Jong-Young Kwak

The Acquisition of Antigen Cross-Presentation Function by Newly Formed Dendritic Cells

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8+ DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8+ DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.

Fucoidan as a Marine Anticancer Agent in Preclinical Development

Fucoidan is a fucose-containing sulfated polysaccharide derived from brown seaweeds, crude extracts of which are commercially available as nutritional supplements. Recent studies have demonstrated antiproliferative, antiangiogenic, and anticancer properties of fucoidan in vitro. Accordingly, the anticancer effects of fucoidan have been shown to vary depending on its structure, while it can target multiple receptors or signaling molecules in various cell types, including tumor cells and immune cells. Low toxicity and the in vitro effects of fucoidan mentioned above make it a suitable agent for cancer prevention or treatment. However, preclinical development of natural marine products requires in vivo examination of purified compounds in animal tumor models. This review discusses the effects of systemic and local administration of fucoidan on tumor growth, angiogenesis, and immune reaction and whether in vivo and in vitro results are likely applicable to the development of fucoidan as a marine anticancer drug.

15d-PGJ2 Induces Apoptosis by Reactive Oxygen Species–mediated Inactivation of Akt in Leukemia and Colorectal Cancer Cells and Shows In vivo Antitumor Activity

Purpose: Recent studies have shown that 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a natural ligand for peroxisome proliferator–activated receptor-γ (PPARγ), inhibits cell proliferation and induces apoptosis. The specific molecular mechanisms underlying this effect remain to be elucidated. We examined whether 15d-PGJ2 has antitumor activity in vitro and in vivo, and investigated the underlying mechanism. Experimental Design: We examined 15d-PGJ2–induced apoptosis in human leukemia cells in the context of mitochondrial injury, oxidative damage, and signaling pathway disturbances. In addition, we investigated the antitumor effect of 15d-PGJ2 in a mouse CT-26 s.c. tumor model and HL-60 leukemia xenograft model. Results: 15d-PGJ2 induced apoptosis in leukemia and colorectal cancer cells in a dose-dependent manner and led to generation of reactive oxygen species (ROS) through mitochondria and NADPH oxidase activation, activation of JNK, and inactivation of Akt, a serine/threonine-specific protein kinase. Constitutive activation of Akt for an engineered myristoylated protein prevented 15d-PGJ2–mediated apoptosis but not ROS generation. Collectively, these findings suggest a hierarchical model of apoptosis induced by 15d-PGJ2 in human leukemia cells: oxidative injury represents a primary event resulting in Akt inactivation, which in turn leads to mitochondrial injury and apoptosis. Moreover, 15d-PGJ2 markedly reduced growth of mouse CT-26 s.c. tumors and HL-60 xenograft tumors and down-regulated p-Akt and Akt expression in vivo. Conclusions: These results suggest that Akt inactivation through ROS production may contribute to 15d-PGJ2–induced apoptosis in leukemia and colorectal cancer cell lines and that 15d-PGJ2 may have therapeutic relevance in the treatment of human leukemia and colorectal cancer. (Clin Cancer Res 2009;15(17):5414–25)

Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+ DC and type I helper T cell-dominant immune state.

Dendritic cells (DC) are known to not only induce the activation of T cells, but are also associated with the polarization of T cells. This study investigated whether or not proteoglycan (PG) isolated from Phellinus linteus induces the phenotypic and functional maturation of CD11c+ DC in vitro and in vivo. PG was found to induce the phenotypic and functional maturation of bone marrow‐derived DC via Toll‐like receptors (TLR) 2 and 4 in vitro. Administration of PG in vivo strongly inhibited the MCA‐102 tumor growth and increase in vivo. The ratio of CD8+ DC to CD8− DC increased, and PG enhanced IL‐12 and IFN‐γ production, and expression of surface molecules including major histocompatibility complexes (MHC) classes I, MHC II, CD80, and CD86 in MCA‐102‐challenged mice. PG also caused a marked increase in the production of Th (helper T cells)‐1 cytokine (IFN‐γ) and a decrease in the production of Th‐2 cytokine (IL‐4) by splenic cells and inguinal lymph node cells in MCA‐102 tumor‐bearing mice. Furthermore, PG stimulated the proliferation of CD4+ and CD8+ T cells. In addition, a combination of PG and tumor lysate‐pulsed DC inhibited completely the growth of MCA‐102 cells in tumor‐bearing mice. These results indicate that the administration of PG inhibited the tumor growth through a mechanism leading to a Th‐1 dominant immune state and the activation of CD11c+CD8+ DC.

Trp-Lys-Tyr-Met-Val-D-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes.

Among the phagocytic leukocytes, monocytes have the important role of clearing out parasitic microorganisms. They accomplish this through production of toxic metabolites of oxygen. Trp‐Lys‐Tyr‐Met‐Val‐D‐Met (WKYMVm), a peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes, including monocytes, binds to a unique cell surface receptor and stimulates superoxide generation, killing of Staphylococcus aureus, and activation of phospholipase D (PLD) in human monocytes. Preincubation of the cells with a PI‐specific phospholipase C (PLC) inhibitor (U‐73122), protein kinase C inhibitor (GF109203X), or intracellular Ca2+ chelator (BAPTA/AM) before the peptide stimulus totally inhibits the peptide‐induced PLD activation and superoxide generation. On the other hand, tyrosine kinase inhibitor genistein only partially inhibits the peptide‐induced processes. The peptide‐induced bacteria killing activity shares regulatory mechanisms for PLD activation with the superoxide generation, which is inhibited in the presence of 1‐butanol. We suggest that the peptide stimulates PLD downstream of PLC activation and PLD activation in turn is essential for the peptide‐induced immunological functions such as the superoxide generation and killing of bacteria by human monocytes. J. Leukoc. Biol. 65:241–248; 1999.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif.

