studies examining the safety, effectiveness, or pharmacokinetics of IVC use in cancer patients.
Results:
Of 897 records, a total of 39 reports of 37 studies were included: 2 randomized controlled trials (RCTs), 15 uncontrolled trials, 6 observational studies, and 14 case reports. IVC dosing ranged from 1 g to more than 200 g ascorbic acid per infusion, typically administered 2 to 3 times weekly. IVC does not appear to increase toxicity or interfere with antitumor effects of gemcitabine/erlotinib therapy or paclitaxel and carboplatin. Based on 1 RCT and data from uncontrolled human trials, IVC may improve time to relapse and possibly enhance reductions in tumor mass and improve survival in combination with chemotherapy. IVC may improve quality of life, physical function, and toxicities associated with chemotherapy, including fatigue, nausea, insomnia, constipation, and depression. Case reports document several instances of tumor regression and long-term disease-free survival associated with use of IVC.
Conclusion:
There is limited high-quality clinical evidence on the safety and effectiveness of IVC. The existing evidence is preliminary and cannot be considered conclusive but is suggestive of a good safety profile and potentially important antitumor activity; however, more rigorous evidence is needed to conclusively demonstrate these effects. IVC may improve the quality of life and symptom severity of patients with cancer, and several cases of cancer remission have been reported. Well-designed, controlled studies of IVC therapy are needed.
Significance:
Ewan Cameron reported that ascorbate, given orally and intravenously at doses of up to 10 g/day, was effective in the treatment of cancer. Double-blind placebo-controlled clinical trials showed no survival advantage when the same doses of ascorbate were given orally, leading the medical and scientific communities to dismiss the use of ascorbate as a potential cancer treatment. However, the route of administration results in major differences in ascorbate bioavailability. Tissue and plasma concentrations are tightly controlled in response to oral administration, but this can be bypassed by intravenous administration. These data provide a plausible scientific rationale for the absence of a response to orally administered ascorbate in the Mayo clinic trials and indicate the need to reassess ascorbate as a cancer therapeutic.
Recent advances:
High dose ascorbate is selectively cytotoxic to cancer cell lines through the generation of extracellular hydrogen peroxide (H2O2). Murine xenograft models confirm a growth inhibitory effect of pharmacological concentrations. The safety of intravenous ascorbate has been verified in encouraging pilot clinical studies.
Critical issues:
Neither the selective toxicity of pharmacologic ascorbate against cancer cells nor the mechanism of H2O2-mediated cytotoxicity is fully understood. Despite promising preclinical data, the question of clinical efficacy remains.
Future directions:
A full delineation of mechanism is of interest because it may indicate susceptible cancer types. Effects of pharmacologic ascorbate used in combination with standard treatments need to be defined. Most importantly, the clinical efficacy of ascorbate needs to be reassessed using proper dosing, route of administration, and controls.
Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provokes an oxidative stress that preferentially kills cancer cells and spares healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.
The side effects of cancer therapy on normal tissues limit the success of therapy. Generation of reactive oxygen species (ROS) has been implicated for numerous chemotherapeutic agents including doxorubicin (DOX), a potent cancer chemotherapeutic drug. The production of ROS by DOX has been linked to DNA damage, nuclear translocation of p53, and mitochondrial injury; however, the causal relationship and molecular mechanisms underlying these events are unknown. The present study used wild-type (WT) and p53 homozygous knock-out (p53(-/-)) mice to investigate the role of p53 in the crosstalk between mitochondria and nucleus. Injecting mice with DOX (20 mg/kg) causes oxidative stress in cardiac tissue as demonstrated by immunogold analysis of the levels of 4-hydroxy-2'-nonenal (4HNE)-adducted protein, a lipid peroxidation product bound to proteins. 4HNE levels increased in both nuclei and mitochondria of WT DOX-treated mice but only in nuclei of DOX-treated p53((-/-)) mice, implicating a critical role for p53 in causing DOX-induced oxidative stress in mitochondria. The stress-activated protein c-Jun amino-terminal kinase (JNKs) was activated in response to increased 4HNE in WT mice but not p53((-/-)) mice receiving DOX treatment, as determined by co-immunoprecipitation of HNE and pJNK. The activation of JNK in DOX treated WT mice was accompanied by Bcl-2 dissociation from Beclin in mitochondria and induction of type II cell death (autophagic cell death), as evidenced by an increase in LC3-I/LC-3-II ratio and γ-H2AX, a biomarker for DNA damage. The absence of p53 significantly reduces mitochondrial injury, assessed by quantitative morphology, and decline in cardiac function, assessed by left ventricular ejection fraction and fraction shortening. These results demonstrate that p53 plays a critical role in DOX-induced cardiac toxicity, in part, by the induction of oxidative stress mediated retrograde signaling.
Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.
The tumor suppressor p53 is a multifunctional, highly regulated, and promoter-specific transcriptional factor that is uniquely sensitive to DNA damage and cellular stress signaling. The mechanisms by which p53 directs a damaged cell down either a cell growth arrest or an apoptotic pathway remain poorly understood. Evidence suggests that the in vivo functions of p53 seem to balance the cell-fate choice with the type and severity of damage that occurs. The concept of antirepression, or inhibition of factors that normally keep p53 at bay, may help explain the physiological mechanisms for p53 activation. These factors also provide novel chemotherapeutic targets for the reactivation of p53 in tumors harboring a wild-type copy of the gene.
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.
Tumor suppressor p53 is reported to be an attractive immunotherapy target because it is mutated in approximately half of human cancers, resulting in inactivation and often an accumulation of the protein in the tumor cells. Only low amounts of protein are detectable in normal tissues. The differential display of antigen in normal versus tumor tissues has been reported to create an opportunity to target p53 by immunotherapy. We sought to determine the relationship between p53 expression and its recognition by cognate T cells in human tumors including common epithelial malignancies. Inasmuch as nonsense or missense p53 mutations may disrupt processing and presentation, we studied tumors with either identified wild-type or mutated p53, based on our gene-sequencing studies or published data. T cells transduced with a high-affinity, p53(264-272)-reactive T cell receptor (TCR) derived from HLA-A2.1 transgenic mice recognized a wide panel of human tumor lines. There was no significant correlation between p53 expression in tumors and recognition by the anti-p53 TCR-transduced T cells. This conclusion was based on the study of 48 cell lines and is in contrast to several prior studies that used only a limited number of selected cell lines. A panel of normal cells was evaluated for recognition, and some of these populations were capable of stimulating anti-p53 T cells, albeit at low levels. These studies raise doubts concerning the suitability of targeting p53 in the immunotherapy of cancer patients.
The MDM2 gene is amplified and/or overexpressed in about 10% of glioblastomas and constitutes one of a number of ways the p53 pathway is disrupted in these tumours. MDM2 encodes a nuclear phosphoprotein that regulates several cell proteins by binding and/or ubiquitinating them, with p53 being a well-established partner. MDM2 has two promoters, P1 and P2 that give rise to transcripts with distinct 5' untranslated regions. Transcription from P2 is believed to be controlled by p53 and a single-nucleotide polymorphism (SNP309, T>G) in P2 is reported to be associated with increased risk for, and early development of, malignancies. The use of P1 and P2 has not been investigated in gliomas. We used RT-PCR to study P1- and P2-MDM2 transcript expression in astrocytic tumours, xenografts and cell lines with known MDM2, TP53 and p14(ARF) gene status. Both promoters were used in all genetic backgrounds including the use of the P2 promoter in TP53 null cells, indicating a p53-independent induction of transcription. Transcripts from the P1 promoter formed a greater proportion of the total MDM2 transcripts in tumours with MDM2 amplification, despite these tumours having two wild-type TP53 alleles. Examination of SNP309 in glioblastoma patients showed a borderline association with survival but no apparent correlation with age at diagnosis nor with TP53 and p14(ARF) status of their tumours. Our findings also indicate that elevated MDM2 mRNA levels in tumours with MDM2 amplification are preferentially driven by the P1 promoter and that the P2 promoter is not only regulated by p53 but also by other transcription factor(s).
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.
The inactivation of the p53 gene in a large proportion of human cancers has inspired an intense search for the encoded protein's physiological and biological properties. Expression of p53 induces either a stable growth arrest or programmed cell death (apoptosis). In human colorectal cancers, the growth arrest is dependent on the transcriptional induction of the protein p21WAF1/CIP1 , but the mechanisms underlying the development of p53-dependent apoptosis are largely unknown. As the most well documented biochemical property of p53 is its ability to activate transcription of genes, we examined in detail the transcripts induced by p53 expression before the onset of apoptosis. Of 7,202 transcripts identified, only 14 (0.19%) were found to be markedly increased in p53-expressing cells compared with control cells. Strikingly, many of these genes were predicted to encode proteins that could generate or respond to oxidative stress, including one that is implicated in apoptosis in plant meristems. These observations stimulated additional biochemical and pharmacological experiments suggesting that p53 results in apoptosis through a three-step process: (1) the transcriptional induction of redox-related genes; (2) the formation of reactive oxygen species; and (3) the oxidative degradation of mitochondrial components, culminating in cell death.
