The enhanced tumor inhibitory effects of gefitinib and L-ascorbic acid combination therapy in non-small cell lung cancer cells.

Despite documentation of successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with lung cancer, the response rate of patients treated with this therapy remains low. The present study investigated whether L-ascorbic acid serves an adjuvant role in vitro when combined with the EGFR tyrosine kinase inhibitor gefitinib (Iressa®) in lung cancer cell lines. A total of three human lung cancer cell lines were used. The antiproliferative effects and changes in the cell cycle and expression of intracellular signaling molecules, including extracellular signal-regulated kinases (Erk), signal transducer and activator of transcription 3 (Stat3) and protein kinase B (Akt), were measured in cells treated with gefitinib and/or L-ascorbic acid at various concentrations. When combined with gefitinib, L-ascorbic acid exhibited an additive effect on cell proliferation in all gefitinib-sensitive and gefitinib-resistant cell lines. A decrement of ~40% was observed with a low dose 0.5 mM L-ascorbic acid and gefitinib in the relatively gefitinib-resistant A549 cell line (85.6±5.4% with gefitinib alone vs. 52.7±7.3% with combination therapy; P=0.046). The downregulation of intracellular signaling cascades, including EGFR, Akt, Erk and Stat3, was also observed. L-Ascorbic acid serves an adjuvant role when administered in combination with gefitinib; however, the degree of inhibition of cell proliferation differs between lung cancer cell lines.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro.

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers.

Red ginseng and vitamin C increase immune cell activity and decrease lung inflammation induced by influenza A virus/H1N1 infection.

OBJECTIVES: Because red ginseng and vitamin C have immunomodulatory function and anti-viral effect, we investigated whether red ginseng and vitamin C synergistically regulate immune cell function and suppress viral infection. METHODS: Red ginseng and vitamin C were treated to human peripheral blood mononuclear cells (PBMCs) or sarcoma-associated herpesvirus (KSHV)-infected BCBL-1, and administrated to Gulo(-/-) mice, which are incapable of synthesizing vitamin C, with or without influenza A virus/H1N1 infection. KEY FINDINGS: Red ginseng and vitamin C increased the expression of CD25 and CD69 of PBMCs and natural killer (NK) cells. Co-treatment of them decreased cell viability and lytic gene expression in BCBL-1. In Gulo(-/-) mice, red ginseng and vitamin C increased the expression of NKp46, a natural cytotoxic receptor of NK cells and interferon (IFN)-γ production. Influenza infection decreased the survival rate, and increased inflammation and viral plaque accumulation in the lungs of vitamin C-depleted Gulo(-/-) mice, which were remarkably reduced by red ginseng and vitamin C supplementation. CONCLUSIONS: Administration of red ginseng and vitamin C enhanced the activation of immune cells like T and NK cells, and repressed the progress of viral lytic cycle. It also reduced lung inflammation caused by viral infection, which consequently increased the survival rate.

Hepatoprotective effect of vitamin C on lithocholic acid-induced cholestatic liver injury in Gulo(-/-) mice.

Prevention and restoration of hepatic fibrosis from chronic liver injury is essential for the treatment of patients with chronic liver diseases. Vitamin C is known to have hepatoprotective effects, but their underlying mechanisms are unclear, especially those associated with hepatic fibrosis. Here, we analyzed the impact of vitamin C on bile acid induced hepatocyte apoptosis in vitro and lithocholic acid (LCA)-induced liver injury in vitamin C-insufficient Gulo(-/-) mice, which cannot synthesize vitamin C similarly to humans. When Huh-BAT cells were treated with bile acid, apoptosis was induced by endoplasmic reticulum stress-related JNK activation but vitamin C attenuated bile acid-induced hepatocyte apoptosis in vitro. In our in vivo experiments, LCA feeding increased plasma marker of cholestasis and resulted in more extensive liver damage and hepatic fibrosis by more prominent apoptotic cell death and recruiting more intrahepatic inflammatory CD11b(+) cells in the liver of vitamin C-insufficient Gulo(-/-) mice compared to wild type mice which have minimal hepatic fibrosis. However, when vitamin C was supplemented to vitamin C-insufficient Gulo(-/-) mice, hepatic fibrosis was significantly attenuated in the liver of vitamin C-sufficient Gulo(-/-) mice like in wild type mice and this hepatoprotective effect of vitamin C was thought to be associated with both decreased hepatic apoptosis and necrosis. These results suggested that vitamin C had hepatoprotective effect against cholestatic liver injury.

Vitamin C Deficiency Causes Severe Defects in the Development of the Neonatal Cerebellum and in the Motor Behaviors of Gulo(-/-) Mice.

