Article

High concentrations of ascorbic acid induces apoptosis of human gastric cancer cell by p38-MAP kinase-dependent up-regulation of transferrin receptor

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

We investigated the molecular mechanism by which ascorbic acid (AA) induces apoptosis in human gastric cancer cells, AGS cells. High concentration (more than 5mM) of AA increased cellular iron uptake by increasing transferrin receptor (TfR) expression and induced AGS cell apoptosis which was inhibited by catalase. Interestingly, p38 mitogen-activated protein kinase (MAPK) inhibitor inhibited the upregulation of TfR and increased cell survival by AA. TfR-siRNA-transfected cells reduced apoptosis by AA. H(2)O(2) increased TfR expression in AGS cells. Taken together, we concluded that high concentration of AA, through H(2)O(2), induces apoptosis of AGS cells by p38-MAPK-dependent upregulation of TfR.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The cytotoxic activity of AA appears to be predominantly seen in cancer cells, but not in normal cells [9,12]. The exact type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial [13][14][15][16][17][18][19][20][21][22][23][24][25]. In this study, we sought to investigate the anticancer effect of AA in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. ...
... It is known that oxidative stress can cause the activation of the MAPKs, i.e., JNK, ERK, and p38-MAPK, and, subsequently, results in caspase activation and apoptotic cell death [13,14]. Han et al. reported that p38-MAPK, but not JNK or ERK, mediates AA-induced cytotoxicity in human gastric cancer cells [15]. In this study, therefore, we examined whether MAPK signaling pathways are involved in AA-induced cell death in MIA-PaCa-2 cells. ...
... The type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial. While some of the studies reported that apoptotic cell death is induced by treatment with AA [15,[23][24][25][26][27], some other studies reported that the AA-induced cell death is a caspase-independent event [5,[28][29][30][31][32][33]. Recently, the role of autophagy in AA-induced cell death has also been suggested [5,19,34]. ...
Article
Full-text available
The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophagosome formation contributes to ascorbate-induced pancreatic cancer cell death. Georg Thieme Verlag KG Stuttgart · New York.
... In addition, Fe(II) catalyzes the formation of OH • and OH − during the interaction between H2O2 and O2 •− (Haber-Weiss reaction) ( Figure 1) [75]. Ascorbate can efficiently reduce free iron, thus recycling the cellular Fe(II)/Fe(III) to produce more OH • from H2O2 than can be generated during the Fenton reaction, which ultimately leads to lipid, protein, and DNA oxidation [76][77][78]. It is worth noting that ascorbate recycling across the cell membrane might boost Vit-C-stimulated iron absorption from low-molecular-weight ironcitrate complexes. ...
... Cancers with high amounts of iron might be more susceptible to mega-dose Vit-C than non-cancer cells, because they may produce higher amounts of H2O2 and OH • via LIP. Other studies found that higher levels of ROS inside mitochondrial lung cells, glioblastoma, and gastric tumor cells increased the amount of LIP inside the cells through transferrin receptor (TfR); thus, enhancing the tumor cell susceptibility to Vit-C [78]. This correlation may explain why the co-delivery of Prussian blue and Vit-C managed to suppress the formation of peroxide and obliterate the anti-cancer activity of Vit-C [99]. ...
Article
Full-text available
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.
... AA is a well-known water-soluble vitamin that possesses a variety of physiological properties (Bijur et al., 1997). In human plasma, it acts as a scavenger of free radicals and protects against lipid peroxidation (Ha et al., 2009). AA is a conventional antioxidant, but it is also capable of acting in a prooxidant manner, which is strongly dependent on its dose. ...
... It has been established that a high dose of AA induces sister chromatid exchange in vitro and that the mutagenic activity of AA is enhanced by transition metals (Bijur et al., 1997). Ha et al. (2009) studied the molecular mechanisms by which AA induces apoptosis in human gastric cancer (AGS) cells and found that its application in concentrations higher than 5 mM using H 2 O 2 induces apoptosis in AGS cells by p38-MAPK-dependent regulation of TfR. It has been established that HL-60 cells are able to transport only the oxidized form of AA (Guaiquil et al., 1997;Witenberg et al., 1999), namely, DHA, which was also produced during the synthesis of AAgNPs and was confirmed by the recorded SERS spectra ( Figure 2). ...
Article
The properties of silver nanoparticles (AgNPs) synthesized using compounds exhibiting biological activity seem to constitute an interesting issue worthy of examination. In these studies, two types of AgNPs were synthesized by a chemical reduction method using well-known antioxidants: gallic acid (GA) and ascorbic acid (AA). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that the AgNPs were spherical. The average size was equal to 26 ± 6 nm and 20 ± 7 nm in the case of ascorbic acid-silver nanoparticles (AAgNPs) and gallic acid-silver nanoparticles (GAAgNPs), respectively. Surface-enhanced Raman spectroscopy (SERS) confirmed that the AgNPs were not stabilized by pure forms of applied antioxidants. Changes in mitochondrial activity and secretion of inflammatory and apoptosis mediators after the exposure of human promyelocytic (HL-60) and histiocytic lymphoma (U-937) cells to the AgNPs were studied to determine the impact of stabilizing layers on nanoparticle toxicity. The GAAgNPs were found to be more toxic for the cells than the AAgNPs. Their toxicity was manifested by a strong reduction in mitochondrial activity and induction of the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and caspase-9. The addition of pure antioxidants to the AgNP suspensions was found to influence their toxicity. There was a significant positive effect in the case of the mixture of AA with AAgNPs and GA with GAAgNPs. The results obtained suggest that the presence of stabilizing agents adsorbed on the surface of AgNPs is the main factor in shaping their toxicity. Nevertheless, the toxic effect can be also tuned by the introduction of free antioxidant molecules to the AgNP suspensions.
... TZT overcomes the oxidative stress induced inhibition of cell proliferation. Vitamin C at high concentrations is known to induce oxidative stress and thereby inhibit cell proliferation [49][50][51][52] . Vitamin C undergoes oxidation in the culture medium to produce hydrogen peroxide in a controlled manner dependent on the ascorbate concentration, time of incubation and percentage of serum used in media. ...
... Higher concentrations of vitamin C induces accumulation of hydrogen peroxide which in turn promotes its cytotoxic effects resulting in cellular damage and cell death. [49][50][51][52] To assess the ability of TZT to inhibit such cytotoxic effects at molecular level, the status of poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in vitamin C and TZT treated cells was investigated. Cleavage of PARP-1 by caspases is considered as a hallmark of apoptosis. ...
Article
Full-text available
A pharmacophoric motif decorated with supramolecular functionalities (TZT) was designed for potential interaction with biological targets. Main insights of this work are correlation of supra functionalities and binding ability of TZT to restructure proteins for bioactivity modulation as promising perspective in the field of cellular protection from oxidative stress. To investigate bio-availabity and role of TZT in obliterating oxidative stress at molecular level, its binding propensity with Bovine serum albumin (BSA) and Catalase (BLC) was characterized by various biophysical methods. The binding constants of TZT with BSA (Kb= 2.09×105 M-1) and BLC (Kb= 2.349×105 M-1) indicate its considerable interaction with these proteins. TZT efficiently triggers favourable structural changes in BLC, thereby enhancing its enzyme activity in a dose dependent manner. The enzyme kinetics parameters of TZT binding to BLC were quantified using Michaelis–Menten model. The molecular docking results and experimental trend of Km values suggest increased substrate availability as possible reason of enhanced BLC activity. Furthermore, physiological relevance of this interaction was investigated by ability of TZT to attenuate oxidative stress induced inhibition of cell proliferation. Treatment with TZT led to reversal of inhibition in A549 cell proliferation effectuated by high concentrations of vitamin C. At molecular level, TZT was found to inhibit apoptotic cell death induced by accumulation of hydrogen peroxide as evaluated from PARP cleavage status.
... Moreover, knockdown of TfR1 expression by a specific siRNA significantly decreased the cell death caused by erastin ( Figure 4B), indicating that TfR1 is involved in erastin-induced cell death. Oncogenic RAS and MAPK signaling have been proposed to enrich the cellular iron pool mainly by upregulation of TfR1 [26][27][28]. Our prevous findings indicated that JNK and p38, but not the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, were responsible for erastin-induced HL-60/ NRAS Q61L cells death [5]. ...
... However, our previous findings indicated that JNK and p38, but not the ERK/MAPK pathway, were responsible for erastin-induced HL-60/NRAS Q61L cells death [5]. Previously, oncogenic RAS and MAPK signaling have been proposed to enrich the cellular iron pool mainly by upregulation of TfR1 [26][27][28]. Here, we showe that oncogenic RAS and JNK and p38 MAPK signaling were involved in erastin-induced TfR1 expression. Moreover, we provide evidence that knockdown of HMGB1 inhibited erastin-induced TfR1 expression and JNK or p38 phosphorylation, whereas overexpression of HMGB1 increased erastin-induced TfR1 expression. ...
