Gi Young Kim

Up-regulation of Nrf2-mediated heme oxygenase-1 expression by eckol, a phlorotannin compound, through activation of Erk and PI3K/Akt.

The aim of the present study was to examine the cytoprotective effect of eckol, a phlorotannin found in Ecklonia cava and to elucidate underlying mechanisms. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a role in cytoprotection against oxidative stress. Eckol-induced HO-1 expression both at the level of mRNA and protein in Chinese hamster lung fibroblast (V79-4) cells, resulting in increased HO-1 activity. The transcription factor NF-E2-related factor 2 (Nrf2) is a critical regulator of HO-1, achieved by binding to the antioxidant response element (ARE). Eckol treatment resulted in the enhanced level of phosphorylated form, nuclear translocation, ARE-binding, and transcriptional activity of Nrf2. Extracellular regulated kinase (Erk) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt) contributed to ARE-driven HO-1 expression. Eckol activated both Erk and Akt, and treatments with U0126 (an Erk kinase inhibitor), LY294002 (a PI3K inhibitor), specific Erk1 siRNA, and Akt siRNA suppressed the eckol-induced activation of Nrf2, resulting in a decrease in HO-1 expression. ZnPP (a HO-1 inhibitor), HO-1 siRNA, and Nrf2 siRNA markedly abolished the cytoprotective effect of eckol against hydrogen peroxide-induced cell damage. Likewise, U0126 and LY294002 inhibited the eckol-induced cytoprotective effect against oxidative cell damage. These studies demonstrate that eckol attenuates oxidative stress by activating Nrf2-mediated HO-1 induction via Erk and PI3K/Akt signaling.

Epigenetic changes induced by oxidative stress in colorectal cancer cells: methylation of tumor suppressor RUNX3.

Runt domain transcription factor 3 (RUNX3) is a tumor suppressor that is silenced in cancer via hypermethylation of its promoter. This study investigated the mechanisms involved in reactive oxygen species (ROS)-induced silencing of RUNX3 in terms of epigenetic alteration since the effects of oxidative stress in tumor suppressor gene transcription are largely unknown. RUNX3 mRNA and protein expressions were down-regulated in response to hydrogen peroxide (H2O2) in the human colorectal cancer cell line SNU-407. This down-regulation was abolished with pretreatment of the ROS scavenger, N-acetylcysteine (NAC). Moreover, methylation-specific PCR data revealed that H2O2 treatment increased RUNX3 promoter methylation; however, NAC and the cytosine methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC), decreased it, suggesting that an epigenetic regulatory mechanism by ROS-induced methylation may be involved in RUNX3 silencing. H2O2 treatment resulted in DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) up-regulation with increased expression and activity, increased binding of DNMT1 to HADC1, and increased DNMT1 binding to the RUNX3 promoter. In addition, 5-Aza-dC treatment prevented the decrease in RUNX3 mRNA and protein levels by H2O2 treatment. Additionally, H2O2 treatment inhibited the nuclear localization and expression of RUNX3, which was abolished by NAC treatment. Furthermore, the down-regulation of RUNX3 expression by H2O2 also influenced cell proliferation. Taken together, the data suggested that ROS silenced the tumor suppressor, RUNX3, by epigenetic regulation and may therefore be associated with the progression of colorectal cancer.

Implication of intracellular ROS formation, caspase-3 activation and Egr-1 induction in platycodon D-induced apoptosis of U937 human leukemia cells.

