Do-Gon Ryu

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.

Genistein protects pancreatic β cells against cytokine-mediated toxicity

In the past few decades, the use of genistein as an anti-inflammatory agent has gained much attention. Our current study focuses on the preventive effects of genistein on cytokine-induced pancreatic beta-cell damage. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin (IL)-1beta and interferon (IFN)-gamma induced cell damage, which was correlated with nitric oxide (NO) production. Genistein completely prevented cytokine-mediated cytotoxicity and NO production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism of genistein inhibition of iNOS gene expression appeared to involve the inhibition of NFkappaB activation. The cytokine induced increases in NFkappaB binding activity, nuclear p50 and p65 subunit levels, and IkappaBalpha degradation in cytosol compared to unstimulated cells; genistein abolished all of these parameters. The cytoprotective effects of genistein are also mediated through the suppression of ERK-1/2 and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. In a second set of experiments, rat islets were used. The findings on beta-cell protective effects of genistein were essentially the same as for the RIN cell data, namely genistein prevented cytokine-induced NO production, iNOS expression, ERK-1/2 activation, JAK/STAT activation, and impairment of glucose-stimulated insulin secretion. Collectively, these results suggest that genistein might be used to preserve functional beta-cell mass.

Flavonoids Protect Against Cytokine-induced Pancreatic β-cell Damage Through Suppression of Nuclear Factor κb Activation

Objectives: In the past few decades, the use of natural compounds, such as flavonoids, as anti-inflammatory agents has gained much attention. Our current study focuses on the preventive effects of quercetin, apigenin, and luteolin on cytokine-induced β-cell damage. Methods: Pancreatic β-cells or islets were treated with cytokine mixtures in the presence or absence of flavonoids and the inhibitory effect of flavonoids against cytokine toxicity was determined. Results: Treatment of RINm5F (RIN) rat insulinoma cells with interleukin 1β (IL-1β) and interferon γ (IFN-γ) induced cell damage. Quercetin, apigenin, and luteolin completely protected against IL-1β- and IFN-γ-mediated cytotoxicity in RIN cells. Incubation with quercetin, apigenin, and luteolin resulted in a significant reduction in IL-1β- and IFN-γ-induced nitric oxide production, a finding that correlated well with reduced levels of the inducible form of NO synthase messenger RNA and protein. The molecular mechanism by which quercetin, apigenin, and luteolin inhibited inducible NO synthase gene expression appeared to involve the inhibition of nuclear factor κB (NF-κB) activation. The IL-1β- and IFN-γ-stimulated RIN cells showed increases in NF-κB binding activity, p50 and p65 subunit levels in nucleus, and IκBα degradation in cytosol compared with unstimulated cells. Quercetin, apigenin, and luteolin also prevented IL-1β- and IFN-γ-mediated inhibition of insulin secretion. Conclusion: Quercetin, apigenin, and luteolin inhibited cytotoxicity in RIN cells and attenuated the decrease of glucose-stimulated insulin secretion in islets by IL-1β and IFN-γ.

Activation of caspase cascades in Korean mistletoe (Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells.

Mistletoe lectins are of high biological activity and exert cytotoxic effects. We have previously shown that Korean mistletoe, Viscum album var. coloratum, lectin-II specifically induces apoptotic cell death in cancer cells, not normal lymphocytes. The destructive mechanism by mistletoe lectins on tumor cells was mediated by activation of c-JUN N-terminal kinase (JNK)/stress-activated protein kinase. Herein, we investigated the involvement of caspase cascade and its proteolytic cleavage effects on biosubstrates of human myeloleukemic U937 cells by D-galactoside and N-acetyl-galactosamine-specific Korean mistletoe lectin-II. Mistletoe lectin-II induced ladder pattern DNA fragmentation and activation of caspase-3, -8, and -9 of U937 cells, but not caspase-1 protease, in a time- and dose-dependent manner. Consistent with catalytic activation of protease, both poly(ADP-ribose) polymerase (PARP) and protein kinase C-delta (PKC-delta) are also cleaved in mistletoe lectin-II-treated U937 cells. An inhibitor of caspase-3-like protease, DEVD-CHO peptide, significantly inhibited mistletoe lectin-II-induced apoptosis, PARP cleavage, and fragmentation of DNA. These results provide the evidence that Korean mistletoe lectin-II induces apoptotic death of U937 cells via activation of caspase cascades.