Article
Ferroptosis is emerging as a new form of regulated cell death driven by oxidative injury promoting lipid peroxidation in an iron-dependent manner. High mobility group box 1 (HMGB1) plays an important role in leukemia pathogenesis and chemotherapy resistance. The mechanisms of ferroptosis in tumor pathogenesis and treatment have been a recent research focus but the role of HMGB1 in regulating ferroptosis especially in leukemia still remains largely unknown. Here, we shown that HMGB1 is a critical regulator of eratin-induced ferroptosis in HL-60 cell line expressing NRASQ61L (HL-60/NRASQ61L). Erastin enhanced ROS levels, thereby promoting cytosolic translocation of HMGB1 and enhancing cell death. Knockdown of HMGB1 decreased erastin-induced ROS generation and cell death in an iron-mediated lysosomal pathway in HL-60/NRASQ61L cells. Knockdown of HMGB1 or rat sarcoma (RAS), or pharmacological inhibition of JNK and p38 decreased TfR1 levels in HL-60/NRASQ61L cells. Importantly, these data were further supported by our in vivo experiment, in which xenografts formed by HMGB1 knockdown HL-60/NRASQ61L cells had lower PTGS2 and TfR1 expression than that in control mice. Taken together, these results suggest that HMGB1 is a novel regulator of ferroptosis via the RAS-JNK/p38 pathway and a potential drug target for therapeutic interventions in leukemia.
... In addition to antioxidant activity, ascorbic acid can affect cell growth by altering cell proliferation and inducing cell death in various cell systems (Brigelius-Flohe & Flohe, 1996; Sakagami & Satoh, 1997). Although, the effect of vitamin C in cancer treatment remains controversial, numerous studies have reported that ascorbate induces cell cycle arrest and apoptosis in various tumour cells (Ha et al., 2009; Hahm et al., 2007; Kang et al., 2005). However, the mechanism underlying the anticancer role of vitamin C has not been fully elucidated. ...
... While cancer cell invasion and metastasis is known to be triggered by multiple genes and the involvement of proteins has been revealed, the molecular mechanism of carcinogenesis and metastasis of gastric cancer remains unclear. Moreover, previous studies have suggested that anticancer mechanisms of vitamin C include cell cycle arrest following apoptotic death in various cancerous cells (Ha et al., 2009; Hahm et al., 2007; Kang et al., 2005). Yet, the mechanisms underlying gastric cancer cell death in response to vitamin C have not been reported. ...
Article
Ascorbic acid (vitamin C) is an essential component of most living cells. Apart from antioxidant activity, it has been reported to inhibit cancer cell growth in vitro in human cancer cells. However, the cellular mechanism underlying anticancer activity has not been fully elucidated. In this study, vitamin C showed a cytotoxic effect on human gastric cancer cell line AGS (LD50 300μg/ml). Further, flow cytometry analysis showed that vitamin C increased the sub-G1 (apoptosis) population and apoptosis confirmed by fluorescein isothiocyanate-Annexin V double staining in AGS cells. Moreover, specific immuno-blotting revealed the expression of the phosphorylated form of Bad (S136), 14-3-3σ, pro-caspases-3, -6, -8, and-9 protein levels were significantly decreased and Bax/Bcl-xL ratio was increased in a dose-dependent manner. Also, wound healing assay results showed that vitamin C inhibited AGS cell proliferation. These findings suggest that vitamin C induces apoptosis and might be a potential therapeutic agent for gastric cancer.
... Ascorbic acid could control level of oxidative stress by ROS and RNS quenching or stimulating which circumstance of cells can be verified vitamin C function. The previous studies have suggested ascorbic acid act as pro-drug for formation of 22 HO and accomplish as a therapeutic agent against cancer related disease [26]. ...
... Ascorbic acid could control level of oxidative stress by ROS and RNS quenching or stimulating which circumstance of cells can be verified vitamin C function. The previous studies have suggested ascorbic acid act as pro-drug for formation of 22 HO and accomplish as a therapeutic agent against cancer related disease [26]. ...
Preprint
Full-text available
Since the last decades, there have been numerous reports about the interaction of magnetic field (MF) and cold atmospheric plasma (CAP) with the biological systems, separately. In this manuscript, we have investigated the combined effect of CAP with the static magnetic field (SMF) as an effective method for cancer cells treatment. MDA-MB-231 breast cancer cells were cultured and treated with CAP in different input power and different exposure time in the presence and absence of the SMF. Vitamin C is used in medium, and cell viability is investigated in the presence and absence of this antioxidant compound. The MTT assay has been employed to measure cell survival, and then T-test and one-way ANOVA are used to assess the significance level of quantitative data. In order to determine the migration rate of cancer cells, wound healing assay has been carried out. Results show that presence of the SMF and vitamin C as well as increasing the input power has a significant role on the attenuation of the survival and migration rate of the cells. The results of the present investigation will greatly contribute to improve the CAP efficiency in cancer therapy through using the SMF and vitamin C as a complement to conventional CAP therapies.
... With regard to the effects of vitamin C in cancer cells, it is known that it acts as an antioxidant at physiological levels and it could be linked to pro-oxidant effects at pharmacological doses which promote the death of cancer cells (Chen et al. 2005(Chen et al. , 2007. Various in vitro studies have already shown that high concentrations of vitamin C led to apoptosis of cancer cells (Chen et al. 2005;Ha et al. 2009;Harakeh et al. 2007;Hong et al. 2007;Kim et al. 2012;Obara and Harasawa 2008). So far, several mechanisms have been described in vitro which give a more detailed insight into the molecular pathways which contribute to the induction of apoptosis (Irimie et al. 2019). ...
Article
Full-text available
Background Vitamin C, also called ascorbic acid, is a water-soluble antioxidant and free radical scavenger. It is required in the body for numerous metabolic functions and is involved in the development of proteins and connective tissue. Methods In April 2020, a systematic search was carried out on five electronic databases (Medline, Embase, Cochrane, Cinahl, PsycINFO) to find studies on the use, efficacy and safety of a complementary therapy with vitamin C in oncological patients. Results Out of the initial 23,195 search results, 21 studies with 1961 patients were included in this review. Five of the included studies ( n = 417) were randomized controlled trials (RCTs). The remaining 16 studies belonged to a lower class of evidence. The patients who were treated with vitamin C suffered from various malignant diseases, some in an advanced and palliative stage. Vitamin C was applied intravenously or orally. It was either the only treatment or was combined with chemo- or radiotherapy. Endpoints included the development of the disease-related symptoms, quality of life, mortality, progression-free survival and safety of vitamin C. The studies were of moderate quality and showed either no effect of vitamin C or a positive trend, although this has rarely been statistically proven in group comparisons. No or only slight side effects with both oral and intravenous administration of vitamin C were reported. Conclusion Oral intake of vitamin C does not appear to have any effect in patients with malignancies. Data are heterogeneous for intravenous administration. There are no RCTs with statistical group comparisons.
... In particular, they contain vitamin A, vitamin C, vitamin E (a family of eight tocotrienols and tocopherols), carotenoids and a wide range of polyphenolic compounds, such as the flavonoid and anthocyanin family. These chemical substances prevent the attack of free radicals, reducing the risk of cancer and mortality (Harakeh et al., 2007;Ha et al., 2009;Du et al., 2012;Forrester et al., 2018). Vitamin C or L-Ascorbic acid (AsA) is a naturally occurring organic compound with antioxidant properties, found to protect cellular components from free radical damage. ...
Article
Full-text available
L-Ascorbic acid (AsA; vitamin C), is a common antioxi-dant in nature. In plants, it plays an important role in photosynthesis as it eliminates reactive oxygen species (ROS) generated during electron transport. Unlike animals that use D-Glucuronate as a precursor, plants produce AsA by four alternative routes using Myo-inositol, L-Gulose, D-Mannose/L-Galactose and D-Galacturonate as main precursors. Humans cannot produce AsA due to the absence of the enzyme that catalyzes the last biosynthetic step, so they need plant sources. Epidemiological studies suggest that the consumption of AsA is important for health; therefore, increasing the vitamin C content of crops could be an important goal. Many approaches have been taken to increase the vitamin C content in plants, but challenges remain. Here, we examine the biosynthesis and recycling pathways of AsA, the close connection with AsA and abiotic and biotic stresses, as well as different strategies for increasing its content in plants.
... Furthermore, they are the main cellular source of oxygen radicals as well as indicate an ideal intracellular point of free radical attack (Lobo et al., 2010). A study by Ha et al. (2009) reported that ascorbic acid induces apoptosis in human adenocarcinoma gastric cancer cells by producing H 2 O 2 . Ascorbic acid at a concentration greater than 1 mM can generate H 2 O 2 by producing the ascorbate radical causing the death of cancer cells. ...
Chapter
Ascorbic acid (AA) is a well-known antioxidant found in fruits and vegetables and is effective to protect tissues from oxidative injury. It is an essential antioxidant able to quench reactive oxygen species (ROS), however, its transport across the mitochondria and the functions are inadequately studied. The mitochondrial fraction of ascorbic acid is of critical importance for the regulation of the redox status of these organelles and for cell survival. AA induces apoptotic cell death at higher concentrations by means of its prooxidant activity and at lower concentrations; it prevents the activation of oxidant-induced apoptosis via its antioxidant properties. This chapter deals with the effects of ascorbic acid on mitochondria as evidenced by in vitro, ex vivo, and in vivo experimental models with a special focus on the mechanism of action by which ascorbic acid modulate mitochondria function.
... Furthermore, ascorbic acid is readily oxidised by dissolved oxygen to produce the pro-oxidant dehydroascorbic acid in neutral or alkaline solutions. Both dehydroascorbic acid and ascorbic acid can induce apoptosis in eukaryotic cells in a prooxidant environment (Ha et al. 2009;Hong et al. 2007). It is therefore possible that ascorbic acid plays a direct role in inhibiting G. duodenalis proliferation. ...