Platycodon D is a major constituent of triterpene saponins found in the root of Platycodon grandiflorum, Platycodi Radix, which is widely used in traditional Oriental medicine for the treatment of many chronic inflammatory diseases. The results of previous studies have shown that this compound has in vitro growth-inhibitory activity in human cancer cells, however, the mechanism by which this action occurs is poorly understood. In this study, we examined the effects of platycodon D on the production of reactive oxygen species (ROS) and evaluated the association of these effects with apoptotic tumor cell death using a human leukemic U937 cell line. The results of this study demonstrate that platycodon D mediates ROS production, and that this mediation is followed by a decrease in mitochondrial membrane potential (MMP, DJm), activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Both the cytotoxic effects and apoptotic characteristics induced by platycodon D treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the observed cytotoxic effect. Additionally, the transcription factor early growth response-1 (Egr-1) gene was transcriptionally activated and the levels of non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) protein were elevated in platycodon D-treatedU937 cells. However, the quenching of ROS generation in response to treatment with a ROS scavenger, N-acetyl-L-cysteine, reversed the platycodon D-induced apoptosis effects via inhibition of Egr-1 activation, ROS production, MMP collapse, and the subsequent activation of caspase-3. Although further studies are needed to demonstrate that increased expression of Egr-1 by platycodon D leads directly to NAG-1 induction and subsequent apoptosis, our observations clearly indicate that ROS induced through Egr-1 activation are involved in the early molecular events involved in the platycodon D-induced apoptotic pathway.

Oxidative stress causes epigenetic alteration of CDX1 expression in colorectal cancer cells.

The intestine-specific transcription factor, caudal type homeobox-1 (CDX1), is a candidate tumor suppressor gene that plays key roles in regulating intestinal epithelial differentiation and proliferation. It is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines by promoter hypermethylation. Since the effects of oxidative stress on the transcription of tumor suppressor genes are largely unknown, this study explored the epigenetic alterations that occur during reactive oxygen species (ROS)-induced silencing of CDX1 in colorectal cancer cells. Oxidative stress by hydrogen peroxide (H2O2) down-regulated CDX1 mRNA levels and protein expression in the human colorectal cancer cell line, T-84. This down-regulation was abolished by pretreatment with the ROS scavenger, N-acetylcysteine. In addition, the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC) markedly attenuated the decrease in mRNA and protein expression levels induced by H2O2. Moreover, methylation-specific PCR data revealed that H2O2 treatment increased CDX1 promoter methylation, and treatment with 5-Aza-dC reversed this effect, suggesting that an epigenetic regulatory mechanism triggered by ROS-induced methylation may be involved in CDX1 expression. Furthermore, H2O2 treatment resulted in up-regulation of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) expression and activity, and enhanced the association between DNMT1 and HDAC1. Taken together, these results suggest that ROS-induced oxidative stress silences the tumor suppressor CDX1 through epigenetic regulation, and may therefore be associated with the progression of colorectal cancer.

Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells, including colon, prostate, breast and leukemia. Among them, aspirin, a classical NSAID, shows promise in cancer therapy in certain types of cancers. We hypothesized that aspirin might affect the growth of liver cancer cells since liver is the principal site for aspirin metabolism. Therefore, we investigated the effects of aspirin on the HepG2 human hepatocellular carcinoma cell line in vitro and the HepG2 cell xenograft model in BALB/c nude mice. We found that treatment with aspirin inhibited cell growth and induced apoptosis involving both extrinsic and intrinsic pathways as measured by DNA ladder formation, alteration in the Bax/Bcl-2 ratio, activation of the caspase activities and related protein expressions. In vivo antitumor activity assay also showed that aspirin resulted in significant tumor growth inhibition compared to the control. Oral administration of aspirin (100 mg/kg/day) caused a significant reduction in the growth of HepG2 tumors in nude mice. These findings suggest that aspirin may be used as a promising anticancer agent against liver cancer.

Induction of Egr-1 Is Associated with Anti-Metastatic and Anti-Invasive Ability of β-Lapachone in Human Hepatocarcinoma Cells

β-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of β-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. β-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that β-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in β-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that β-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.

