Seong-Il Suh

The green tea polyphenol (−)-epigallocatechin gallate attenuates β-amyloid-induced neurotoxicity in cultured hippocampal neurons

Abstract Previous evidence has indicated that the neuronal toxicity of amyloid β (βA) protein is mediated through oxygen free radicals and can be attenuated by antioxidants and free radical scavengers. Recent studies have shown that green tea polyphenols reduced free radical-induced lipid peroxidation. The purpose of this study was to investigate whether (−)-epigallocatechin gallate (EGCG) would prevent or reduce the death of cultured hippocampal neuronal cells exposed to βA because EGCG has a potent antioxidant property as a green tea polyphenol. Following exposure of the hippocampal neuronal cells to βA for 48 hours, a marked hippocampal neuronal injuries and increases in malondialdehyde (MDA) level and caspase activity were observed. Co-treatment of cells with EGCG to βA exposure elevated the cell survival and decreased the levels of MDA and caspase activity. Proapoptotic (p53 and Bax), Bcl-XL and cyclooxygenase (COX) proteins have been implicated in βA-induced neuronal death. However, in this study the protective effects of EGCG seem to be independent of the regulation of p53, Bax, Bcl-XL and COX proteins. Taken together, the results suggest that EGCG has protective effects against βA-induced neuronal apoptosis through scavenging reactive oxygen species, which may be beneficial for the prevention of Alzheimers disease.

Protective effects of the green tea polyphenol (−)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils

Recent studies have shown that green tea polyphenols reduce free radical-induced lipid peroxidation. Oxygen free radical injury plays an important role in neuronal damage induced by brain ischemia and reperfusion. The purpose of this study was to examine whether (-)-epigallocatechin gallate (EGCG) would reduce neuronal damage after transient global ischemia in the gerbils because EGCG has a potent antioxidant property as a green tea polyphenol. To produce transient global ischemia, both common carotid arteries were occluded for 3 min with microaneurysmal clips. The gerbils were treated with EGCG (10, 25, or 50 mg/kg, i.p.) immediately after ischemia. Neuronal cell damage in the hippocampal CA1 region was evaluated quantitatively 5 days after ischemia by a blinded investigator. EGCG at the dose of 10 mg/kg failed to reduce hippocampal neuronal damage. However, EGCG when administered at the dose of 25 or 50 mg/kg significantly reduced hippocampal neuronal damage in a dose-dependent manner (P<0.001, respectively). The present results show that the green tea polyphenol, EGCG, has a neuroprotective effect against neuronal damage following global ischemia in the gerbils.

Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-γ-induced upregulation of B7-H1 (CD274)

Majority of cancer cells upregulate co‐inhibitory molecule B7‐H1 which confers resistance to anti‐tumor immunity, allowing cancers to escape from host immune surveillance. We addressed the molecular mechanism underlying the regulation of cancer‐associated B7‐H1 expression in response to interferon‐γ (IFN‐γ). Using promoter constructs in luciferase assay, the region between 202 and 320 bp from the translational start site is responsible for B7‐H1 expression. Electrophoretic mobility shift assay, site‐directed mutagenesis and knockdown experiment using siRNA revealed that interferon regulatory factor‐1 (IRF‐1) is primarily responsible for the constitutive B7‐H1 expression as well as for the IFN‐γ‐mediated B7‐H1 upregulation in a human lung cancer cell line A549. Additionally, AG490, a Janus activated kinase/signal transducer and activator of transcription inhibitor, greatly abolished the responsiveness of A549 cells to IFN‐γ by reducing the IRF‐1 transcription. Our findings support a critical role of IRF‐1 in the regulation of constitutive and IFN‐γ‐induced expression of B7‐H1 in cancer cells.

Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC δ signal transduction

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase in MMP-9 expression and activity. These effects of resveratrol are dose dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection utilizing MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via an activator protein-1 and nuclear factor-κB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-δ activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-δ may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.

Curcumin inhibits the expression of COX-2 in UVB-irradiated human keratinocytes (HaCaT) by inhibiting activation of AP-1: p38 MAP kinase and JNK as potential upstream targets

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.

Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells

Resveratrol, a natural product derived from grapes, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces antiproliferation and arrests the S phase in human histiocytic lymphoma U937 cells. Resveratrol induces arrest in the S phase at low concentrations (30-60 microM), but high concentrations do not induce S phase accumulation in U937 cells. Removal of resveratrol from the culture medium stimulates U937 cells to reenter the cell cycle synchronously, as judged by the expression patterns of cyclin E, A and by fluorescent activated cell sorting analysis. These data demonstrate that resveratrol causes S phase arrest and reversible cell cycle arrest. Thus, resveratrol provides an important new cell cycle blocker as well as a cancer chemopreventive agent.

Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular glutathione depletion

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.

Manganese induces inducible nitric oxide synthase (iNOS) expression via activation of both MAP kinase and PI3K/Akt pathways in BV2 microglial cells

It is well documented that manganese neurotoxicity induces clinical symptoms similar to those of idiopathic Parkinsons disease. Although microglial cytotoxic mediator-induced neurotoxicity is suggested, the mechanism by which manganese up-regulates cytotoxic mediator, such as nitric oxide (NO), remains poorly understood. Therefore, in this study, we investigated the mechanism of manganese on induction of iNOS in microglial cells. iNOS promoter/luciferase assay revealed that manganese (500 (M) regulated the iNOS expression at the transcriptional level. Immunoblot analysis also revealed that phosphorylation levels of ERK, JNK MAPKs and Akt (PKB, PI 3-kinase downstream effector), were increased. Both protein and mRNA levels of iNOS expression were abrogated by specific inhibitors, SP600125 (JNK inhibitor, 20 microM), PD98059 (ERKs inhibitor, 50 microM), or LY294002 (PI 3-kinase inhibitor, 20 microM), but not by SB203580 (20 microM), a p38 specific inhibitor. These data lead to the conclusion that manganese regulates the iNOS expression at the transcriptional level in BV2 microglial cells and the increased iNOS protein expression is mediated via both JNK-ERK MAPK and PI3K/Akt signaling pathways, but not via p38 MAPK pathway. Increased iNOS protein level was also found in RAW264.7 murine macrophage cells.

Immunosuppressant PG490 (triptolide) induces apoptosis through the activation of caspase-3 and down-regulation of XIAP in U937 cells

PG490 (triptolide) is a natural, biologically active compound extracted from Chinese herb Tripterygium wilfordii. It possesses potent anti-inflammatory and immunosuppressive properties. The mechanism by which triptolide initiates apoptosis remains poorly understood. In the present report, we investigated the effect of triptolide on the apoptotic pathway in U937 human promonocytic cells. We show that triptolide inhibits U937 cells growth by inducing apoptosis. Following treatment of U937 cells with 25 nM triptoride for 24 hr, morphological features of apoptosis and DNA fragmentation were observed. Caspase inhibitors significantly reduced triptolide-induced caspase-3 activation. In addition, apoptosis triggered by triptolide was not associated with the generation of reactive oxygen species, which was not affected by the antioxidant N-acetylcysteine (NAC). The data collectively indicate that the cytotoxic effect of triptolide in U937 cells is attributable to apoptosis mediated by the caspase-3 activation pathway that may be associated with XIAP down-regulation.

Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells

Se-methylselenocysteine (Se-MSC) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis, but its mechanism of action is still not well understood. The present study was designed to assess the mechanism of Se-MSC on the induction of apoptosis in SKOV-3 ovarian cancer cells. Se-MSC displayed strong inhibitory effects on cell proliferation and viability of SKOV-3 cells in dose and time dependent manners and induced apoptosis. Investigation of the mechanism of Se-MSC-induced apoptosis revealed that treatment with Se-MSC produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase and phospholipase C-gamma1 proteins. However, SKOV-3 cells treated with Se-MSC did not demonstrate cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the caspase inhibitors (z-VAD-fmk and DEVD-CHO) prevented Se-MSC-induced apoptosis. These results suggested that Se-MSC induces apoptosis through cytochrome c-independent caspase-3 activation in SKOV-3 cells. In late stage of apoptosis, p18kDa fragment of Bax was generated with the down-regulation of the expressions of survivin, X-linked inhibitor of apoptosis protein, and human inhibitor of apoptosis protein 1 following Se-MSC treatment, suggesting that the modulation of Bax and IAP (inhibitors of apoptosis) family proteins play some role in Se-MSC-mediated apoptosis. Pre-treatments of z-VAD-fmk and the calpain inhibitor, calpeptin inhibited Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be a caspase-dependent one. Taken together, the chemopreventive effects of Se-MSC may be related in part to the caspase-3 activation, the down-regulation of IAP family proteins, and Bax cleavage mediated by caspase-dependent calpain activation.