Article
Full-text available
Giardiasis, one of the most common causes of diarrhoeal disease, is caused by gastrointestinal protozoal parasites of the genus Giardia. Metronidazole is the most commonly used drug to treat giardiasis. However, metronidazole resistance is increasingly common, making the development of new anti-giardial drugs a high priority. A panel of 11 compounds previously identified in T. ferdinandiana fruit extracts were investigated for the ability to inhibit G. duodenalis proliferation. Eight of the 11 compounds inhibited the growth of all three G. duodenalis strains. 2,3-Dihydroxyphenyl B-D-glucopyranosiduronic acid (DPGA) was the most potent anti-giardial compound, with IC50 values as low as 126 μM (38 μg/mL). Notably, DPGA inhibited a metronidazole-resistant G. duodenalis strain with similar activity as determined for the metronidazole-sensitive strains. Furthermore, the activity of DPGA was greatly potentiated when it was tested in combination with ascorbic acid, to approximately 17 μM (5 μg/mL) for the metronidazole-sensitive G. duodenalis strains and 40 μM (12 mg/mL) for the resistant strain. The T. ferdinandiana tannins (gallic acid and chebulic acid) were moderate inhibitors of G. duodenalis growth when tested in combination with ascorbic acid, although they had only low levels of activity when tested alone. All of the tested compounds (and their combinations with ascorbic acid) displayed low toxic effects and all compounds are conformed to Lipinski's rules of 5 with few violations, indicating their potential as drug leads and chemotherapies for the treatment and prevention of giardiasis.
... Ascorbic acid controls the level of oxidative stress by ROS and RNS quenching or stimulating depending on the cell state [27]. The previous studies suggested that ascorbic acid acts as a pro-drug for the formation of H 2 O 2 and as a therapeutic agent against cancer-related disease [28]. ...
Article
Full-text available
In the last decades, there have been numerous reports about the separate interactions of a magnetic field and cold atmospheric plasma (CAP) with the biological systems. We have investigated the combined effect of CAP with the static magnetic field (SMF) as an effective method for cancer cells treatment. MDA-MB-231 breast cancer cells were cultured and treated with CAP with different input power and exposure times in the presence and absence of the SMF. Vitamin C was also used in medium, and cell viability was investigated in the presence and absence of this antioxidant compound. The MTT assay was employed to measure cell survival, and a T-test or one-way ANOVA was used to assess the significance level of quantitative data. In order to determine the migration rate of cancer cells, wound healing assay was carried out. Results show that the presence of the SMF and vitamin C as well as increasing the input power significantly decrease the survival and migration rate of the cells. The results of the present investigation will greatly contribute to improve the CAP efficiency in cancer therapy by using the SMF and vitamin C as a complement to conventional CAP therapies.
... Experimentally, elevated concentrations of vitamin C induce apoptosis in gastric tumor cells mediated by p38 MAP-kinase (mitogen-activated protein kinase) 13 , however, no clinical intervention trials evaluating this aspect. There are retrospective studies that demonstrate a risk association between low vitamin C consumption and gastric cancer (OR 0.40, 95% CI 0.19-0.83) ...
Article
Full-text available
Vitamin C has been widely studied in medicine and although the importance of its deficiency with scurvy was recognized, the optimization of its use as a therapeutic resource has not been included in protocols or clinical practice guidelines. The pharmacokinetics and biology of vitamin C indicates the systemic effects it has, and based on it, research has been developed in recent years that supports its parenteral use in some diseases. The available evidence indicates that the benefit of its use does not extend to several diseases but to some such as cancer, whose preliminary report is promising, with adequate tolerability at high doses, but still needs to complete the prospective follow-up of the intervention.
... The fact that in our study only the erythroid lineage was affected by FeAS, indicates that the dose utilized is not toxic for all cells. In addition, although cells exposed to high concentrations of ascorbic acid (>5 mM) can undergo apoptosis [42], ascorbic acid concentrations utilized in our experimental conditions are not toxic and did not affect the cytokine-dependent growth of HSCs. ...
Article
Full-text available
Iron overload is the accumulation of excess iron in the body that may occur as a result of various genetic disorders or as a consequence of repeated blood transfusions. The surplus iron is then stored in the liver, pancreas, heart and other organs, which may lead to chronic liver disease or cirrhosis, diabetes and heart disease, respectively. In addition, excessive iron may impair hematopoiesis, although the mechanisms of this deleterious effect is not entirely known. In this study, we found that ferrous ammonium sulfate (FeAS), induced growth arrest and apoptosis in immature hematopoietic cells, which was mediated via reactive oxygen species (ROS) activation of p38MAPK and JNK pathways. In in vitro hematopoiesis derived from embryonic stem cells (ES cells), FeAS enhanced the development of dysplastic erythroblasts but inhibited their terminal differentiation; in contrast, it had little effect on the development of granulocytes, megakaryocytes, and B lymphocytes. In addition to its directs effects on hematopoietic cells, iron overload altered the expression of several adhesion molecules on stromal cells and impaired the cytokine production profile of these cells. Therefore, excessive iron would affect whole hematopoiesis by inflicting vicious effects on both immature hematopoietic cells and stromal cells.
... ROS play an important role in Cd-induced testicular oxidative stress injury (Sen Gupta et al. 2004b, Siu et al. 2009). Numerous studies have demonstrated that p38 MAPK activation can promote cellular oxidation reactions and enhance oxidative damage (Ha et al. 2009, Peus et al. 1999, Usatyuk et al. 2003. Moreover, it has been revealed that the activation of p38 MAPK is an important target in oxidative stress-induced heart damage (Consoli et al. 2013, Ruan et al. 2015. ...
Article
Full-text available
Ascorbic acid (AA), one of the best-known reactive oxygen species (ROS) scavengers, exhibits numerous functions such as antioxidant, anti-cancer, and anti-inflammatory effects. Increasing evidence demonstrates that oxidative stress plays an important role in testicular toxicity. In the present study, we investigated the protective effect of AA against cadmium (Cd)-induced blood-testis barrier (BTB) disruption. Sprague-Dawley (SD) rats were divided into four groups: the Cd-treated group received a single dose (s.c.) of 2 mg/kg BW cadmium chloride; the AA antagonism group received an injection of AA at a dose of 400 mg/kg BW (200 mg 24 h prior to Cd treatment and 200 mg 24 h following Cd treatment); and the control groups received an equal volume of saline or an equal dose of AA. As expected, ROS expression was upregulated in the Cd-treated rats, accompanied by an increase in malondialdehyde (MDA). Interestingly, AA suppressed Cd-induced oxidative stress by decreasing the levels of ROS and MDA and increasing the activity of superoxide dismutase (SOD) and catalase (CAT). In addition, AA also reduced BTB disruption by inhibiting TGF-β3 activation and p38 MAPK phosphorylation. Significant decreases in occludin and claudin-11 expression were observed in the Cd-treated rats, whereas AA administration attenuated this effect. Moreover, testicular histopathology and transmission electron microscopy further demonstrated the protective effects of AA against Cd-induced BTB damage. In conclusion, the results of the present study suggest that AA protects BTB destruction via the inhibition of oxidative stress and the TGF-β3/p38 MAPK signalling pathway in the testis of Cd-exposed rats.
... The transferrin receptor (TfR) is a protein of approximately 95 kDa that has an established role in the uptake of iron-loaded transferrin into a variety of cell types including AGS cells [26] whereas ferritin is recognized as an intracellular iron storage protein [27]. RT-PCR revealed an increase in TfR gene expression in uninfected and H. pylori-infected cells over time that was not discernibly different between the two conditions ( Fig 1A; broken line; left and middle). ...
Article
Full-text available
The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.
... While recent studies showed that high dose of vitamin C exhibit anticancer effect only administered intravenously (approximately 40-400 mg/kg) or intraperitoneally (4 g/kg) (Padayatty et al., 2004;Chen et al., 2008;Verrax & Calderon, 2009;Takemura et al., 2010). Since then, numerous in vitro studies on both human and animal tumors have been carried out to elucidate the role of ascorbic acid in the prevention of different types of cancer, including studies of prostate (Pollard et al., 2010), pancreatic (Du et al., 2010, Espey et al., 2011, hepatocellular (Lin & Chuang, 2010), ovarian (Chen et al., 2008), colon (Ha et al., 2009;Pathi et al., 2011), mesothelioma (Takemura et al., 2010), neuroblastoma (Hardaway et al., 2012), and malignant melanoma (Kang et al., 2003;Miles et al., 2015) cell lines. Nevertheless, the in vivo application of ascorbate is currently limited by the very high blood concentrations that are required to achieve therapeutic levels in tumors (Ohno et al., 2009). ...
Article
Full-text available
A co-loaded drug delivery system based on ascorbyl palmitate that can transport various functional drugs to their targets within a tumor represents an attractive strategy for increasing the efficiency of anticancer treatment. In this study, we developed a dual drug delivery system to encapsulate ascorbyl palmitate (AP) and paclitaxel (PTX) for synergistic cancer therapy. AP, which is a vitamin C derivative, and PTX were incorporated into solid lipid nanoparticles (AP/PTX-SLNs), which were used to treat murine B16F10 melanoma that had metastasized to the lungs of mice. These nanoparticles were spherical with an average size of 223 nm as measured by transmission electron microscope and dynamic light scattering. In vitro cytotoxicity assays indicated that the AP/PTX-SLNs with an AP/PTX mass ratio of 2/1 provided the optimal synergistic anticancer efficacy. In vivo, AP/PTX-SLNs were revealed to be much more effective in suppressing tumor growth in B16F10-bearing mice and in eliminating cancer cells in the lungs than single drug (AP or PTX)-loaded SLNs via a synergistic effect through reducing the Bcl-2/Bax ratio. Furthermore, no marked side effects were observed during the treatment with the AP/PTX-SLNs, indicating that the co-delivery system with ascorbyl palmitate holds promising clinical potential in cancer therapy.
... Apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) protein family and also by certain caspases, which are a family of cysteine proteases (18). Apoptosis plays a pivotal role and prevents carcinogenesis by suppressing abnormal cell development or by removing mutated/damaged cells (19). Therefore, apoptosis is essential to the anticancer properties of numerous anticancer agents. ...
Article
Full-text available
Lonicera japonica Thunb. (L. japonica T.) has historically been used in Korean herbal medicine due to its anticancer and protective effects on the respiratory system. In the present study, the polyphenolic compounds in L. japonica T. were investigated using high-performance liquid chromatography coupled with tandem mass spectrometry, and its anticancer effects on A549 non-small-cell lung cancer cells were studied. Polyphenolic compounds potentially inhibit A549 cells in a dose-dependent manner. Flow cytometry and western blot analysis demonstrated that polyphenolic compounds induce apoptosis by regulating the protein expression levels of caspases, poly-(ADP-ribose) polymerase and the B-cell lymphoma-2-associated X-protein/B-cell lymphoma-extra large ratio. Furthermore, polyphenolic compounds inhibited mitochondrial membrane potential activity. Caspase-3 activity was increased in a dose-dependent manner and polyphenolic compounds inhibited the activation of protein kinase B by dephosphorylation. These results suggest that polyphenolic compounds in A549 cells indicate the anticancer activity through the induction of apoptosis.
... A previous report demonstrated that vitamin C produces H 2 O 2 , thus inducing the apoptosis of human adenocarcinoma gastric cancer cells from the AGS cell line (7). A concentration of vitamin C >1 mM can produce H 2 O 2 , causing the death of cancer cells by producing the ascorbate radical (4). ...
Article
Full-text available
It has been demonstrated that vitamin C exhibits anti-cancer activity in various tumor cell lines; however, its specific mechanism of action remains unknown. Although the diagnosis and therapy of cancer patients have markedly improved in recent years, safer and more cost-effective treatments are still required. Therefore, the present study examined the effect of vitamin C on the induction of cell death in gastric cancer and its underlying mechanism of action. It was observed that the cytotoxicity of vitamin C on the human gastric cancer cell line AGS is dependent on the apoptotic pathway, including caspase cascades, but not on the necroptotic pathway. It was demonstrated that the vitamin C-induced calcium influx and ROS generation have critical roles in the induction of apoptosis. Furthermore, vitamin C treatment depleted adenosine triphosphate (ATP) production in AGS cells, and the autophagy pathway may be involved in this process. Taken together, the current study suggests that a high dose of vitamin C may induce gastric cancer cell apoptosis through the dysfunction of mitochondria, including calcium influx, reactive oxygen species generation and ATP depletion.
... Some study results implicated JNK and P38 signaling activation induced breast cancer cells apoptosis (Uehara et al. 2012;Brosseau et al. 2010;Koyuturk et al. 2007). Other research showed JNK and P38 signaling activation induced the apoptosis of gastric cancer cells (Lin et al. 2007;Ha et al. 2009;Tseng et al. 2004). ...
Article
Full-text available
Background Lung adenocarcinoma can easily cause malignant pleural effusion which was difficult to discriminate from benign pleural effusion. Now there was no biomarker with high sensitivity and specificity for the malignant pleural effusion. Purpose This study used proteomics technology to acquire and analyze the protein profiles of the benign and malignant pleural effusion, to seek useful protein biomarkers with diagnostic value and to establish the diagnostic model. Methods We chose the weak cationic-exchanger magnetic bead (WCX-MB) to purify peptides in the pleural effusion, used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain peptide expression profiles from the benign and malignant pleural effusion samples, established and validated the diagnostic model through a genetic algorithm (GA) and finally identified the most promising protein biomarker. Results A GA diagnostic model was established with spectra of 3930.9 and 2942.8 m/z in the training set including 25 malignant pleural effusion and 26 benign pleural effusion samples, yielding both 100 % sensitivity and 100 % specificity. The accuracy of diagnostic prediction was validated in the independent testing set with 58 malignant pleural effusion and 34 benign pleural effusion samples. Blind evaluation was as follows: the sensitivity was 89.6 %, specificity 88.2 %, PPV 92.8 %, NPV 83.3 % and accuracy 89.1 % in the independent testing set. The most promising peptide biomarker was identified successfully: Isoform 1 of caspase recruitment domain-containing protein 9 (CARD9), with 3930.9 m/z, was decreased in the malignant pleural effusion. Conclusions This model is suitable to discriminate benign and malignant pleural effusion and CARD9 can be used as a new peptide biomarker.
... Thephenomenon has been illustrated in as tudyb yP adayatta and co-workers where intravenous administration of 1.25 go fv itamin Cr esulted in ap lasma concentration >1 mmol/l (Padayatty et al., 2004). These extreme supraphysiological levels of plasma ascorbate are evaluated for the treatment of cancer because several in vitro data suggest that millimolar concentrations of vitamin Ca re cytotoxic to cancer cells (Leung et al., 1993;Lin et al., 2006;Ha et al., 2009). Emerging data suggest that vitamin Cinfusion might promote apoptosis in cancer cells in vivo.H owever,c ontrolled clinical trials are required to elaborate on aconsistent effect in cancer patients. ...
... Moreover, the oxidative damage caused by ROS and reactive nitrogen species is controlled by cellular iron homeostasis [45], implying a potential link between oxidative stress and TfR1-mediated iron uptake. Recent studies also have reported that ROS induce the expression of TfR1 by activating p38-MAP kinase [46], thus facilitating iron uptake and the Fenton reaction, which consequently results in more toxic ROS. However, oxidative damage alone is not sufficient to explain all the anticancer activities of ART [47,48]. ...
Article
Full-text available
Despite advances in the development of molecularly targeted therapies, metastatic renal cell carcinoma (RCC) is still incurable. Artesunate (ART), a well-known anti-malarial drug with low toxicity, exhibits highly selective anti-tumor actions against various tumors through generation of cytotoxic carbon-centered free radical in the presence of free iron. However, the therapeutic efficacy of ART against metastatic RCC has not yet been fully elucidated. In the analysis on a dataset from The Cancer Genome Atlas (TCGA) (n = 469) and a tissue microarray set from Samsung Medical Center (n = 119) from a cohort of patients with clear cell RCC (ccRCC), up-regulation of transferrin receptor 1 (TfR1), which is a well-known predictive marker for ART, was correlated with the presence of distant metastasis and an unfavorable prognosis. Moreover, ART exerted potent selective cytotoxicity against human RCC cell lines (Caki-1, 786-O, and SN12C-GFP-SRLu2) and sensitized these cells to sorafenib in vitro, and the extent of ART cytotoxicity correlated with TfR1 expression. ART-mediated growth inhibition of human RCC cell lines was shown to result from the induction of cell cycle arrest at the G2/M phase and oncosis-like cell death. Furthermore, ART inhibited cell clonogenicity and invasion of human RCC cells and anti-angiogenic effects in vitro in a dose-dependent manner. Consistent with these in vitro data, anti-tumor, anti-metastatic and anti-angiogenic effects of ART were also validated in human 786-O xenografts. Taken together, ART is a promising novel candidate for treating human RCC, either alone or in combination with other therapies.
... In the present study, the effects of a combined treatment of 5 mM AsA and X-ray irradiation on epithelial cancer cells and sarcoma cells were examined. Several previous studies have also used 5 mM AsA in their investigations to determine its direct effect in vitro and by diffusion into the human body (15)(16)(17). ...
Article
Our previous studies demonstrated that the combination of treatment with ascorbic acid (AsA) and X‑ray irradiation results in increased apoptosis in HL60 cells. The present study was performed to investigate the effects of the combined use of AsA and X‑ray irradiation on epithelial cancer and sarcoma cells, and its potential use in future clinical treatment. X‑ray irradiation combined with AsA treatment resulted in increased suppression of cell growth of HT1080, SAS and A549 cells in vitro compared with X‑ray irradiation alone. The combined treatment also suppressed tumor growth in implanted HT‑1080 cells in vivo. Using annexin V/propidium iodide staining and the detection of activated caspase 3, it was found that X‑ray irradiation increased the apoptotic rate of HT1080 cells and resulted in G2/M arrest. However, apoptosis in the HT1080 cells treated with 5 mM AsA remained unchanged, and no changes were observed in the G2/M fraction. By contrast, AsA treatment caused increased suppression of proliferation compared with X‑ray irradiation. These results suggested that 5 mM AsA slowed the cell cycle and reduced tumor growth. Therefore, X‑ray irradiation combined with AsA treatment may be effective against epithelial cancer and sarcoma cells.
... The crocetin-treated cells were harvested, lysed, and their protein concentrations were quantified by the BCA assay (Ha et al. 2009), using specific antibodies to Bax and Bcl-2; βactin was used as a loading control. The equal amounts of protein from each sample were separated by electrophoresis on a 12% denaturing SDS gel. ...
... DVDMs triggers mitochondrial-dependent apoptosis via rOs therapy. 36,37 Here we observed an increase in expression of phospho-p38 MAPK immediately after SDT, whereas in the presence of the ROS scavenger, N-acetylcysteine, expression of this protein was markedly suppressed. ...