The p53 tumor suppressor protein is a short-lived protein, which is stabilized in response to cellular stress. The ubiquitination and degradation of p53 are largely controlled by Mdm2, an oncogenic E3 ligase. Stress signals lead to p53 stabilization either by induction of covalent modifications in Mdm2 and p53, or through altered protein-protein interactions. Mdm2 also harbors a post-ubiquitination function, probably enabling efficient targeting of ubiquitinated p53 to the proteasome. p53 ubiquitination is associated with its export from the nucleus into the cytoplasm. However, the exact site of degradation of p53 is presently under debate. p53 may be targeted by other E3 ligases besides Mdm2, as well as by non-proteasomal mechanisms. Despite extensive information about p53 degradation, many important aspects remain unresolved.
The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype
in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with
the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and
cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each
case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting
mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous
ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role
of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate
with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely
to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that
do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.
Vitamin C at high concentrations is toxic to cancer cells in vitro. Early clinical studies of vitamin C in patients with terminal cancer suggested clinical benefit, but 2 double-blind, placebo-controlled trials showed none. However, these studies used different routes of administration.
To determine whether plasma vitamin C concentrations vary substantially with the route of administration.
Dose concentration studies and pharmacokinetic modeling.
Academic medical center.
17 healthy hospitalized volunteers.
Vitamin C plasma and urine concentrations were measured after administration of oral and intravenous doses at a dose range of 0.015 to 1.25 g, and plasma concentrations were calculated for a dose range of 1 to 100 g.
Peak plasma vitamin C concentrations were higher after administration of intravenous doses than after administration of oral doses (P < 0.001), and the difference increased according to dose. Vitamin C at a dose of 1.25 g administered orally produced mean (+/-sd) peak plasma concentrations of 134.8 +/- 20.6 micromol/L compared with 885 +/- 201.2 micromol/L for intravenous administration. For the maximum tolerated oral dose of 3 g every 4 hours, pharmacokinetic modeling predicted peak plasma vitamin C concentrations of 220 micromol/L and 13 400 micromol/L for a 50-g intravenous dose. Peak predicted urine concentrations of vitamin C from intravenous administration were 140-fold higher than those from maximum oral doses.
Patient data are not available to confirm pharmacokinetic modeling at high doses and in patients with cancer.
Oral vitamin C produces plasma concentrations that are tightly controlled. Only intravenous administration of vitamin C produces high plasma and urine concentrations that might have antitumor activity. Because efficacy of vitamin C treatment cannot be judged from clinical trials that use only oral dosing, the role of vitamin C in cancer treatment should be reevaluated.
Paradoxically, reactive oxygen species (ROS) can promote normal cellular proliferation and carcinogenesis, and can also induce apoptosis of tumor cells. In this report, we study the contribution of ROS to various cellular signals depending on the nature and the level of ROS produced. In nontransformed NIH 3T3 cells, ROS are at low levels and originate from NADPH oxidase. Hydrogen peroxide (H(2)O(2)), controlled by the glutathione system, is pivotal for the modulation of normal cell proliferation. In CT26 (colon) and Hepa 1-6 (liver) tumor cells, high levels of ROS, close to the threshold of cytotoxicity, are produced by mitochondria and H(2)O(2) is controlled by catalase. N-acetylcysteine, which decreases H(2)O(2) levels, inhibits mitogen-activated protein kinase and normal cell proliferation but increases tumor cell proliferation as H(2)O(2) concentration drops from the toxicity threshold. In contrast, antioxidant molecules, such as mimics of superoxide dismutase (SOD), increase H(2)O(2) levels through superoxide anion dismutation, as well as in vitro proliferation of normal cells, but kill tumor cells. CT26 tumors were implanted in mice and treated by oxaliplatin in association with one of the three SOD mimics manganese(III)tetrakis(4-benzoic acid) porphyrin, copper(II)(3,5-diisopropylsalicylate)2, or manganese dipyridoxyl diphosphate. After 1 month, the volumes of tumors were respectively 35%, 31%, and 63% smaller than with oxaliplatin alone (P < 0.001). Similar data were gained with Hepa 1-6 tumors. In conclusion, antioxidant molecules may have opposite effects on tumor growth. SOD mimics can act in synergy with cytotoxic drugs to treat colon and liver cancers.