The flavonoid morin from Moraceae induces apoptosis by modulation of Bcl-2 family members and Fas receptor in HCT 116 cells

It is evident based on literature that flavonoids from fruit can safely modulate cancer cell biology and induce apoptosis. Therefore, we investigated the anticancer activity of morin, a flavonoid which is plentiful in twigs of mulberry focusing on apoptosis, and its mechanisms. Morin upregulated the Fas receptor, and activates caspase-8, -9 and -3 in HCT-116 cells. Morin also activates Bid, and induced the loss of mitochondrial membrane potential (MMP, ∆Ψm) with Bax protein activation and cytochrome c release. In addition, morin induced ROS generation which was not blocked by N-acetylcysteine. Morin also suppressed Bcl-2 and cIAP-1, anti-apoptotic proteins, which may contribute to augmentation of morin-triggered apoptosis. As an upstream signaling pathway, suppressed Akt activity by morin was associated to apoptosis. This study suggests that morin induces caspase-dependent apoptosis through extrinsic pathway by upregulating Fas receptor as well as through the intrinsic pathway by modulating Bcl-2 and IAP family members, and ROS generation, and that Akt is the critical upstream signaling that regulates the apoptotic effect of morin in human colon cancer HCT-116 cells.

Ganoderma lucidum ethanol extract inhibits the inflammatory response by suppressing the NF-κB and toll-like receptor pathways in lipopolysaccharide-stimulated BV2 microglial cells

Ganoderma lucidum is a traditional Oriental medicine that has been widely used as a tonic to promote longevity and health in Korea and other Asian countries. Although a great deal of work has been carried out on the therapeutic potential of this mushroom, the pharmacological mechanisms of its anti-inflammatory actions remain unclear. In this study, we evaluated the inhibitory effects of G. lucidum ethanol extract (EGL) on the production of inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of EGL on the LPS-induced activation of nuclear factor kappaB (NF-κB) and upregulation of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Elevated levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokine production were detected in BV2 microglia following LPS stimulation. We identifed that EGL significantly inhibits the excessive production of NO, PGE2 and pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor-α in a concentration-dependent manner without causing cytotoxicity. In addition, EGL suppressed NF-κB translocation and transcriptional activity by blocking IκB degradation and inhibiting TLR4 and MyD88 expression in LPS-stimulated BV2 cells. Our results indicate that the inhibitory effects of EGL on LPS-stimulated inflammatory responses in BV2 microglia are associated with the suppression of the NF-κB and TLR signaling pathways. Therefore, EGL may be useful in the treatment of neurodegenerative diseases by inhibiting inflammatory mediator responses in activated microglia.

Induction of apoptosis by sanguinarine in C6 rat glioblastoma cells is associated with the modulation of the Bcl-2 family and activation of caspases through downregulation of extracellular signal-regulated kinase and Akt.

Sanguinarine is a benzophenanthridine alkaloid that is derived from the root of Sanguinaria canadensis and other poppy fumaria species, and is known to have antimicrobial, antiinflammatory and antioxidant properties. This study investigated the possible mechanisms through which sanguinarine exerts its antiproliferative action in cultured C6 rat glioblastoma cells. The exposure of C6 cells to sanguinarine resulted in growth inhibition and the induction of apoptosis in a dose-dependent manner, as measured by the MTT assay, fluorescence microscopy, agarose gel electrophoresis and annexin-V-based assay. The sanguinarine treatment induced the proteolytic activation of caspases and ICAD/DFF45, which was associated with the modulation of the Bcl-2 family, concomitant degradation of poly(ADP ribose) polymerase and phospholipase C-&ggr;1 protein, and DNA fragmentation. z-DEVD-fmk, a caspase-3-specific inhibitor, blocked poly(ADP ribose) polymerase degradation, DNA fragmentation and increased the survival rate of sanguinarine-treated C6 cells. Moreover, the activity of extracellular signal-regulated kinase and Akt was downregulated in sanguinarine-treated cells, and PD98059, a specific extracellular signal-regulated kinase inhibitor, and phosphatidylinositol 3′-kinase/Akt inhibitors, LY294002 and wortmanin, sensitized the cells to sanguinarine-induced apoptosis, indicating that the downregulation of the extracellular signal-regulated kinase and Akt signaling pathway may play a key role in sanguinarine-induced apoptosis in C6 cells.

Morin, a Flavonoid from Moraceae, Induces Apoptosis by Induction of BAD Protein in Human Leukemic Cells

Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨm) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.