Article
Full-text available
Background Sonodynamic therapy (SDT) is a promising method that uses ultrasound to activate certain chemical sensitizers for the treatment of cancer. The purpose of this study was to investigate the sonoactivity of a novel sensitizer, sinoporphyrin sodium (DVDMS), and its sonotoxicity in an esophageal cancer (ECA-109) cell line. Methods The fluorescence intensity of DVDMS, hematoporphyrin, protoporphyrin IX, and Photofrin II was detected by fluorescence microscopy and flow cytometry. Generation of singlet oxygen was measured using a 1, 3-diphenylisobenzofuran experiment. A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used to examine cell viability. Production of reactive oxygen species (ROS) and destabilization of the mitochondrial membrane potential were assessed by flow cytometry. Apoptosis was analyzed using Annexin-PE/7-amino-actinomycin D staining. Confocal microscopy was performed to assess mitochondrial damage and identify release of cytochrome C after treatment. Western blots were used to determine expression of oxidative stress-related and apoptosis-associated protein. Ultrastructural changes in the cell were studied by scanning electron microscopy. Results DVDMS showed higher autofluorescence intensity and singlet oxygen production efficiency compared with other photosensitizers in both cancerous and normal cells. Compared with hematoporphyrin, DVDMS-mediated SDT was more cytotoxic in ECA-109 cells. Abundant intracellular ROS was found in the SDT groups, and the cytotoxicity induced by SDT was effectively remitted by ROS scavengers. DVDMS located mainly to the mitochondria of ECA-109 cells, which were seriously damaged after exposure to SDT. Release of cytochrome C, an increased rate of apoptosis, and activated apoptosis protein were detected in the SDT group. In addition, relatively severe cell damage was observed on scanning electron microscopy after treatment with DVDMS and SDT. Conclusion These results suggest that DVDMS could be activated by ultrasound, and that DVDMS mediates SDT-induced mitochondrial-dependent apoptosis in ECA-109 cells via production of ROS.
... A recent study shows that intravenous administration of ascorbic acid can be used as a way to selectively kill cancer cells. The apoptotic effect of ascorbic acid on human gastric cancer cells is iron-mediated oxidative stress, which increases upregulated expression of transferrin receptor (Ha et al., 2009). Therefore, we suggest that HMGB3 may also be involved in gastric cancer cell proliferation, migration, and apoptosis by interacting with those DEGs. ...
Article
Gastric cancer is a major health problem worldwide; it is the second most common cause of cancer death in the world. Recent studies indicate that the high-mobility group (HMG) of chromosomal proteins is associated with cancer progression. However, HMGB3 has been little studied. We analyzed the co-expression network between HMGB3 and differentially-expressed genes in the GSE17187 database, identifying the relevant transcription factors, and the conserved domain of HMGB3 to understand the underlying regulation mechanisms involved in gastric cancer. Thirty-one relationships between 11 differentially-expressed genes were included in a co-expression network; many of these genes have been identified as related to cancer, including TBX5 and TFR2. Further analysis identified nine transcription factors, these being GATA3, MZF1, GATA1, GATA2, SRY, REL, NFYB, NFYC, and NFYA, which could interact with HMGB3 to regulate target gene expression and consequently regulate gastric cancer cell proliferation, migration and invasion. The HMG-box domain was very similar in various species, with only a few amino acid changes, indicating conserved functions in HMG-box. This information helps to provide insight into the molecular mechanisms of HMGB3 in human gastric cancer.
... The crocetin-treated cells were harvested, lysed, and their protein concentrations were quantified by the BCA assay (Ha et al. 2009), using specific antibodies to Bax and Bcl-2; ␤-actin was used as a loading control. The equal amounts of protein from each sample were separated by electrophoresis on a 12% denaturing SDS gel. ...
Article
This study investigated the therapeutic effect of crocetin, a carotenoid derived from saffron, on gastric adenocarcinoma (AGS) cells and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced gastric cancer in rats. An MTT assay showed a significant dose- and time-dependent inhibition of AGS cell proliferation as a result of crocetin administration. Flow cytometry and caspases activity assays revealed apoptosis had been induced in these cells; RT-PCR and Western blot analyses revealed the suppression of Bcl-2 and up-regulation of Bax expression in AGS cells treated with crocetin. These changes were not observed in normal human fibroblast (HFSF-PI3) cells. Pathological study of the tumor tissue in MNNG-induced gastric cancer in rats indicated the dose-dependent inhibition of tumor progression. In addition, crocetin reversed some changed biochemical parameters, including serum antioxidant activity and lactate dehydrogenase in rat serum. The present study demonstrates the antioxidant, anti-proliferative, and apoptotic activities of crocetin against gastric cancer that may benefit human stomach cancer treatment.
... Vitamin C (ascorbic acid) is an essential nutrient of most living tissues, and readily acts as a strong reducing agent [4]. Epidemiological studies have reported that vitamin C deficiency in humans are linked to more severe H. pylori-associated gastritis and a gastric cancer risk is also higher [5]. ...
Article
Full-text available
Vitamin C (ascorbic acid) is an essential nutrient of most living tissues that readily acts as a strong reducing agent, which is abundant in fruits and vegetables. Although, it inhibits cell growth in many human cancer cells in vitro, treatment in cancer is still controversial. Hence, the purpose of this study was to investigate the molecular mechanism of the inhibitory effect of vitamin C on AGS cell growth, and protein profiles in AGS cells after exposure to vitamin C treatment, by using proteomic tools. Vitamin C showed a cytotoxic effect on AGS cells (IC50 300mug/mL) and, 20 differentially expressed proteins (spot intensities which show >=2 fold change and statistically significant, p<0.05 between the control and vitamin-C treated group) were successfully identified by assisted laser desorption/ ionization-time of flight/mass spectrometry (MALDI-TOF/MS). Of the 20 proteins, six were up-regulated and fourteen were down-regulated. Specifically, 14-3-3sigma, 14-3-3epsilon, 14-3-3delta, tropomyosin alpha-3 chain and tropomyosin alpha-4 chain were down-regulated and peroxiredoxin-4 and thioredoxin domain-containing proteins 5 were up-regulated. The identified proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism. Further, the expressions of 14-3-3 isoforms were verified with immuno-blotting analysis. Our proteome results suggest that the apoptosis related proteins were involved in promoting and regulating cell death of AGS cells, and might be helpful to understand the molecular mechanism of vitamin C on AGS cell growth inhibition.
... The AKT family members, AKT 1, 2, and 3, have been shown to be over expressed and constitutively activated in human tumors such as breast, pancreatic, ovarian, prostatic and gastric carcinomas [126,127]. Inhibitory effects of ascorbyl stearate was also reported in a panel of human ovarian and pancreatic cancer cells suggesting that the anti-proliferative effect and induction of apoptosis in these cells were mediated through cell cycle arrest and modulation of the IGF-I receptor and PI3K/AKT2 survival pathways [128]. In the same study the authors reported an anti-tumorigenic effect of Asc-S in vivo using a human ovarian carcinoma xenograft nude mouse (C57BL/6) model. ...
Article
Full-text available
Chemoprevention, which is referred to as the use of nontoxic natural or synthetic chemicals to intervene in multistage carcinogenesis has since decades attracted a considerable interest in plant-derived chemical constituents often termed as "phytochemicals" or sometimes as "Nutraceuticals" in case they are derived from dietary sources. A comprehensive search of the literature show that such an interest in natural product pharmacology has surged in the last 25 years and particularly risen at exponential rates since the last one decade. Phytochemicals such as curcumin (from spice turmeric), resveratrol (from red wine) and genistein (from soy) share the major efforts as indicated by overwhelming publications, despite skepticism concerning their bioavailability. Ascorbic acid (AA), the popular anti-oxidant in fruits and vegetables, has even a longer historical perspective than these dietary agents as for more than 35 years; there had been lingering questions about the efficacy of AA in cancer therapy. The footprints of AA from "scurvy" to "cancer" though complex seems to carry potential provided the puzzle could be set right. The use of AA in cancer treatment has been debated extensively as evident from the literature but surprisingly the complementing early phase bench work on the mechanistic studies for anticancer action was rather retarded. Proposed mechanisms of action for AA in the prevention and treatment of cancer includes antioxidant as well as pro-oxidant properties, stimulation of the immune system, altering carcinogen metabolism, enhancement of collagen synthesis necessary for tumor encapsulation and interference with cancer cell signaling. The observation that the intravenous administration of AA enhances its bioavailability to the extent of deriving pharmacological benefits against cancer has in recent years partially supported the clinical plausibility (efficacy) of AA towards realizing its translational advantage. Here, we provide an overview of AA with regard to its potential in the management of cancer disease.
... E make it possible to achieve both a preven- tive and therapeutic effect, cancelling the possibility of damage caused by the free radicals ( Launoy et al. 1998;Shimizu et al. 2004;Elangovan et al. 2008;Neuzil et al. 2002;Frei et al. 2008). (Cameron et al. 1979;Murata et al. 1982;Frei et al. 2008;Ha et al. 2009) ? MLT (Di Bella et al. 1971;Di Bella et al. 1974;Di Bella et al.1976;Di Bella et al. 1977;Kvetno? ...
