Sang-Seok Oh

Vibrio vulnificus VvhA induces NF-κB-dependent mitochondrial cell death via lipid raft-mediated ROS production in intestinal epithelial cells

The Gram-negative bacterium Vibrio vulnificus produces hemolysin (VvhA), which induces cytotoxicity in mammalian cells. However, our understanding of the cytotoxic mechanism and the modes of action of VvhA are still fragmentary and incomplete. The recombinant protein (r) VvhA (50 pg/ml) significantly induces necrotic cell death and apoptosis in human intestinal epithelial (INT-407) cells. The apoptotic cell death induced by rVvhA is highly susceptible to the sequestration of cholesterol by methyl-β-cyclodextrin, whereas for necrotic cell death, this shows a marginal effect. We found that rVvhA induces the aggregation of lipid raft components coupled with NADPH oxidase enzymes, in which rVvhA increased the interaction of NADPH oxidase 2 (NOX2, gp91phox) with a cytosolic protein NCF1 (p47phox) to facilitate the production of reactive oxygen species (ROS). rVvhA uniquely stimulated a conventional PKC isoform PKCα and induced the phosphorylation of both ERK and JNK, which are responsible for the activation of transcription factor NF-κB. rVvhA induced an NF-κB-dependent imbalance of the Bcl-2/Bax ratio, the release of mitochondrial cytochrome c, and caspase-3/-9 activation during its promotion of apoptotic cell death. In addition, rVvhA has the ability to inhibit the expression of cell cycle-related proteins, such as CDK2, CDK4, cyclin D1, and cyclin E. These results demonstrate that rVvhA induces NF-κB-dependent mitochondrial cell death via lipid raft-mediated ROS production by the distinct activation of PKCα and ERK/JNK in intestinal epithelial cells.

6-Acetonyl-5,6-dihydrosanguinarine (ADS) from Chelidonium majus L. triggers proinflammatory cytokine production via ROS-JNK/ERK-NFκB signaling pathway.

Chelidonium majus L. is an herbal plant that is commonly used in Western phytotherapy and traditional Chinese medicine for diuretic, antitussive, eye-regenerative, anti-osteoporotic, and radioprotective purposes. In this study, we purified 6-acetonyl-5,6-dihydrosanguinarine (ADS) from C. majus and investigated its immune-stimulatory effect. We found that ADS has the potential to induce the inflammatory cytokines TNF-α, IL-6, and IL-8 in macrophages and dendritic cells (DCs), that NFκB activation is a critical mediator of ADS-induced cytokine production, and that the activation of NFκB was dependent on reactive oxygen species (ROS). ADS induced phosphorylation of ERK and JNK, which was also associated with NFκB activation; phosphorylarion and cytokine production were inhibited by ROS scavenger and by specific MAPK inhibitors. Taken together, the results suggest that ADS from C. majus, as a positive immune modulator, induces inflammatory cytokines that might improve immunity, via the ROS-ERK/JNK-NFκB pathway.

NDRG2 correlated with favorable recurrence-free survival inhibits metastasis of mouse breast cancer cells via attenuation of active TGF-β production

N-myc downstream-regulated gene 2 (NDRG2) has been studied for its inhibitory effects against growth and metastasis of many tumor cell types. In this study, we showed NDRG2 expression was correlated with favorable recurrence-free survival of patients with breast cancer and inhibited metastasis of breast cancer cells (4T1). NDRG2 expression was examined in 189 breast carcinoma tissues and paired normal breast tissues using immunohistochemistry. Histological and clinicopathological data were correlated using Pearsons chi-square test of independence. NDRG2 expression in human breast cancer tissues was inversely associated with lymph node metastasis and pTNM stage. Furthermore, patients with breast cancer with a high level of NDRG2 expression showed favorable recurrence-free survival (P = 0.038). To study the effect of NDRG2 on metastasis in vivo, we established an NDRG2-overexpressing mouse breast cancer cell line (4T1-NDRG2) and measured the metastasis and survival of 4T1-NDRG2 tumor-bearing mice. To test whether transforming growth factor β (TGF-β)- mediated metastasis of 4T1 was inhibited by NDRG2 expression, TGF-Smad-binding element (SBE)-luciferase activity and/or measurement of active TGF-β were performed in cell or tumor tissue level. 4T1-NDRG2 cells grew gradually and showed less metastatic activity in vivo and low invasiveness in vitro. 4T1-NDRG2 cells showed lower SBE-luciferase activity and lower level of active autocrine TGF-β than 4T1-Mock did. Correctly, our data show that NDRG2 significantly suppress tumor metastasis by attenuating active autocrine TGF-β production, and the attenuation might be typically associated with the favorable recurrence-free survival of patients clinically.

NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34(+) progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.

Arachidonic acid promotes skin wound healing through induction of human MSC migration by MT3-MMP-mediated fibronectin degradation

Arachidonic acid (AA) is largely released during injury, but it has not been fully studied yet how AA modulates wound repair with stem cells. Therefore, we investigated skin wound-healing effect of AA-stimulated human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in vivo and its molecular mechanism in vitro. We found that transplantation of hUCB-MSCs pre-treated with AA enhanced wound filling, re-epithelization, and angiogenesis in a mouse skin excisional wound model. AA significantly promoted hUCB-MSCs migration after a 24 h incubation, which was inhibited by the knockdown of G-protein-coupled receptor 40 (GPR40). AA activated mammalian target of rapamycin complex 2 (mTORC2) and Aktser473 through the GPR40/phosphoinositide 3-kinase (PI3K) signaling, which was responsible for the stimulation of an atypical protein kinase C (PKC) isoform, PKCζ. Subsequently, AA stimulated phosphorylation of p38 MAPK and transcription factor Sp1, and induced membrane type 3-matrix metalloproteinase (MT3-MMP)-dependent fibronectin degradation in promoting hUCB-MSCs motility. Finally, the silencing of MT3-MMP in AA-stimulated hUCB-MSCs failed to promote the repair of skin wounds owing to impaired cell motility. In conclusion, AA enhances skin wound healing through induction of hUCB-MSCs motility by MT3-MMP-mediated fibronectin degradation, which relies on GPR40-dependent mTORC2 signaling pathways.

Adenovirally delivered IFN-β exerts antitumor effects through transient T-lymphocyte depletion and Ag-specific T-cell proliferation

Type I interferons (IFNs), including IFN-β, are known to enhance antigen (Ag) presentation and to promote the expansion, survival and effector function of CD8+ cytotoxic lymphocytes (CTL) during viral infections. Furthermore, IFN-β is a potent candidate for antitumor drugs; however, recombinant IFN-β is too unstable for use in tumor therapy in vivo. In this study, we therefore examined the efficacy and mechanism of exogenous IFN-β as a biomolecule for tumor therapy, using adenovirus encoding IFN-β (Ad-IFNβ) as a therapeutic agent in a mouse model. Ag104Ld and 4T1 tumor cells exposed to Ad-IFNβ showed growth retardation and cell death in vitro, and tumor growth as well as tumor metastasis was inhibited in vivo. The Ad-IFNβ-mediated antitumor effect was dependent on CD8+ T cells in vivo, rather than on a direct cytotoxic effect of Ad-IFNβ. Transient T lymphocyte depletion was observed in tumor tissue after intratumoral injection with Ad-IFNβ. Despite the T lymphocyte depletion, the proliferation of Ag-specific CD8+ T cells was increased in Ad-IFNβ-treated mice compared to control virus-treated mice. These results suggest that IFN-β might contribute to the inhibition of tumor growth by depleting Ag-nonspecific T lymphocytes and enhancing proliferation of Ag-specific CD8+ T cells.

Depigmentation of α-melanocyte-stimulating hormone-treated melanoma cells by β-mangostin is mediated by selective autophagy

Melanogenesis is a key pathway for the regulation of skin pigmentation and the development of skin‐lightening/skin‐whitening drugs or cosmetics. In this study, we found that β‐mangostin from seedcases of Garcinia mangostana inhibited α‐melanocyte‐stimulating hormone (α‐MSH)‐mediated melanogenesis in B16F10 melanoma cells and a three‐dimensional human skin model. β‐Mangostin significantly inhibited the protein level of tyrosinase induced by α‐MSH in UPS (ubiquitin proteasome system)‐independent and lysosome‐dependent manner. The inhibition of autophagy by 3‐methyladenine treatment or ATG5 knockdown effectively recovered premelanosome protein as well as tyrosinase degraded by the β‐mangostin treatment. However, rapamycin, a representative non‐selective autophagy inducer, triggered autophagy in α‐MSH‐stimulated cells, which was characterized by a considerable decrease in p62, but it was unable to inhibit melanogenesis. Melanosome‐engulfing autophagosomes were observed using transmission electron microscopy. Furthermore, previously formed melanin could be degraded effectively in an autophagy‐dependent manner in β‐mangostin‐treated cells. Taken together, our results suggest that β‐mangostin inhibits the melanogenesis induced by α‐MSH via an autophagy‐dependent mechanism, and thus, the depigmentation effect of β‐mangostin may depend on autophagy targeted at the melanosome rather than non‐selective autophagy.

Ciglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Ligand, Inhibits Proliferation and Differentiation of Th17 Cells

Peroxisome proliferator-activated receptor gamma (PPARγ) was identified as a cell-intrinsic regulator of Th17 cell differentiation. Th17 cells have been associated with several autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease (IBD), and collagen-induced arthritis. In this study, we confirmed PPARγ-mediated inhibition of Th17 cell differentiation and cytokine production at an early stage. Treatment with ciglitazone, a PPARγ ligand, reduced both IL-1β-mediated enhancement of Th17 differentiation and activation of Th17 cells after polarization. For Th17 cell differentiation, we found that ciglitazone-treated cells had a relatively low proliferative activity and produced a lower amount of cytokines, regardless of the presence of IL-1β. The inhibitory activity of ciglitazone might be due to decrease of CCNB1 expression, which regulates the cell cycle in T cells. Hence, we postulate that a pharmaceutical PPARγ activator might be a potent candidate for treatment of Th17-mediated autoimmune disease patients.

NDRG2 contributes to cisplatin sensitivity through modulation of BAK-to-Mcl-1 ratio

The downregulation of N-Myc downstream-regulated gene 2 (NDRG2) is known to be associated with the progression and poor prognosis of several cancers. Sensitivity to anti-cancer may be associated with a good prognosis in cancer patients, and NDRG2, which is induced by p53, sensitizes the cells to chemotherapy. However, the unique function of NDRG2 as an inducer of apoptosis under chemotreatment has not been sufficiently studied. In this study, we investigated the role of NDRG2 in chemo-sensitivity, focusing on cisplatin in U937 histiocytic lymphoma, which has the loss-of-functional mutation in p53. NDRG2 promoted the sensitivity to cisplatin through the modulation of the BAK-to-Mcl-1 ratio. The degradation of Mcl-1 and increase in BAK were mediated by JNK activation and the eIF2α/p-eIF2α pathway, respectively, which depended on PKR activation in NDRG2-overexpressed U937 (U937-NDRG2) cells. NOX5 was highly expressed in U937-NDRG2 cells and contributed to ROS production after cisplatin treatment. ROS scavenging or NOX5-knockdown successfully inhibited the sensitivity of U937-NDRG2 cells to cisplatin. Taken together, these findings indicate that NDRG2 contributed to the increased sensitivity to ciplatin through the modulation of Bak-to-Mcl-1 ratio regulated by NOX5-ROS-PKR pathway; therefore, we suggest that NDRG2 may be a molecular target for improving the efficacy of drug treatment in cancer patients.

Cytotoxic effects of kazinol A derived from Broussonetia papyrifera on human bladder cancer cells, T24 and T24R2

BACKGROUND Broussonetia papyrifera (B. papyrifera), also known as paper mulberry, has been used as a traditional medicine for the treatment of several diseases, including ophthalmic disorders and impotency. However, the biological activity of kazinol A (1) among flavonols isolated from B. papyrifera has not been identified. PURPOSE We identified a candidate metabolite for anti-human bladder cancer treatment from B. papyrifera and investigated the possible molecular mechanisms underlying its cytotoxic effects in T24 and cisplatin-resistant T24R2 human bladder cancer cells. METHODS T24 and T24R2 cells were treated with five flavonols from B. papyrifera and their cytotoxic effects were determined using MTT assay, cell cycle analysis, mitochondrial membrane potential, and propidium iodide staining. Autophagy rate was calculated by counting LC3-GFP dots in the cells. All related protein expressions were analyzed by immunoblotting. RESULTS Compound 1 showed relatively higher cytotoxicity in the human bladder cancer cells, T24 and T24R2, rather than other tissues-originated cancer cells. Compound 1 significantly attenuated cell growth through G0/1 arrest mediated by a decrease in cyclin D1 and an increase of p21. Apoptosis and autophagy induced by compound 1 treatment was accompanied by a modulation of the AKT-BAD pathway and AMPK-mTOR pathway, respectively. CONCLUSIONS Our results suggest that compound 1 induces cytotoxic effects in human bladder cancer cells, including the cisplatin-resistant T24R2. Compound 1 may be a candidate for the development of effective anti-cancer drug on human urinary bladder cancer.