Young Il Hwang

Immunohistochemical study of the distribution of sodium-dependent vitamin C transporters in adult rat brain

Sodium‐dependent vitamin C transporters (SVCTs) is known to transport the reduced form of ascorbic acid into the cell, whereas the oxidized form of vitamin C (VC) is moved through a facilitative sugar transporter, such as glucose transporter (GLUT). With regard to the distribution of SVCT1 and ‐2 within the various organs, they were reported to be expressed in different types of cells. Especially in the central nervous system, only SVCT2 mRNA was expressed mainly in neurons and some types of neuroglial cells. However, data on the expression of SVCT proteins in the brain are scant. Therefore, we tried to develop comprehensive data on the distribution of SVCT proteins in adult rat brain by using immunohistochemical techniques for the first time. In our study, SVCT2 immunoreactivities (IRs) were intensely localized in the neurons of cerebral cortex, hippocampus, and Purkinje cells of cerebellum, and much weaker SVCT2 IRs were found in the other brain regions. Judging from double‐immunohistochemical data, most of the cells expressing SVCT2 IRs were likely to be neurons or microglia, even though the cells in choroids plexus or ependymal cells around the ventricles also exhibited SVCT2 IRs. Complete mapping of the distribution of SVCT2 IRs was available by using a semiquantitative method. The subcellular localization of SVCT proteins is necessary for understanding the exact role of the protein, so the current overall mapping of SVCT IRs in the rat brain could be the basis for further studies on related subjects.

Sodium Ascorbate (Vitamin C) Induces Apoptosis in Melanoma Cells via the Down-Regulation of Transferrin Receptor Dependent Iron Uptake

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.

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway.

Abstractl-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.

The enhanced IL-18 production by UVB irradiation requires ROI and AP-1 signaling in human keratinocyte cell line (HaCaT)

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human keratinocyte cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N-acetyl-L-cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.

The Molecular Mechanisms of Vitamin C on Cell Cycle Regulation in B16F10 Murine Melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose‐dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53‐p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C‐induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53‐p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K‐family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C‐induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2‐p53‐p21Waf1/Cip1 pathway. J. Cell. Biochem. 102: 1002–1010, 2007.

Role of follicular dendritic cells in the apoptosis of germinal center B cells

Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas-ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas-FasL interaction.

Vitamin C downregulates interleukin-18 production by increasing reactive oxygen intermediate and mitogen-activated protein kinase signalling in B16F10 murine melanoma cells.

We recently reported that interleukin-18 (IL-18) is highly expressed in malignant skin tumours such as melanomas, and may play a key role in the malignancy of such tumours. This study was designed to investigate the mechanisms of IL-18 regulation by vitamin C in B16F10 murine melanoma cells. Cells were treated with vitamin C, and the expression of IL-18 was measured by reverse transcription-polymerase chain reaction and intracellular flow cytometry analysis. Decreased IL-18 production and a significant reduction in IL-18 mRNA transcript were detected in cells treated with vitamin C. The effect of vitamin C treatment was blocked by the antioxidant N-acetyl-l-cysteine, suggesting that vitamin C affects IL-18 expression by up-regulating intracellular reactive oxygen intermediate (ROI) levels. To investigate whether the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the downregulation of IL-18 production, cells were pretreated with SB203580, an inhibitor of p38 MAPK, prior to the addition of vitamin C. This pretreatment blocked the decrease in IL-18 production. However, vitamin C treatment enhanced the expression of phosphorylated p38 MAPK. Taken together, we conclude that vitamin C increases intracellular ROI levels, and regulates IL-18 production through the MAPK signalling pathway.

Immunohistochemical localization of sodium-dependent L-ascorbic acid transporter 1 protein in rat kidney

Recently, two l-ascorbic acid transporters were identified; sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2. The previous study suggested that SVCT protein might be present on the apical membrane in the straight segment (S3) of proximal tubule. In the present study, SVCT1 immunoreactivity (IR) was observed in the brush border of proximal straight tubules in the medullary ray of renal cortex and the outer stripe of outer medulla, while SVCT2 IR was not localized in any region of the kidney. Since the mechanism of VC reabsorption in the kidney has not been fully elucidated up to the present time, it is meaningful to demonstrate the exact cellular distribution of SVCT protein in the kidney.

The Anti-inflammatory Effect of GV1001 Mediated by the Downregulation of ENO1-induced Pro-inflammatory Cytokine Production

GV1001 is a peptide derived from the human telomerase reverse transcriptase (hTERT) sequence that is reported to have anti-cancer and anti-inflammatory effects. Enolase1 (ENO1) is a glycolytic enzyme, and stimulation of this enzyme induces high levels of pro-inflammatory cytokines from concanavalin A (Con A)-activated peripheral blood mononuclear cells (PBMCs) and ENO1-expressing monocytes in healthy subjects, as well as from macrophages in rheumatoid arthritis (RA) patients. Therefore, this study investigated whether GV1001 downregulates ENO1-induced pro-inflammatory cytokines as an anti-inflammatory peptide. The results showed that GV1001 does not affect the expression of ENO1 in either Con A-activated PBMCs or RA PBMCs. However, ENO1 stimulation increased the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and these cytokines were downregulated by pretreatment with GV1001. Moreover, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB were activated when ENO1, on the surface of Con A-activated PBMCs and RA PBMCs, was stimulated, and they were successfully suppressed by pre-treatment with GV1001. These results suggest that GV1001 may be an effective anti-inflammatory peptide that downregulates the production of pro-inflammatory cytokines through the suppression of p38 MAPK and NF-κB activation following ENO1 stimulation.

CM1 ligation initiates apoptosis in a caspase 8-dependent manner in Ramos cells and in a mitochondria-controlled manner in Raji cells

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.