Yeong Seok Kim

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.

Endoplasmic Reticulum Stress-Mediated Apoptosis of EBV-Transformed B Cells by Cross-Linking of CD70 Is Dependent upon Generation of Reactive Oxygen Species and Activation of p38 MAPK and JNK Pathway

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70+ tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca2+ chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca2+, but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

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.

Interleukin-18 increases metastasis and immune escape of stomach cancer via the downregulation of CD70 and maintenance of CD44

Cancer cells metastasize to the other site after escaping from the immune system and CD70, CD44 and vascular endothelial growth factor (VEGF) play important roles in this process. It is recently reported that interleukin (IL)-18 is closely related with the pathogenesis of skin tumor. Therefore, we investigated the role of endogenous IL-18 from stomach cancer on the immune escape mechanism and metastasis via the regulation of CD70, CD44 and VEGF expression. IL-18 and IL-18R expressions were not only investigated on tumor tissues (n = 10), and sera (n = 20) from stomach cancer patients, but also on human stomach cancer cell lines. IL-18 and IL-18R expressions were found on stomach cancer cell lines and tumor tissues. In addition, IL-18 levels were elevated in sera from cancer patients (P < 0.05), compared with sera from normal individuals. Changes in CD70, CD44 and VEGF expression by flow cytometry, immunoblotting and enzyme-linked immunosorbent assay and immune susceptibility by (51)Cr-release assay were investigated, after silencing or neutralization of endogenous IL-18. CD70 expression was increased and it increases immune susceptibility of cancer cells. In contrast, CD44 and VEGF expression was decreased and it suppresses neovascularization and the metastasis of stomach cancer. After inoculation of IL-18 small interfering RNA (siRNA)-transfected stomach cancer cells into Balb/C (nu/nu) mice, regression of tumor mass was determined by measuring of tumor size. And the number and location of metastatic lesions were investigated by hematoxylin and eosin staining. The regression of tumor mass and the suppression of metastasis were observed in the mice, which are injected with IL-18 siRNA-transfected cell lines. Our data suggest that endogenous IL-18 might facilitate stomach cancer cell immune escape by suppressing CD70 and increasing metastatic ability by upregulating CD44 and VEGF.

Cross-Linking of B7-H1 on EBV-Transformed B Cells Induces Apoptosis through Reactive Oxygen Species Production, JNK Signaling Activation, and fasL Expression

B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.

ROS-mediated JNK/p38-MAPK activation regulates Bax translocation in Sorafenib-induced apoptosis of EBV-transformed B cells

Sorafenib (SRF) is a multi-kinase inhibitor that has been shown to have antitumor activity against several types of cancers, but the effect of SRF on EBV-transformed B cells is unknown. We report that SRF can induce the apoptosis of EBV-transformed B cells through JNK/p38-MAPK activation. SRF triggered the generation of reactive oxygen species (ROS), translocation of Bax into the mitochondria, disruption of mitochondrial membrane potential, activation of caspase-9, caspase-3 and PARP, and subsequent apoptosis. Moreover, we found that SRF exposure activated the phosphorylation of JNK and p38-MAPK and suppressed the phosphorylation of PI3K-p85 and Akt. N-acetyl-l-cysteine (NAC) inhibited the activation of JNK and p38-MAPK. SP600125 and SB203580 blocked apoptosis and mitochondrial membrane disruption but did not affect ROS production after SRF treatment. These findings provide novel insights into the molecular mechanisms driving SRF-mediated cell death and suggest that SRF could be a potential therapeutic drug for the treatment of EBV-related malignant diseases.

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.

Selenium Inhibits Metastasis of Murine Melanoma Cells through the Induction of Cell Cycle Arrest and Cell Death

Background Melanoma is the most fatal form of skin cancer due to its rapid metastasis. Recently, several studies reported that selenium can induce apoptosis in melanoma cells. However, the precise mechanism remains to be elucidated. In this study, we investigated the effect of selenium on cell proliferation in murine melanoma and on tumor growth and metastasis in C57BL/6 mice. Methods Cell proliferation was measured by MTT assay in selenium-treated melanoma cells. Cell cycle distribution was analysized by staining DNA with propidum iodide (PI). mRNA and protein expression related to cell cycle arrest was measured by reverse transcription PCR and western blot. Tumor growth and metastasis was measured by in vivo model. Results Selenium was suppressed the proliferation of melanoma cells in a dose dependent manner. The growth inhibition of melanoma by selenium was associated with an arrest of cell cycle distribution at G0/G1 stage. The mRNA and protein level of CDK2/CDK4 was suppressed by treatment with selenium in a time-dependent manner. In vivo, tumor growth was not suppressed by selenium; however tumor metastasis was suppressed by selenium in mouse model. Conclusion These results suggest that selenium might be a potent agent to inhibit proliferative activity of melanoma cells.

Selenium inhibits migration of murine melanoma cells via down-modulation of IL-18 expression

Melanoma is an aggressive form of skin cancer due to its rapid metastasis. Recently, several studies have reported that selenium can prevent metastasis of melanoma cells, but the mechanism of this anti-metastatic ability is not fully understood. In this study, we investigated the effect of selenium on cell migration in melanoma and on tumor metastasis in mice. Interestingly, tumor metastasis was suppressed by selenium in a mouse model. Cell migration was measured by a wound-healing assay using selenium-treated melanoma cells. Treatment with a non-cytotoxic concentration of selenium suppressed migration of melanoma cells in a dose-dependent manner. In addition, we found decreased HIF-1α and VEGF expression in selenium-treated melanoma cells as compared to non-treated control cells. Mechanistically, our studies show that selenium inhibits IL-18 gene expression in a dose-dependent manner. IL-18 protein level was suppressed by treatment with selenium. The wound-healing assay revealed that the anti-metastatic effect of selenium was abrogated by treatment with exogenous IL-18. These results suggest that selenium might be a potent inhibitor of the metastatic capacity of melanoma cells, via down-modulation of IL-18 expression.

Reactive oxygen species and p38 MAPK regulate Bax translocation and calcium redistribution in salubrinal-induced apoptosis of EBV-transformed B cells.

Salubrinal is a specific eIF2α phosphatase inhibitor that inhibits ER stress-mediated apoptosis. However, maintaining hyper-phosphorylated eIF2α state with high doses of salubrinal treatment promotes apoptosis in some cancer cells. In this report, we found that salubrinal induced apoptosis of EBV-transformed B cells. Notably, salubrinal induced ROS generation and p38 MPAK activation, which then induced expression of FasL. Moreover, salubrinal subsequently led to activation of caspases, calcium redistribution, Bax translocation, cytochrome c release, and apoptosis. These findings suggest that salubrinal may be a novel therapeutic approach for EBV-associated malignant diseases.