AIMS: The developing brain of a neonate is particularly susceptible to damage by vitamin C deficiency because of its rapid growth and immature antioxidant system. Cognitive impairment and sensory motor deficits are found in the adult brain upon vitamin C deficiency. Therefore, the aim of this study was to clarify the role of vitamin C in its own right and its related mechanisms in Gulo(-/-) mice incapable of synthesizing vitamin C. RESULTS: When vitamin C supplementation was ceased for 2 weeks until delivery, stillbirths and a significant reduction in neonatal mice were observed and the growth of neonates was remarkably decreased. In addition, intraparenchymal hemorrhages were found in most of the brains, especially in the stillborn neonates. In addition, the levels of malondialdehyde (MDA) and 8-isoprostanes were increased and structural abnormalities were found in the cortex, hippocampus, and cerebellum. Especially, vitamin C deficiency caused the failure of or a delay in the formation of cerebellar fissures accompanied by abnormal foliation and altered Purkinje cell alignment. In the developed adult brains from vitamin C-deficient Gulo(-/-) mice, the levels of glutathione, MDA, nitrate, IL-6, TNF-α, and Bax were increased and the expression of the GABRA6 and calbindin-28k was decreased. Due to atrophy of the granule and Purkinje cells, the motor behavior of vitamin C-deficient Gulo(-/-) mice declined. INNOVATION AND CONCLUSION: Vitamin C deficiency during gestation induces intraparenchymal hemorrhages and severe defects in the development of the cerebellum. In fully developed brains, it induces the functional impairment by altering the cellular composition in the cerebellum.

Chronic vitamin C insufficiency aggravated thioacetamide-induced liver fibrosis in gulo-knockout mice.

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.

Vitamin C prevents stress-induced damage on the heart caused by the death of cardiomyocytes, through down-regulation of the excessive production of catecholamine, TNF-α, and ROS production in Gulo(-/-)Vit C-Insufficient mice.

It is thought that vitamin C has protective roles on stress-induced heart damage and the development of cardiovascular diseases, but its precise role and mechanisms are unclear. In the present study, we investigated the specific mechanisms by which vitamin C leads to protecting the heart from stress-induced damage in the Gulo(-/-) mice which cannot synthesize vitamin C like humans. By exposure to stress (1h/day), the heartbeat and cardiac output in vitamin C-insufficient Gulo(-/-) mice were definitely decreased, despite a significant increase of adrenaline (ADR) and noradrenaline (NA) production. A change of cardiac structure caused by the death of cardiomyocytes and an increased expression of matrix metalloprotease (MMP)-2 and -9 were also found. Moreover, lipid peroxidation and the production of tumor necrosis factor-alpha (TNF-α) in the heart were increased. Finally, all vitamin C-insufficient Gulo(-/-) mice were expired within 2 weeks. Interestingly, all of the findings in vitamin C-insufficient Gulo(-/-) mice were completely prevented by the supplementation of a sufficient amount of vitamin C. Taken together, vitamin C insufficiency increases the risk of stress-induced cardiac damage with structural and functional changes arising from the apoptosis of cardiomyocytes.

In vivo consequence of vitamin C insufficiency in liver injury: vitamin C ameliorates T-cell-mediated acute liver injury in gulo(-/-) mice.

AIM: l-ascorbic acid (vitamin C) insufficiency is considered one of the major risk factors for the development of liver disease. However, its specific effects and related mechanisms in vivo are largely unknown. The objective of this study was to investigate the in vivo protective role of vitamin C and its related mechanisms in liver injury with Gulo(-/-) mice that cannot synthesize vitamin C like humans due to the lack of l-gulonolactone-γ-oxidase (Gulo), an essential enzyme for vitamin C synthesis. RESULTS: When liver injury was induced in Gulo(-/-) mice by injection of concanavalin A (Con A), there was greater extensive liver damage accompanied by an increased number of apoptotic hepatocytes in vitamin C-insufficient Gulo(-/-) mice. Additionally, the plasma and hepatic levels of the proinflammatory cytokines, such as TNF-α and IFN-γ, were much higher in the vitamin C-insufficient Gulo(-/-) mice than in the control mice. Moreover, increased numbers of liver-infiltrating T-cells in the vitamin C-insufficient Gulo(-/-) mice were related to the increased hepatic levels of IFN-inducible factor (IP-10). Although the vitamin C-insufficient Gulo(-/-) mice had higher amounts of interleukin-22 (IL-22), a hepatoprotective cytokine, a defect in IL-22Rα expression and its downstream STAT3 activation in hepatocytes were found. INNOVATION: We first demonstrate the novel in vivo action mechanisms of vitamin C on the prevention of disease development in the liver, through the regulation of excessive immune activation and maintenance of the IL-22Rα signaling pathways. CONCLUSION: These results suggest that severe liver damage induced by inflammation could be prevented by sufficient supplementation with vitamin C.

