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.

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.

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.