Ga Bin Park

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.

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.

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.

The Epstein-Barr Virus Causes Epithelial–Mesenchymal Transition in Human Corneal Epithelial Cells Via Syk/Src and Akt/Erk Signaling Pathways

PURPOSE Although Epstein-Barr virus (EBV)-associated keratitis is rare, it can cause acute corneal necrosis and neovascularization. We aimed to examine the signaling mechanism by which EBV causes epithelial-mesenchymal transition (EMT) in human corneal epithelial cells (HCECs) in vitro. METHODS The cellular response to EBV was assessed by real-time PCR, Western blot, migration assay, invasion assay, inhibitor assay, and ELISA assay. RESULTS A model of EBV-induced EMT was established in HCECs. The EBV induced morphologic changes in the cells; the loss of epithelial markers E-cadherin, ZO-1, and β-catenin; and an increase in the mesenchymal markers N-cadherin, Vimentin, Snail, and TCF8/Zeb1. The EBV infection also led to the nuclear translocation of Snail and TCF8/Zeb1; enhanced the secretion of IL-6, IL-8, VEGF, TGF-β1, TNF-α, and MCP-1; and upregulated the expression of MMP2 and MMP9. The EBV-infected HCECs exhibited increased migration and invasiveness compared to uninfected HCECs. We measured the involvement of Syk, Src, PI3K/Akt, and Erk signaling, but not Smad, in EMT by EBV-induced TGF-β1. We demonstrated that treatment with TGF-β1, TGF-β receptors, Syk, or Src inhibitor blocked TGF-β1, Syk, or Src signaling activation, and EMT development by EBV. Moreover, these inhibitors prevented PI3K/Akt and Erk activation. CONCLUSIONS An EBV infection in HCECs can lead to a mesenchymal fibroblast-like morphology, and cause EMT through the activation of PI3K/Akt and Erk by TGF-β1-mediated Syk and Src signaling. This phenomenon may have implications for EBV-associated keratitis and molecular approaches to treatment.

Cell cycle arrest induced by engagement of B7-H4 on Epstein-Barr virus-positive B-cell lymphoma cell lines.

B7‐H4 is a recently discovered B7 family member that has inhibitory effects on T‐cell immunity. However, the reverse signalling mechanism of the B7‐H4‐expressing cells remains unclear. Previous work has shown that B7‐H4 expression was enhanced on B cells following Epstein–Barr virus (EBV) infection, and engagement of cell‐surface‐expressed B7‐H4 induces cell death of EBV‐transformed B cells. Here we found that B7‐H4 was constitutively expressed on EBV‐positive lymphoma cells, Raji and IM‐9 cells, but was not expressed on EBV‐negative lymphoma cells (Ramos). Engagement of B7‐H4 significantly reduced cell growth of Raji and IM‐9 cells and resulted in cell cycle arrest at G0–G1 phase in a dose‐ and time‐dependent manner. To clarify the mechanism of cell cycle arrest via activation of B7‐H4, cell cycle regulatory factors were examined by reverse transcription–polymerase chain reaction and immunoblotting. We found that B7‐H4 triggered down‐regulation of CDK4/6 and up‐regulation of p21 expression at both protein and RNA levels. Furthermore, CDK2 and cyclin E/D expression was down‐regulated by B7‐H4 triggering. Additionally, the down‐regulation of phospho‐AKT and phospho‐cyclin E were clearly detected in B7‐H4‐activated Raji cells, but the phosphorylation of p53 was constitutively maintained. These results indicate that B7‐H4‐mediated signalling on EBV‐positive B‐cell lymphoma cells modulates the cell cycle through down‐regulation of the AKT pathway. Consequently, B7‐H4 may be a new potential target for use in EBV‐positive lymphoma therapy.

ROS and ERK1/2-mediated caspase-9 activation increases XAF1 expression in dexamethasone-induced apoptosis of EBV-transformed B cells

Dexamethasone (Dex) inhibits the growth of diverse types of cancer cells and is utilized clinically for the therapy of hematological malignancies. In this study, we investigated the molecular mechanisms of Dex action in the apoptosis of Epstein-Barr virus (EBV)-transformed B cells. We showed that Dex inhibited the proliferation of EBV-transformed B cells and induced apoptosis by activating caspase-9, -3 and -8. While activation of caspase-9 was triggered as early as 2 h after Dex treatment, cleavage of caspase-8 was deferred and was found 8 h after the exposure. Dex-dependent activation of caspase-8 was blocked by the specific caspase-9 inhibitor, z-LEHD-fmk. Moreover, Dex significantly increased the expression of X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) and induced the translocation of XAF1 into the cytosol. Cytosolic XAF1 with Puma induced the translocation of Bax into mitochondria. Dex led to up-regulation of reactive oxygen species (ROS) generation and the phosphorylation of ERK1/2 after the exposure. We speculated that ROS generation might be the first event of Dex-induced apoptosis because ROS inhibitor NAC abrogated ROS production and ERK1/2 activation, but PD98059 did not block ROS production. NAC and PD98059 also suppressed the translocation of XAF1, Puma and Bax into mitochondria. These results demonstrated that Dex-mediated activation of caspase-9 via ROS generation and ERK1/2 pathway activation resulted in the activation of caspase-8 and the increment of XAF1, thereby induced apoptosis of EBV-transformed B cells. These findings suggest that Dex constitutes a probable therapy for EBV-associated hematological malignancies.

Silencing of galectin-3 represses osteosarcoma cell migration and invasion through inhibition of FAK/Src/Lyn activationand β-catenin expression and increases susceptibilityto chemotherapeutic agents

Galectin-3 is involved in tumor cell proliferation, adhesion, angiogenesis and metastasis. Galectin-3 promotes β-catenin/Wnt signaling, and β-catenin-related oncogenesis has been frequently reported in osteosarcoma. However, the correlation between galectin-3 and β‑catenin signaling in osteosarcoma is poorly defined. We hypothesized that galectin-3 may control the migration and invasion of cancer cells and that silencing of galectin-3 would therefore, suppress motility in osteosarcoma cells. In the present study, we show that galectin-3 silencing in cultured human osteosarcoma cells had decreased cell migration and invasion capabilities; reduced the expression and activation of FAK, Src, Lyn, PI3K/Akt, ERK1/2 and β-catenin, which are key mediators of invasion; inhibited the expression and secretion of VEGF, MCP-1, IL-8, IL-6, MMP2/9 and phospho-Stat3; and potentiated sensitivity to cisplatin. Our results suggest that galectin-3 may be a feasible therapeutic target for osteosarcoma.