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.
It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.
Ascorbate (ascorbic acid, vitamin C), in pharmacologic concentrations easily achieved in humans by i.v. administration, selectively kills some cancer cells but not normal cells. We proposed that pharmacologic ascorbate is a prodrug for preferential steady-state formation of ascorbate radical (Asc•−) and H2O2 in the extracellular space compared with blood. Here we test this hypothesis in vivo. Rats were administered parenteral (i.v. or i.p.) or oral ascorbate in typical human pharmacologic doses (≈0.25–0.5 mg per gram of body weight). After i.v. injection, ascorbate baseline concentrations of 50–100 μM in blood and extracellular fluid increased to peaks of >8 mM. After i.p. injection, peaks approached 3 mM in both fluids. By gavage, the same doses produced ascorbate concentrations of <150 μM in both fluids. In blood, Asc•− concentrations measured by EPR were undetectable with oral administration and always <50 nM with parenteral administration, even when corresponding ascorbate concentrations were >8 mM. After parenteral dosing, Asc•− concentrations in extracellular fluid were 4- to 12-fold higher than those in blood, were as high as 250 nM, and were a function of ascorbate concentrations. By using the synthesized probe peroxyxanthone, H2O2 in extracellular fluid was detected only after parenteral administration of ascorbate and when Asc•− concentrations in extracellular fluid exceeded 100 nM. The data show that pharmacologic ascorbate is a prodrug for preferential steady-state formation of Asc•− and H2O2 in the extracellular space but not blood. These data provide a foundation for pursuing pharmacologic ascorbate as a prooxidant therapeutic agent in cancer and infections.
• ascorbic acid
• cancer
• vitamin C
• pharmacokinetics
Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.
Ascorbyl stearate (Asc-S) is a synthetic ester of ascorbic acid that has been shown to significantly reduce the mutagenic effects of alkylating agents and hepatocarcinogenesis in vivo. We have previously demonstrated that Asc-S inhibits ovarian carcinoma cell proliferation through modulation of the cell cycle. This study was designed to further elucidate the mechanisms underlying such regulation.
Wild type p53-expressing cell lines (Ov2008 and C13) were used to evaluate the contributions of p53 to Asc-S-induced G2/M arrest. Cell cycle analysis was performed by flow cytometry. Variation of p53, p21, and GADD45 was evaluated by Western blot and RT-PCR. Knockdown of endogenous p53 was achieved by siRNA.
The expression of p53 downstream genes, p21 and GADD45 was upregulated whereas 14-3-3sigma was unaffected. Phosphorylation of Cdc2 at residue tyrosine-15 was also induced by Asc-S treatment. However, pSilencer-p53-siRNA only partially rescued the Asc-S induced G2/M arrest.
These data show that the anti-proliferative activity of Asc-S on ovarian cancer cells is due in part to G2/M arrest modulated by a p53-dependent pathway.
The tumor suppressor protein p53 is a redox-active transcription factor that organizes and directs cellular responses in the face of a variety of stresses that lead to genomic instability. One of the most important questions in the study of p53 is how selective transactivation of certain p53 target genes is achieved. Reactive oxygen species (ROS), generated by cells as products or by-products, can function either as signaling molecules or as cellular toxicants. Cellular generation of ROS is central to redox signaling. Recent studies have revealed that each cellular concentration and distribution of p53 has a distinct cellular function and that ROS act as both an upstream signal that triggers p53 activation and a downstream factor that mediates apoptosis. Here, we examine the newly discovered role of p53 in regulating cellular ROS generation and how ROS modulate selective transactivation of p53 target genes. The focus is on interlinks between ROS and p53.
Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.