The analysis of vitamin C concentration in organs of gulo(-/-) mice upon vitamin C withdrawal.

BACKGROUND: Vitamin C is an essential nutrient for maintaining human life. Vitamin C insufficiency in the plasma is closely related with the development of scurvy. However, in vivo kinetics of vitamin C regarding its storage and consumption is still largely unknown. METHODS: We used Gulo(-/-) mice, which cannot synthesize vitamin C like human. Vitamin C level in plasma and organs from Gulo(-/-) mice was examined, and it compared with the level of wild-type mice during 5 weeks. RESULTS: The significant weight loss of Gulo(-/-) mice was shown at 3 weeks after vitamin C withdrawal. However, there was no differences between wild-type and vitamin C-supplemented Gulo(-/-) mice (3.3 g/L in drinking water). The concentration of vitamin C in plasma and organs was significantly decreased at 1 week after vitamin C withdrawal. Vitamin C is preferentially deposited in adrenal gland, lymph node, lung, and brain. There were no significant changes in the numbers and CD4/CD8 ratio of splenocytes in Gulo(-/-) mice with vitamin C withdrawal for 4 weeks. And the architecture of spleen in Gulo(-/-) mice was disrupted at 5 weeks after vitamin C withdrawal. CONCLUSION: The vitamin C level of Gulo(-/-) mice was considerably decreased from 1 week after vitamin C withdrawal. Vitamin C is preferentially stored in some organs such as brain, adrenal gland and lung.

Depletion of ascorbic acid impairs NK cell activity against ovarian cancer in a mouse model.

Ascorbic acid (Vitamin C) administration has been used to prevent infectious diseases in public or as a therapeutic agent by the physicians in treatment of several diseases. Ascorbic acid is also involved in immune cell functions and immune responses, although the mechanisms by which it exerts effects on immune cells against cancer cells are not fully understood at the normal plasma level. In this study, we used the mice lacking l-gulono-γ-lactone oxidase (Gulo), the enzyme required for the biosynthesis of ascorbic acid, to characterize the effects of ascorbic acid on NK cell cytotoxicity against ovarian cancer cells, MOSECs (murine ovarian surface epithelial cells). Gulo(-/-) mice depleted of ascorbic acid survived for a shorter time than the normal control or Gulo(-/-) mice supplemented with ascorbic acid after tumor challenge regardless of treatment with IL-2. CD69 and NKG2D expression was clearly reduced in NK cells isolated from mice depleted of ascorbic acid as compared to that in the normal control and the mice supplemented with ascorbic acid. We also observed that IFN-γ secretion by NK cells isolated from Gulo(-/-) mice depleted of ascorbic acid was decreased after NK cells were co-cultured with MOSECs. Furthermore, the mRNA expression of perforin and granzyme B genes was also significantly decreased in NK cells isolated from mice depleted of ascorbic acid. Taken together, our results suggest that ascorbic acid at the normal plasma concentration has an essential role in maintaining the NK cytotoxicity against cancer cells.

Iris Nertschinskia ethanol extract differentially induces cytotoxicity in human breast cancer cells depending on AKT1/2 activity.

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

MicroRNA expression profiles are altered by gonadotropins and vitamin C status during in vitro follicular growth.

MicroRNAs (miRs) are known to repress target genes at posttranscriptional level and play important roles in the maturation of cells. However, the expression profiles of miRs during follicular maturation have not been fully elucidated. This study was designed to investigate the expression profiles of miRs in murine follicles according to human chorionic gonadotropin (hCG) treatment and vitamin C status during in vitro culture. Ovaries were removed from the 12-day-old wild-type and vitamin C-deficient (L-gulonogammalactone oxidase knockout, Gulo-/-) C57BL6 mice. Preantral follicles were isolated and cultured in 20 µL droplets of culture media supplemented with follicle-stimulating hormone and luteinizing hormone (FSH + LH). After their full maturation, follicles were divided into 2 groups: with and without hCG treatment. Real-time polymerase chain reaction (PCR) was performed using oocytes and granulosa cells (G-cells) to evaluate the miRs known to be expressed mainly in the mouse ovary. After the addition of hCG, miR profiles showed divergent changes between oocytes and G-cells. These profiles significantly differed from those of hCG(-) group. Compared to wild type, Gulo-/- mice showed altered miR profiles in matured oocytes and G-cells. Conclusively, hCG supplementation and vitamin C status alter the miR expression profiles in oocytes and G-cells during in vitro growth of murine follicles.