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1α, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1α were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1α were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1α were specific targets for Chk1 and/or Chk2 at least in vitro.

The mechanism of fucoidan-induced apoptosis in leukemic cells: involvement of ERK1/2, JNK, glutathione, and nitric oxide.

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti‐tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan‐mediated signaling pathways. Fucoidan induced apoptosis of HL‐60, NB4, and THP‐1 cells, but not K562 cells. Fucoidan treatment of HL‐60 cells induced activation of caspases‐8, ‐9, and ‐3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan‐induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen‐activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH2‐terminal kinase (JNK). The phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan‐treated HL‐60, NB4, and THP‐1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan‐induced apoptosis was inhibited by addition of glutathione and/or L‐NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine‐[R,S]‐sulfoximine rendered HL‐60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan‐treated HL‐60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan‐induced apoptosis of human leukemic cells.

Differential effects of triterpene glycosides, frondoside A and cucumarioside A2-2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells

Frondoside A is a pentaoside having an acetyl moiety at the aglycon ring and xylose as a third monosaccharide residue. Cucumarioside A2‐2 is a pentaoside having glucose as a third monosaccahride unit. We compared the effects of frondoside A and A2‐2 for cell death‐inducing capability with close attention paid to structure–activity relationships. Both frondoside A and A2‐2 strongly induced apoptosis of leukemic cells. Frondoside A‐induced apoptosis was more potent and rapid than A2‐2‐induced apoptosis. A2‐2‐induced but not frondoside A‐induced apoptosis was caspase‐dependent. This suggests that holothurians may induce apoptosis of leukemic cells caspase‐dependently or ‐independently, depending on the holothurian structure.

Proteoglycan isolated from Phellinus linteus activates murine B lymphocytes via protein kinase C and protein tyrosine kinase

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes. Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not been known. In the present study, we investigated whether proteoglycan (PL) isolated from P. linteus has an effect on the immunomodulatory activities of the murine splenic lymphocytes (MSLs). Treatment with PL caused a four-fold augmentation in [3H]thymidine incorporation compared to untreated control group in MSLs. Flow cytometric analysis indicated that the affected cell population was mainly CD19(+) cells, but not CD3(+) cells. These data suggested that the main target of PL was the B cells, but not T cells. PL also enhanced the expression of co-stimulatory molecules, CD80 and CD86, in murine B cells in a time-dependent manner. Accordingly, we investigated if intracellular [Ca(2+)] and reactive oxygen intermediates (ROI) were the principal downstream components that contributed to PL-induced activation, with respect to both increases of proliferation and induction of co-stimulatory molecules. However, PL has no influence on the [Ca(2+)] concentration and the ROI formation in murine B cells, whereas the genistein, protein tyrosine kinase (PTK) inhibitor or staurosporine, protein kinase C (PKC), blocked the proliferation and the induction of co-stimulatory molecules, CD80 and CD86, in B cells stimulated with PL. Taken together, these data suggest that PL is a biological response modifier that stimulates proliferation and expression of co-stimulatory molecules in B cells, probably by regulating PTK and PKC signaling pathways.

Ligand of scavenger receptor class A indirectly induces maturation of human blood dendritic cells via production of tumor necrosis factor-α

Dendritic cells (DCs) are the most potent antigen-presenting cells for naive T cells. In this study, scavenger receptor class A type I and type II (SR-A) were shown to be expressed by peripheral blood DCs (PBDCs) and monocyte-derived DCs (MDDCs). In addition, the binding of anti-SR-A antibody to these cells was lower in the presence of fucoidan, an SR-A agonist. Treatment of these DCs with fucoidan or anti-SR-A antibody markedly increased the surface expression of costimulatory molecules CD83 and major histocompatibility complex class II on the CD11c(high)CD123(low) myeloid subset of PBDCs. Furthermore, fucoidan-treated PBDCs produced tumor necrosis factor-alpha (TNF-alpha) but not IL-12p70. In addition, fucoidan-induced maturation was eliminated by pretreatment with TNF-alpha-neutralizing antibody. Finally, interferon-gamma secretion and T-cell proliferation were enhanced by coculture of T cells with fucoidan-matured PBDCs. Specific inhibitors of p38 MAPK and glycogen synthase kinase 3 suppressed TNF-alpha production and maturation of fucoidan-treated PBDCs. Moreover, MDDCs lacking SR-A failed to up-regulate CD83 expression, TNF-alpha production, and phosphorylation of p38 MAPK and glycogen synthase kinase 3-beta in the presence of fucoidan. Taken together, these results suggest that ligation of SR-A leads to induction of TNF-alpha, which subsequently induces PBDC maturation, thereby leading to enhanced T-cell stimulatory capacity.