D4-GDI is cleaved by caspase-3 during daunorubicin-induced apoptosis in HL-60 cells

Daunorubicin, an anti-cancer drug, is known to induce apoptosis in HL-60 cells in a dose-dependent manner through the activation of caspase-3 (CPP32). Caspase-3 selective inhibitor, Ac-DEVD-CHO, prevented both the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). D4-GDI is a GDP dissociation inhibitor for the Ras-related Rho family GTPase in hematopoietic cells. Here we report that D4-GDI is a substrate for the caspase-3. D4-GDI was cleaved to a 23 kDa fragment by daunorubicin treatment in HL-60 cells with kinetics that parallel the onset of apoptosis. D4-GDI cleavage as well as DNA fragmentation was inhibited by treatment with Ac-DEVD-CHO but not with Ac-YVAD-CHO, a caspase-1 inhibitor. These data suggest that D4-GDI of Rho family GTPase may be regulated during apoptosis through the caspase-3 mediated cleavage of the GDI protein.

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic β-cells through suppression of NF-κB activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced β-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of β-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1β and IFN-γ resulted in a reduction of cell viability. CRE completely protected IL-1β and IFN-γ-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1β and IFN-γ-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-κ B activation. The IL-1β and IFN-γ-stimulated RIN cells showed increases in NF-κ B binding activity and p65 subunit levels in nucleus, and IκBα degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1β and IFN-γ-treated islets.

Herba houttuyniae extract induces apoptotic death of human promyelocytic leukemia cells via caspase activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release

Herba houttuyniae has been used as a constituent of herval medicine prescriptions for the treatment of inflammation, cancer, and other diseases. In the present study, we investigated the cellular effects of herba houttuyniae extract (HHE) and the signal pathways of HHE-induced apoptosis in HL-60 human promyelocytic leukemia cell line. HHE treatment caused apoptosis of cells as evidenced by discontinuous fragmentation of DNA, the loss of mitochondrial membrane potential, release of mitochondrial cytochrome c into the cytosol, activation of procaspase-9 and caspase-3, and proteolytic cleavage of poly(ADP-ribose) polymerase. Pretreatment of Ac-DEVD-CHO, caspase-3 specific inhibitor, or cyclosporin A, a mitochondrial permeability transition inhibitor, completely abolished HHE-induced DNA fragmentation. Together, these results suggest that HHE possibly causes mitochondrial damage leading to cytochrome c release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HL-60 cells.

Ethanol Extract of Alismatis rhizome Inhibits Adipocyte Differentiation of OP9 Cells

The rhizome of Alisma orientale (Alismatis rhizome) has been used in Asia for promoting diuresis to eliminate dampness from the lower-jiao and to expel heat. In this study, an ethanol extract of the rhizome of Alisma orientale (AOE) was prepared and its effects on adipocyte differentiation of OP9 cells were investigated. Treatment with AOE in a differentiation medium for 5 days resulted in dose-dependent inhibition of lipid droplet formation in OP9 cells. Furthermore, AOE significantly inhibited adipocyte differentiation by downregulating the expression of the master transcription factor of adipogenesis, peroxisome proliferation-activity receptor γ (PPARγ), and related genes, including CCAAT/enhancer binding protein β (C/EBPβ), fatty acid-binding protein (aP2), and fatty acid synthase (FAS). AOE exerted its inhibitory effects primarily during the early adipogenesis stage (days 1-2), at which time it also exerted dose-dependent inhibition of the expression of C/EBPβ, a protein related to the inhibition of mitotic clonal expansion. Additionally, AOE decreased the expression of autophagy-related proteins, including beclin 1, and the autophagy-related genes, (Atg) 7 and Atg12. Our results indicate that AOEs inhibitory effects on adipocyte differentiation of OP9 cells are mediated by reduced C/EBPβ expression, causing inhibition of mitotic clonal expansion and autophagy.

Induction of apoptosis by takrisodokyeum through generation of hydrogen peroxide and activation of caspase-3 in HL-60 cells.

Takrisodokyeum (TRSDY), a Chinese herbal medicine, has been known to exert anti-tumoral activity in Korea. However, its molecular mechanism of action is not understood. In this study, we found that TRSDY induced apoptosis in HL-60 cells as evidenced by both a characteristic ladder pattern of discontinuous DNA fragments and an increase of annexin V+/PI- stained cell population. Our data demonstrated that TRSDY-induced apoptotic cell death was accompanied by activation of caspase-3 and cleavages of its substrates, poly(ADP-ribose) polymerase (PARP) and RhoGDP dissociation inhibitor (RhoGDI-2; also called D4-GDI) in a time- and concentration-dependent manner. Caspase-3 inhibitor, but not caspase-1 inhibitor, prevented TRSDY-induced apoptosis. Furthermore, treatment with TRSDY increased the production of intracellular hydrogen peroxide and pretreatment of cells with anti-oxidants conferred complete protection against hydrogen peroxide generation and subsequent caspase-3 activation. Taken together, these results suggest that TRSDY induces hydrogen peroxide generation, which, in turn, causes activation of caspase-3, degradation of PARP and D4-GDI, and eventually leads to apoptotic cell death.