Article
Full-text available
the aim of the Di Bella Method (DBM) is to try to overcome the high toxicity level and the limited efficacy of the current medical treatments for cancer. using Melatonin, Retinoids, and vitamins E, D3, and C, components of the extracellular matrix, the DBM reinforces those means that Physiology considers essential for life. Acting together, these differentiating molecules also have an antiangiogenic and antiproliferative effect. Cabergoline and/or Bromocriptin negatively regulate Prolactin, the ubiquitary mitogenic hormone. This effect is reinforced by Somatostatin and/or its analogues by negatively regulating highly mitogenic molecules such as GH and GH-dependent growth factors. the preliminary results are reported of a retrospective observational study on 553 patients treated with the DBM. These data show that the DBM achieved an evident improvement in the quality of life and a considerable increase in the mean survival rates for every disease and stage with respect to the data available in the literature relative to chemotherapy and/or monoclonal antibodies. The result was achieved without any of the known significant toxic effects of chemotherapy and (albeit to a lesser extent with respect to chemotherapy) of monoclonal antibodies. The invalidating causes which removed all scientific credibility from the DBM experiments carried out in Italy in 1998 are also reported. I considered it of use to inform the scientific community of the rationale, the mechanism of action, the scientific basis and clinical findings of the DBM in order to encourage interest in the prospects opened up by the DBM through innovative formulations of the vitamins and Melatonin and the use of biological molecules with a high degree of antitumoural efficacy and low toxicity such as Somatostatin and its analogues.
... In addition daily pharmacologic ascorbate treatment showed significantly decreased growth of ovarian, pancreatic, and glioblastoma tumors in xenograft mice. Other studies [100,101] also showed that anti-proliferative effects of pharmacologic ascorbate may act through inhibition of angiogenesis or inhibition of genes necessary to cell cycle progression in two tumor models. Two phase 1 clinical trials [102,103] of vitamin C on cancer seemed to indicate well tolerance and safety of high dose iv. ...
Article
Full-text available
Prostate cancer (PC) is the second most common cancer in men worldwide. Its prevention and treatment remain a challenge to clinicians. Here we review the relationship of vitamins to PC risk. Many vitamins and related chemicals, including vitamin A, retinoids, several B vitamins, vitamin C, vitamin D and vitamin E have shown their anti-cancer activities as anti-oxidants, activators of transcription factors or factors influencing epigenetic events. Although laboratory tests including the use of animal models showed these vitamins may have anti-PC properties, whether they can effectively prevent the development and/or progression of PC in humans remains to be intensively studied subjects. This review will provide up-to-date information regarding the recent outcomes of laboratory, epidemiology and/or clinical trials on the effects of vitamins on PC prevention and/or treatment.
Chapter
Gastric cancer remains an important contributor to the global cancer burden ranking the 5th most common and the 4th most deadly cancer, according to the latest cancer statistics. Despite the recent advances in the treatment of gastric cancer, with combinatorial and targeted therapies, the overall survival and cure rates are still poor, in particular for patients with advanced metastatic disease. Several reasons may explain the yet unsatisfactory clinical outcome of gastric cancer disease, from biological to experimental and conceptual, which should be tackled in an integrated manner to allow the development of better therapeutic options. Drug repurposing (DR, also known as drug repositioning) is gaining considerable attention as an additional strategy to the mainstream de novo drug discovery process. DR provides suitable drugs to expand the cancer chemotherapy options because it may explore the vast number of approved agents with known safety profiles. The opportunity to use repurposed drugs is grounded in the progress of the knowledge of cancer “physiology” and the consequent identification of more targetable pathways. It is also fostered by the possibility of the combined use of computational and bioinformatic tools, drug screening automation, sequencing technologies, and chemistry. Herein, after a brief introduction to gastric cancer facts and currently approved therapies, we review the current status of DR in gastric cancer mainly focusing on non-oncological drugs (i.e., drugs approved for diseases other than cancers) that have been under pre-clinical and clinical evaluation for cancer and compare the potential advantages and limitations of DR over the traditionally de novo development process. It will also be described the main strategies used to identify potentially “repurposable” drugs and discussed the challenges ahead for DR in gastric cancer.KeywordsGastric cancerDrug repurposingNon-cancer drugsPre-clinical studiesClinical trials
Article
The lifeless earth was formed around 4.5 billion years ago and the first anaerobic unicellular "organisms" may have appeared half a billion years later. Despite subsequent prokaryotes (bacteria and archaea) evolving quite complex biochemistry and some eukaryote characteristics, the transition from unicellular prokaryotes to multicellular, aerobic eukaryotes took a further 2.5 billion years to begin. The key factor or factors that eventually caused this long-delayed transition is a question that has been a focus of considerable research and a topic of discussion over many years. On the basis of the extensive literature available and consideration of some of the characteristics that distinguish multicellular eukaryotes from prokaryotes, it is proposed that, as well as the development of oxygenic photosynthesis producing high levels of environmental oxygen and the formation of vital organelles such as aerobic adenosine triphosphate-generating mitochondria, the concurrent evolution of the L-ascorbic acid redox system should be considered as a key factor that led to the evolution of multicellular eukaryotes and it remains vitally involved in the maintenance of multicellularity and many other eukaryote characteristics.
Article
Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. This article is protected by copyright. All rights reserved.
Article
Gastric precancerous lesion refers to epithelial dysplasia (atypical hyperplasia or intraepithelial neoplasias), which is associated with increased risk of gastric cancer. It happens to be controlled by multiple factors and/or polygene such as the H. pylori infection, diet and environment which play an important role in the development of gastric precancerous lesions. This article describes the pathogenesis of gastric precancerous lesions as many scholars have studied some of it.
Article
Although cervical cancer incidence and mortality rates in the developed world have significantly declined over the past 30 years, it is still the second leading cause of death in women aged 19-39 years. Novel developments of chemotherapeutic agents for cervical cancer are important in reducing patient mortality rates. Recent chemotherapeutic developments have begun to manipulate the apoptotic pathway. L-ascorbic acid (Vitamin C) is generally considered an antioxidant at normal physiological levels (60–80 μM), but considered a pro-oxidant at much higher concentrations (> 1 mM). Due to its pro-oxidant effects, in the 1970s L-ascorbic acid underwent clinical trials as an anticancer treatment at high intravenous concentrations (1-2 mM), and presented ambiguous results. In the last 5 years, L-ascorbic acid has been re-investigated in in vitro cancerous cell lines, and has exhibited a selective toxicity in cancerous cells. This investigation aimed to characterize the mechanism of cell death induced by...
Article
Photodynamic therapy (PDT) is a promising and noninvasive treatment that can induce apoptosis, autophagy, or both depending on the cell phenotype. In this work, chlorin e6 (Ce6) was used to photosensitize human colorectal cancer SW620 cells. In cells, apparent autophagy and apoptosis with dependence on intracellular reactive oxygen species (ROS) generation were detected. p38MAPK activation followed by ROS generation might be a core component in Ce6 mediate PDT (Ce6-PDT)-induced autophagy and apoptosis signaling pathway. By using p38MAPK siRNA, the results showed a marked enhancement on cell apoptosis in Ce6-PDT with increased annexin (+) apoptotic cells, nuclear condensation, caspase-3, and PARP cleavage. Besides, impairment of p38MAPK also promoted the autophagic response to photodamage as indicated by conversion of LC3 and monodansyl cadaverine (MDC) labeling patterns. It appears that Ce6-PDT induced ROS production involving activation of p38MAPK, probably to prevent SW620 cells from photodamage. Moreover, autophagy inhibitor 3-methyladenine/bafilomycin A1 greatly aggravated Ce6-PDT-induced apoptosis in SW620 cells with knockdown of p38MAPK. Taken together, this study suggests that autophagy could represent a promising field in cancer treatment and p38MAPK may be a potential therapeutic target to enhance the efficacy on clinical evaluation for the treatment of colorectal cancer.
Article
Malignant cells are highly dependent on aerobic glycolysis, which differs significantly from normal cells (Warburg effect). Interference of this metabolic process has been considered as an innovative method for developing selective cancer therapy. Recent study demonstrates glycolysis inhibitor 2-deoxyglucose (2-DG) can potentiate PDT efficacy, whereas the possible mechanisms haven’t been carefully investigated. This study firstly proved the general potentiation of PDT efficacy by 2-DG and 3-bromopyruvate (3-BP) on human breast cancer MDA-MB-231 cells, and carefully elucidated the underlying mechanism in the process. Our results showed both 2-DG and 3-BP could significantly promote a PDT-induced cell cytotoxic effect when compared with either monotherapy. Synergistic potentiation of mitochondria- and caspase- dependent cell apoptosis were observed, including mitochondrial membrane potential (MMP) drop, Bax translocation, caspase-3 activation. Besides, ROS generation and the expression of oxidative stress related proteins such as P38 MAPK phosphorylation and JNK phosphorylation were notably increased after the combined treatment. Moreover, when pretreated with ROS scavenger N-acetylcysteine (NAC), the ROS generation, MMP drop, cell apoptosis and cytotoxicity were differently relieved, suggesting ROS was vertical in the pro-apoptotic process induced by 2-DG/3-BP combined with PDT treatment. These results indicate that by combination with glycolytic antagonists and PDT may be a promising therapeutic strategy to effectively kill cancer cells.
Article
Full-text available
Abstract l-ascorbic acid is an abundant water-soluble nutrient found in vegetables and fruits. It enhances the cell proliferation, which is helpful in wound healing process. However, it is relatively unstable and easily degraded under external environments including acidity, alkalinity, evaporation, heat, oxidization, light or moisture. Its storage remains challenged. This study reported the development of l-ascorbic acid microcapsules using the natural protein, gelatin, and the natural polysaccharide, agar, as the wall protection carrier. The physical properties including entrapment efficiency, particle size, surface morphology, chemical compositions and release profile were identified. The cell proliferation of l-ascorbic acid microcapsules was stronger than the free drug. Significant cell growth in microencapsulated l-ascorbic acid-treated human epithelial HaCaT cells was observed when compared with untreated control. Since cell proliferation and wound repair are closely related, it is believed that l-ascorbic acid microcapsules would effectively increase the potential effect of wound healing activity in human skin.