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 relationship between chemosensitivity and p53 is currently considered from two mutually exclusive points of view: (1) wt p53 increases chemosensitivity due to apoptosis and (2) wt p53 decreases chemosensitivity due to growth arrest and DNA repair. We used p53-expressing adenovirus (Ad-p53) to directly evaluate effect of p53 on sensitivity to anticancer drugs. When p53 was expressed at sublethal levels, it sensitized cells to the DNA-damaging drugs Adriamycin, mitomycin C, actinomycin D, etoposide (VP16), cisplatin and CPT11. This sensitization was observed in cancer cell lines (N=10) regardless of endogenous p53 status and also in normal human lung and skin fibroblasts. The degree of sensitization appeared to be greater in cancer cells with mutant p53. Normal fibroblasts required significantly higher doses of Ad-p53 to affect a drug's sensitivity partly because of their lower infectivity by adenovirus. Wt p53 not only decreased IC50 but also accelerated cell death induced by DNA-damaging drugs. In contrast, sensitization to microtubule-active drugs by p53 was shown only in a few cell lines. We conclude that exogenous wt p53 accelerates cell death induced by DNA damaging agents in both normal and cancer cells and offers no protection from anticancer drugs.
Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.
Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.
p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.
One hundred and fifty patients with advanced cancer participated in a controlled double-blind study to evaluate the effects of high-dose vitamin C on symptoms and survival. Patients were divided randomly into a group that received vitamin C (10 g per day) and one that received a comparably flavored lactose placebo. Sixty evaluable patients received vitamin C and 63 received a placebo. Both groups were similar in age, sex, site of primary tumor, performance score, tumor grade and previous chemotherapy. The two groups showed no appreciable difference in changes in symptoms, performance status, appetite or weight. The median survival for all patients was about seven weeks, and the survival curves essentially overlapped. In this selected group of patients, we were unable to show a therapeutic benefit of high-dose vitamin C treatment.
Ascorbic acid metabolism is associated with a number of mechanisms known to be involved in host resistance to malignant disease. Cancer patients are significantly depleted of ascorbic acid, and in our opinion this demonstrable biochemical characteristic indicates a substantially increased requirement and utilization of this substance to potentiate these various host resistance factors. The results of a clinical trial are presented in which 100 terminal cancer patients were given supplemental ascorbate as part of their routine management. Their progress is compared to that of 1000 similar patients treated identically, but who received no supplemental ascorbate. The mean survival time is more than 4.2 times as great for the ascorbate subjects (more than 210 days) as for the controls (50 days). Analysis of the survival-time curves indicates that deaths occur for about 90% of the ascorbate-treated patients at one-third the rate for the controls and that the other 10% have a much greater survival time, averaging more than 20 times that for the controls. The results clearly indicate that this simple and safe form of medication is of definite value in the treatment of patients with advanced cancer.
In vitro and in vivo results suggest that sodium ascorbate (vitamin C) could be useful in the management of neoplasms provided it is used on a biological rationale. Vitamin C can exert multiple mechanisms of action depending upon the cell type and experimental conditions. Vitamin C may kill certain tumor cells, may increase the cell killing effect of certain tumor therapeutic agents and may stimulate the host's immune system against the residual tumor cells. In vitro data also suggest that the irrational use of vitamin C in the management of neoplasms could be ineffective and even harmful. Further study on the effects of vitamin C in combination with tumor therapeutic agents must be done, using animal tumor models before assaying its role in the management of human neoplasms.
In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.
The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared
in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed
after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued
to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and
relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53,
can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.
p53 is a multifunctional protein which plays a role in modulating gene transcription, policing cell cycle checkpoints, activating apoptosis, controlling DNA replication and repair, maintaining genomic stability and responding to genetic insults. Mutation of the p53 gene confers the single greatest known selective advantage favoring cancer formation. Point mutations result not only in the loss of tumor suppressor functions, but also in the gain of tumor promotion functions. These dual circumstances may be unique to p53 and, in part, could explain the relatively powerful force behind this selection pressure. General mechanisms of gain of function by mutated p53 may include alteration in transcriptional modulation and newly acquired targets for transcriptional regulation and protein binding. Despite the direct significance of p53 mutations, loss of the remaining wild-type allele is usually required for the formation of tumors in the natural setting. Novel applications of the basic scientific knowledge of p53 could lead to an improvement in cancer treatment, hopefully in the not so distant future.
Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This "two-faced" character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.