Mega-dose vitamin C attenuated lung inflammation in mouse asthma model.

Asthma is a Th2-dependent disease mediated by IgE and Th2 cytokines, and asthmatic patients suffer from oxidative stresses from abnormal airway inflammation. Vitamin C is a micro-nutrient functioning as an antioxidant. When administered at a mega-dose, vitamin C has been reported to shift immune responses toward Th1. Thus, we tried to determine whether vitamin C exerted beneficial effects in asthma animal model. Asthma was induced in mice by sensitizing and challenging with ovalbumin. At the time of challenge, 3~5 mg of vitamin C was administered and the effects were evaluated. Vitamin C did not modulate Th1/Th2 balance in asthma model. However, it decreased airway hyperreactivity to methacholine, decreased inflammatory cell numbers in brochoalveolar lavage fluid, and moderate reduction of perivascular and peribronchiolar inflammatory cell infiltration. These results suggest that vitamin C administered at the time of antigen challenge exerted anti-inflammatory effects. Further studies based on chronic asthma model are needed to evaluate a long-term effect of vitamin C in asthma. In conclusion, even though vitamin C did not show any Th1/Th2 shifting effects in this experiment, it still exerted moderate anti-inflammatory effects. Considering other beneficial effects and inexpensiveness of vitamin C, mega-dose usage of vitamin C could be a potential supplementary modality for the management of asthma.

Vitamin C supplementation prevents testosterone-induced hyperplasia of rat prostate by down-regulating HIF-1alpha.

Benign prostatic hyperplasia (BPH) is a disease that impairs the well-being of many aged men. To alleviate BPH symptoms or to find a cure for this disease, key molecules should be identified that control prostate cell proliferation. Recently, HIF-1alpha has attracted attention in this context, because it is highly expressed in hyperplasic prostates and prevents prostate cell death. Thus, given that vitamin C inhibits HIF-1alpha expression in several malignant tumors, we examined its therapeutic potential in BPH. HIF-1alpha was noticeably induced by testosterone in prostate cells, and this HIF-1alpha induction was abolished by vitamin C. Vascular endothelial growth factor (VEGF) promoter activity reporter assays and semi-quantitative RT-PCR revealed that vitamin C inhibited HIF-1-dependent VEGF expression. Furthermore, HIF-1alpha suppression by vitamin C was rescued by knocking down HIF-prolyl hydroxylase-2, suggesting that vitamin C destabilizes HIF-1alpha via prolyl hydroxylation. Moreover, vitamin C treatment abolished cell proliferation induced by testosterone treatment to the control level. These results suggest that vitamin C inhibits testosterone-induced HIF-1alpha expression and by so doing effectively prevents prostate hyperplasia. In male rats, testosterone treatment for 4 weeks induced prostate hyperplasia. Furthermore, HIF-1alpha and VEGF levels were significantly elevated in hyperplasic prostates. In vitamin C-treated rats, however, most prostate hyperplasia parameters and prostrate HIF-1alpha/VEGF levels were markedly reduced. Accordingly, our findings indicate that vitamin C could be further developed clinically for use as an anti-BPH agent.

Mega-dose Vitamin C modulates T cell functions in Balb/c mice only when administered during T cell activation.

Previously we reported that a mega-dose of Vitamin C enhanced the initial stage of delayed-type hypersensitivity reaction in Balb/c mice. In this study its effects were further evaluated as follows. Mice were administered Vitamin C intraperitoneally at 0.625 mg/day or at 5mg/day for variable days before, during, or after being sensitized with DNFB. T cells were isolated in each group and examined. When stimulated antigen-specifically or non-specifically in vitro, mice showed elevated thymidine uptake and a shift of cytokine secretion profiles toward Th1, i.e., elevated levels IL-2, TNF-alpha, and IFN-gamma, and lowered level of the Th2 cytokine IL-4, only when Vitamin C was administered during sensitization. T cells from those mice administered Vitamin C before sensitization or after challenge showed the same T cell properties as those from PBS-treated mice. Mice were also given 0.625 mg/day of Vitamin C during primary and/or secondary immunizations with KLH and secondary specific antibody titers in sera were measured. The total specific antibody titer was lowered in Vitamin C-treated animals whenever treatments were administered, and this was entirely attributed to decreased levels of IgG1 and IgE antibodies. Based on these results, we suggest that an exogenously administered mega-dose of Vitamin C shifts immunity in Balb/c mouse toward Th1 and that these affects occur only when Vitamin C is administered during T cell activation.