Article
Full-text available
Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a
Article
Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H2O2, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 +/- 39 microM H2O2 in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 +/- 17 microM H2O2 is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H2O2 at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H2O2 at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.
Article
Full-text available
We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kühn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 μM H2O2 suffice for complete IRP-1 activation within 60 min when H2O2 is generated extracellularly at steady-state. By contrast, rapid cellular H2O2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H2O2 degradation); in parallel, 2′,7′-dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H2O2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H2O2, but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H2O2. IRP-1 is thus activated both by extra- and intracellular generation of ROIs. While extracellular H2O2 rapidly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activation. IRP-1 thus represents a novel example of an H2O2-regulated protein that responds differentially to alterations of extra- and intracellular H2O2 levels. Our data also suggest that a direct attack on the 4Fe-4S cluster of IRP-1 by H2O2 (or an H2O2-derived reactive species) represents an unlikely explanation for IRP-1 activation by oxidative stress.
Article
Full-text available
Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1–1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.
Article
Full-text available
The temporal disappearance in human blood plasma of endogenous antioxidants in relation to the appearance of various classes of lipid hydroperoxides measured by HPLC postcolumn chemiluminescence detection has been investigated under two types of oxidizing conditions. Exposure of plasma to aqueous peroxyl radicals generated at a constant rate leads immediately to oxidation of endogenous ascorbate and sulfhydryl groups, followed by sequential depletion of bilirubin, urate, and alpha-tocopherol. Stimulating polymorphonuclear leukocytes in plasma initiates very rapid oxidation of ascorbate, followed by partial depletion of urate. Once ascorbate is consumed completely, micromolar concentrations of hydroperoxides of plasma phospholipids, triglycerides, and cholesterol esters appear simultaneously, even though sulfhydryl groups, bilirubin, urate, and alpha-tocopherol are still present at high concentrations. Nonesterified fatty acids, the only lipid class in plasma not transported in lipoproteins but bound to albumin, are preserved from peroxidative damage even after complete oxidation of ascorbate, most likely due to site-specific antioxidant protection by albumin-bound bilirubin and possibly by albumin itself. Thus, in plasma ascorbate and, in a site-specific manner, bilirubin appear to be much more effective in protecting lipids from peroxidative damage by aqueous oxidants than all the other endogenous antioxidants. Hydroperoxides of linoleic acid, phosphatidylcholine, and cholesterol added to plasma in the absence of added reducing substrates are degraded, in contrast to hydroperoxides of trilinolein and cholesterol linoleate. These findings indicate the presence of a selective peroxidase activity operative under physiological conditions. Our data suggest that in states of leukocyte activation and other types of acute or chronic oxidative stress such a simple regimen as controlled ascorbate supplementation could prove helpful in preventing formation of lipid hydroperoxides, some of which cannot be detoxified by endogenous plasma activities and thus might cause damage to critical targets.
Article
Full-text available
Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.
Article
Full-text available
The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number (approximately equal to 150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.
Article
Full-text available
Reactive oxygen intermediates (ROIs), including superoxide anion (O2.-) and hydrogen peroxide (H2O2), are by-products of aerobic metabolism with potential toxicity towards cellular macromolecules, including lipids, proteins and DNA. Excess ROIs, a condition referred to as oxidative stress, is considered to be a major contributor to ageing, degenerative diseases and reperfusion injury. The reactivity of H2O2 with iron (Fenton reaction) intimately connects oxidative stress and cellular iron metabolism. We have found a novel oxidative stress response pathway in mammalian cells which links oxidative stress to the regulation of iron metabolism. Exposure of cells to H2O2 leads to reduced synthesis of the intracellular iron storage protein ferritin and stimulates transferrin receptor (TfR) mRNA expression. Both responses are post-transcriptional and result from induction of iron regulatory protein (IRP) binding to iron-responsive elements (IREs) in ferritin and TfR mRNAs. IRP induction by H2O2 appears to involve the disassembly of its cubane 4Fe-4S cluster and occurs even in the presence of the protein synthesis inhibitor cycloheximide. The induction kinetics by H2O2 far exceed those by iron starvation. The response requires cellular integrity and cannot be elicited in cell extracts. Whereas the activation of IRP by iron depletion is insensitive to okadaic acid, the rapid induction by H2O2 is blocked by this inhibitor of type I/IIa protein phosphatases. Thus okadaic acid separates the activation pathways by iron depletion and oxidative stress, suggesting the involvement of stress-induced kinase/phosphatase pathways in the latter.
Article
Full-text available
Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15–30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1–2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidantN-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5′-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the “classical” Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.
Article
Full-text available
In the past, investigators have successfully used iron chelators to mitigate the cardiotoxicity of doxorubicin (DOX), a widely used anticancer drug that induces reactive oxygen species (ROS), oxidative damage, and apoptosis. Although intracellular iron plays a critical role in initiating DOX-induced apoptosis, the molecular mechanism(s) that link iron, ROS, and apoptosis are still unknown. In this study, we demonstrate that apoptosis results from the exposure of bovine aortic endothelial cells to DOX and that the apoptotic cell death is accompanied by a significant increase in cellular iron (55Fe) uptake and activation of iron regulatory protein-1. Furthermore, DOX-induced iron uptake was shown to be mediated by the transferrin receptor (TfR)-dependent mechanism. Treatment with the anti-TfR antibody (IgA class) dramatically inhibited DOX-induced apoptosis, iron uptake, and intracellular oxidant formation as measured by fluorescence using dichlorodihydrofluorescein. Treatment with cell-permeable iron chelators and ROS scavengers inhibited DOX-induced cellular55Fe uptake, ROS formation, and apoptosis. Based on these findings, we conclude that DOX-induced iron signaling is regulated by the cell surface TfR expression, intracellular oxidant levels, and iron regulatory proteins. The implications of TfR-dependent iron transport in oxidant-induced apoptosis in endothelial cells are discussed.
Article
Full-text available
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.
Article
Full-text available
Iron is an essential nutrient with limited bioavailability. When present in excess, iron poses a threat to cells and tissues, and therefore iron homeostasis has to be tightly controlled. Iron's toxicity is largely based on its ability to catalyze the generation of radicals, which attack and damage cellular macromolecules and promote cell death and tissue injury. This is lucidly illustrated in diseases of iron overload, such as hereditary hemochromatosis or transfusional siderosis, where excessive iron accumulation results in tissue damage and organ failure. Pathological iron accumulation in the liver has also been linked to the development of hepatocellular cancer. Here we provide a background on the biology and toxicity of iron and the basic concepts of iron homeostasis at the cellular and systemic level. In addition, we provide an overview of the various disorders of iron overload, which are directly linked to iron's toxicity. Finally, we discuss the potential role of iron in malignant transformation and cancer.
Article
Full-text available
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.
Article
Full-text available
Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L-gulono-gamma-lactone oxidase (Gulo)-/- mice, which are unable to synthesize ascorbate, to generate ascorbate-deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate-deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate-induced inflammation in Gulo-/- mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate-deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo-/- mice had elevated levels of hypoxia-inducible factor (HIF)-1alpha, a transcription factor regulated by Fe2+-dependent hydroxylases which require ascorbate for optimal activity. HIF-1alpha has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up-regulation of HIF-1alpha blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells.
Article
Full-text available
Neuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred. We could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells. Sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.
Article
Full-text available
Although ascorbate (Vitamin C) has been shown to inhibit cell growth and induce cell death in variety of cancer cells, results reported in other studies are inconsistent with this conclusion. It was previously reported that ascorbate induces apoptosis in human breast cancer cells. However, the molecular mechanism for this is not clear. In this study, we demonstrate that ascorbate induces cell death through the apoptosis-inducing factor (AIF) in the human breast cancer cell lines, SK-BR3 and Hs578T, but not in a normal breast cell line, Hs578. Ascorbate treatment caused the nuclear translocation of AIF, which is retained in the mitochondria in healthy cells, but caspase cleavage is not induced. Moreover, MG132, an inhibitor of AIF release from mitochondria, blocked the induction of cell death. Furthermore, cells that had been treated with human AIF-specific siRNA resisted cell death induced by ascorbate, implying that the translocation of AIF from mitochondria to the nucleus is responsible for ascorbate-mediated cell death. Therefore, these results suggest that ascorbate activates a caspase-independent and AIF-mediated cell death pathway in human breast cancer cells, SK-BR3, and Hs578T.
Article
We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2′,7′-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.
Article
Mitogen-activated protein kinases (MAPKs) were extensively studied in cancer-derived cell lines; however, studies in non-transformed human cells are scarce. In the current paper, we studied the effect of SB203580, a pharmacological inhibitor of p38 MAPK, on activation and inhibition of p38 MAPK transduction partway in primary human hepatocytes (in vitro model of differentiated cells) in comparison with several tumor cell lines (proliferating non-differentiated in vitro model). In addition, we analyzed the effect of SB203580 on extracellular-regulated protein kinase (ERK) and c-jun-N-terminal kinase (JNK) pathways both in primary human hepatocytes and tumor cell lines employing primary antibodies detecting phosphorylated kinases. We show that SB203580 activates ERK and JNK in primary cultures of human hepatocytes. The levels of ERK-P(Thr202/Tyr204), JNK-P(Thr183/Tyr185) and c-Jun-P(Ser63/73), a target down-stream protein of JNK, were increased by SB203580. In contrast, SB203580 activated ERK but not JNK in HepG2, HL-60, Saos-2 and HaCaT human cancer cell lines. We tested, whether the effects of SB203580 are due to metabolism. Using liquid chromatography/mass spectrometry, we found one minor metabolite in human liver microsomes but not in HepG2 cells. These data imply that biotransformation could be responsible for the effects of SB203580 in human hepatocytes. This study is the first report on the effects of MAPK activators (sorbitol, anisomycin, EGF) and MAPK inhibitors in primary human hepatocytes. We observed differential effects of these compounds in primary human hepatocytes and in cancer cells, implying the cell-type specificity and the essential differences between the role and function of MAPKs in normal and cancer cells.