Chemical carcinogenesis follows a multistep process involving both mutation and increased cell proliferation. Oxidative stress can occur through overproduction of reactive oxygen and nitrogen species through either endogenous or exogenous insults. Important to carcinogenesis, the unregulated or prolonged production of cellular oxidants has been linked to mutation (induced by oxidant-induced DNA damage), as well as modification of gene expression. In particular, signal transduction pathways, including AP-1 and NFkappaB, are known to be activated by reactive oxygen species, and they lead to the transcription of genes involved in cell growth regulatory pathways. This review examines the evidence of cellular oxidants' involvement in the carcinogenesis process, and focuses on the mechanisms for production, cellular damage produced, and the role of signaling cascades by reactive oxygen species.
Oxidative stress by increased production of reactive oxygen species such as superoxide has been implicated in the toxicity of PCB's and non-target toxicity of many pesticides. We report the development of a microplate-based method for determination of early stage oxidative stress using an established cell line (EPC) from a skin tumour of carp Cyprinus carpio L. and 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) as a fluorescent probe for detection of reactive oxygen species (ROS) formation. Sublethal concentrations of the herbicide Paraquat, an established redox cycling agent and a crude PCB mixture, Arochlor 1254 elicited a linear increase in ROS formation over 2 h exposure which was some 45- and 10-fold higher, respectively, than attributable to basal respiration, confirming the suitability and response of the test system. Whilst in vivo studies in mammals have implicated early stage oxidative stress in the toxicity of pesticides, we did not observe an increase in ROS production after exposure of EPC cells to sublethal concentrations of Carbaryl, 2,4-DDT, Lindane or Malathion implying that this is not the causative mechanism of acute toxicity in this fish cell line. The apparent involvement of oxidative stress in their mammalian toxicity may therefore be an indirect effect or dependent upon compound metabolism.
Although ascorbate (vitamin C) has been shown to have anti-cancer actions, its effect on human hepatoma cells has not yet been investigated, and thus, the exact mechanism of this action is not fully understood. In this study, the mechanism by which ascorbate induces apoptosis using HepG2 human hepatoblastoma cells is investigated. Ascorbate induced apoptotic cell death in a dose-dependent manner in the cells, was assessed through flow cytometric analysis. Contrary to expectation, ascorbate did not alter the cellular redox status, and treatment with antioxidants (N-acetyl cysteine and N,N-diphenyl-p-phenylenediamine) had no influence on the ascorbate-induced apoptosis. However, ascorbate induced a rapid and sustained increase in intracellular Ca2+ concentration. EGTA, an extracellular Ca2+ chelator did not significantly alter the ascorbate-induced intracellular Ca2+ increase and apoptosis, whereas dantrolene, an intracellular Ca2+ release blocker, completely blocked these actions of ascorbate. In addition, phospholipase C (PLC) inhibitors (U-73122 and manoalide) significantly suppressed the intracellular Ca2+ release and apoptosis induced by ascorbate. Collectively, these results suggest that ascorbate induced apoptosis without changes in the cellular redox status in HepG2 cells, and that the PLC-coupled intracellular Ca2+ release mechanism may mediate ascorbate-induced apoptosis.
Low levels of DNA damage caused by oxidative stress can be repaired, whereas extensive damage usually results in cell death. p53 contributes to both outcomes by stimulating expression of either pro- or antioxidant genes (pages 1306-1313).
The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H(2)O(2). Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2'-deoxycytidine (oh(5)dCyd) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H(2)O(2) (200 microM), resulted in a remarkable sensitization of oh(5)dCyd from 2.7 +/- 0.6 to 40.8 +/- 6.1 lesions /10(6) dCyd (15-fold). In contrast, the level of oxo(8)dGuo increased from 8.4 +/- 0.4 to 12.1 +/- 0.5 lesions/10(6) dGuo (50%). The formation of oh(5)dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H(2)O(2). Additional studies showed that replacement of H(2)O(2) with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H(2)O(2)-induced oxidative DNA damage in cells, leading to elevated levels of oh(5)dCyd and oxo(8)dGuo, with a strong bias toward the formation of oh(5)dCyd.
Reactive oxygen species in oncogenic transformation
- R M Zwacka
Zwacka, R. M. Reactive oxygen species in oncogenic transformation. Biochem. Soc. Trans. 31:1441–1444; 2003.
Relationship of p53 overexpression on cancers and recognition by anti-p53 T cell receptor-transduced T cells
- Theoret
Reactive oxygen species in oncogenic transformation
- Behrend
p53 status and the efficacy of cancer therapy in vivo
- Lowe