Article
The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified New circulator gassed with 95% O2 + 5% CO2 was 1.5 hr.; and when gassed with 20% O2 + 5% CO2 + 75% N2, about 2 hr. In Petri dishes gassed with 20% O2 + 5% CO2 + 75% N2, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at 0 degrees C and about 9% per day when stored at 5 degrees C. When medium with an initial content of 300 microng per ml was stored at room temperature, the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which allows the provision of a relatively constant level of L-ascorbic acid to explant by taking advantage of the slow oxidation of L-ascorbic acid at 0 degrees C.
Article
DNA binds Fe(II) in a manner such that OH formation from H2O2 is enhanced as compared to the amount formed in the absence of DNA. Hydroxyl free radical formation was assayed using the spin-trap DMPO (5,5-dimethyl pyrroline-1-oxide). The maximum ȮH formation occurs at an added Fe(II) per nucleotide ratio of about . The ȮH formed from H2O2 by the DNA-Fe(II) complex attacked DNA producing aldehydes as determined by the thiobarbituric acid method. These data implicate DNA strand breaks induced by ȮH attack.
Article
A cytosolic protein, named iron-responsive element-binding protein (IRE-BP), is sensitive to cellular iron concentration. At low cytosolic iron level, IRE-BP is activated and binds to stem-loop untranslated regions (IRE regions) of transferrin and ferritin mRNAs, activating and inhibiting their translations, respectively. This concerted mechanism permits a fine control of iron homeostasis in the cell. The activity of IRE-BP can be measured by its binding to IRE regions, using a protein band-shift electrophoretic assay. Damage to cells by oxidative stress is known to be mediated by iron. We observed that IRE-BP is rapidly activated by exposure of V79 Chinese hamster ovary cells to H2O2. However, if cell extracts are exposed to H2O2 IRE-BP activation is not observed. Therefore, the activation is not a direct consequence of the H2O2 attack to IRE-BP. The in vivo IRE-BP-activation by H2O2 is not prevented by hydroxyl radical scavengers or by the iron chelator 1,10-phenanthroline, indicating that Fenton reaction is not involved in the process. In fact, simultaneous exposure of cells to H2O2 and 1,10-phenanthroline produces an even stronger activation than exposure to H2O2 alone. The interpretation of the mechanism of IRE-BP activation by oxidative stress is hampered by the fact that the mechanism of IRE-BP modulation by cytosolic iron has not been established. It has been recently shown that the iron-sulfur cluster in IRE-BP must be completely disassembled in order for activation to occur and that this is triggered by low iron in the cell. It is likely that IRE-BP senses Fe(II) and that its oxidation to Fe(III) by H2O2 or chelation by 1,10-phenanthroline set up a program for increasing iron uptake. The physiological consequences of this activation still has to be assessed.
Article
Ascorbic acid and its related compounds were compared for their ascorbyl radical intensity and apoptosis-inducing activity. Sodium L-ascorbate, L-ascorbic acid, D-isoascorbic acid, sodium 6-beta-O-galactosyl-L-ascorbate and sodium 5,6-benzylidene-L-ascorbate, at the concentration of 1-10 mM, induced apoptotic cell death characterized by cell shrinkage, nuclear fragmentation and internucleosomal DNA cleavage in human promyelocytic leukemic HL-60 cells. On the other hand, L-ascorbic acid-2-phosphate magnesium salt and L-ascorbic acid 2-sulfate did not induce any of these apoptosis-associated characteristics. ESR measurements revealed that all the active compounds were progressively degraded, producing the ascorbyl radical (g = 2.0064, hfc = 0.17 mT) in culture medium, whereas the inactive compounds were stable and did not produce the ascorbyl radical. Cytotoxicity began to appear when the radical intensity exceeded a certain threshold level. In the presence of N-acetyl-L-cysteine, both ascorbyl radical intensity and apoptosis-inducing activity were significantly reduced. These data suggest the possible involvement of the ascorbyl radical in apoptosis induction by ascorbic acid-related compounds. Exposure of HL-60 cells to ascorbic acid or its active derivatives resulted in the rapid elevation of intracellular Ca2+ concentration, which might serve as the initial signal leading to the cell death pathway.
Article
Ascorbate acts both as an antioxidant and as an oxidant, depending upon the environment in which the molecule is present. We have reported that millimolar concentrations of ascorbate induced apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and internucleosomal DNA cleavage, in human myelogenous leukemic cell lines. Ascorbate derivatives, which can induce the apoptosis, produced the radical(s), elevated the oxidation potential and stimulated the methionine oxidation in the culture medium, whereas inactive derivatives did not. This suggests that the ascorbate induce the apoptosis by its prooxidant action. The effects of various factors, such as temperature, pH, metal, metal antagonist, redox agent, serum protein, polyphenol and (natural, chemically modified) polysaccharide on the radical intensity and apoptosis-inducing activity of ascorbate are reviewed. Gallate and benzo[a]phenothiazine derivatives, which can induce apoptosis or monocytic differentiation in human myelogenous leukemic cell lines, also produced radicals. These data suggest the significant role of radicals in the initiation of diverse biological activities.
Article
Telomeres in eukaryotic somatic cells are destined to the age-dependent shortening, which has not been demonstrated to correlate to direct lesion of telomeric DNA by reactive oxygen intermediates (ROI); still less explicable is the inhibitory effect of ROI-scavenging on telomere shortening. Here, we succeeded in artificial slowdown of age-dependent telomere shortening to 52-62% of the untreated control, in human vascular endothelial cells, by addition of the oxidation-resistant type of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), which concurrently achieved both extension of cellular life-span and prevention of cell size enlargement indicative of cellular senescence. The results are attributable to a 3.9-fold more marked enrichment of intracellular Asc (Asc(in)) by addition of Asc2P, subsequently dephosphorylated before or during transmembrane influx, than by addition of Asc itself, and also attributed to diminution of intracellular ROI to 53% of the control level by Asc2P; telomerase activity was at a trace level and underwent an age-dependent decline, which was significantly decelerated by Asc2P. Thus, age-dependent telomere-shortening can be decelerated by suppression of intracellular oxidative stress and/or by telomerase retention, both of which are achieved by enriched Asc(in) but not by extracellular Asc overwhelmingly more abundant than Asc(in).
Article
The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 microM extracellular ascorbate, and by pre-incubation of cells with 100 microM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 microM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.
Article
Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H2O2, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 +/- 39 microM H2O2 in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 +/- 17 microM H2O2 is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H2O2 at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H2O2 at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.
Article
Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.
Article
Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H2O2), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H2O2 production. H2O2 generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37 degrees C under 95% air - 5% CO2 was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferriox-amine, but partial inhibition by EDTA and diethylene-triaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37 degrees C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO2. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H2O2 generated by ascorbate and phenolic compounds, but there was still substantial H2O2 generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H2O2 generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H2O2 generation is discussed.
Article
Reactive oxygen species (ROS) have been considered for some time only in the context of oxidative stress-induced cell damage. In this review, we discuss the growing body of evidence that implicates ROS in general, and hydrogen peroxide (H2O2) in particular, in regulatory events underlying synaptic plasticity. H2O2 is regarded in this context as a specific diffusible signaling molecule. The action of H2O2 is assumed to be carried out via the release of calcium ions from internal stores, modulating the activity of specific calcium-dependent protein phosphatases. These phosphatases eventually affect neuronal plasticity. We discuss the role of H2O2 in these systems, stressing the importance of cellular regulation of H2O2 levels that are altered in aging individuals, in the ability to express plasticity. These studies highlight the function of H2O2 in processes of learning and memory and their change in elderly individuals, irrespective of neurodegeneration found in Alzheimer's patients.
Article
The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.
Article
Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose- and time-dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate-induced apoptosis. Indeed, sodium ascorbate-induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down-regulation of TfR expression and this precedes sodium ascorbate-induced apoptosis. Taken together, sodium ascorbate-mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down-regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate-induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.
Article
Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.
Article
In the presence of oxygen, ascorbic acid (AA) is unstable in aqueous media and oxidises to dehydroascorbate (DHA), generating reactive intermediates such as ascorbate free radical and H2O2. It is proposed that the cytotoxicity of AA is due to the extracellular production of H2O2 and that this is mediated by transition metal ions present in cell media. Here we investigate the role of extracellular H2O2 and metal ions in the genotoxicity of AA in cell culture models. Our preliminary results confirmed that physiological concentrations of AA were not toxic to confluent human fibroblasts, although they inhibited the proliferation of cells at low density. No inhibition was observed with ascorbic acid 2-phosphate (AA2P), a vitamin C derivative that remains stable in culture media. Furthermore, high concentrations of AA induced DNA strand breakage in a dose-dependent manner, whereas DHA and AA2P were not genotoxic. The genotoxic effect of AA was transient, required the formation of extracellular H2O2 and the presence of intracellular iron, but not of extracellular transition metal ions. These observations further clarify the pro-oxidant effect of AA solutions in cell culture models. The possibility that intravenous administration of high-dose AA may cause a similar genotoxic effect in vivo is discussed.
Article
Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.
Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells
  • Kotamraju