Hyung Gyun Kim

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.

Protective effect of saponins derived from the roots of Platycodon grandiflorum against carbon tetrachloride induced hepatotoxicity in mice

The purpose of this study was to investigate the protective effects of the saponins isolated from the root of Platycodi Radix (Changkil saponins: CKS) on carbon tetrachloride (CCl(4))-induced hepatotoxicities in mice. Pretreatment with CKS prior to the administration of CCl(4) significantly prevented the increase in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic lipid peroxidation formation. In addition, CKS prevented CCl(4)-induced apoptosis and necrosis, as indicated by a liver histopathologic study and DNA laddering. To determine whether Fas/Fas ligand (FasL) pathway involved in CCl(4)-induced acute liver injury, Fas and FasL proteins and caspase-3, -8 activities were tested by western blotting and ELISA. CKS markedly decreased CCl(4)-induced Fas/FasL protein expression levels and in turn attenuated CCl(4)-induced caspase-3, -8 activities in mouse livers. Additionally, CKS protected the CCl(4)-induced depletion of hepatic glutathione levels. The effect of CKS on CYP2E1, the major isozyme involved in CCl(4) bioactivation, was investigated. Treatment with CKS resulted in a significant decrease in the CYP2E1-dependent hydroxylation of aniline. In addition, CKS exhibited antioxidant effects on FeCl(2)-ascorbate induced lipid peroxidation in liver homogenates, and on superoxide radical scavenging activity. These findings suggest that the protective effects of CKS against CCl(4)-induced acute liver injury possibly involve mechanisms related to its ability to block CYP2El-mediated CCl(4) bioactivation and its free radical scavenging effects, and that is also protects against Fas/FasL pathway mediated apoptosis.

Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2-dependent manner.

The zinc-binding protein metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. In this study, we demonstrate that MT-III prevents the accumulation of reactive oxygen species (ROS) in dopaminergic SH-SY5Y cells challenged with the Parkinsons disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) and ERK kinase/NF-E2-related factor 2 (Nrf2) dependent induction of the stress response protein heme oxygenase-1 (HO-1). Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, MT-III up-regulates HO-1 expression and this expression confers neuroprotection against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that the PI3K and ERK/Nrf2 signaling pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.

Molecular mechanism of suppression of MDR1 by puerarin from Pueraria lobata via NF‐κB pathway and cAMP‐responsive element transcriptional activity‐dependent up‐regulation of AMP‐activated protein kinase in breast cancer MCF‐7/adr cells

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy and its inhibition is an effective way to reverse cancer drug resistance. In the present study, we investigated that puerarin, a natural isoflavonoid from Pueraria lobata, down-regulated MDR1 expression in MCF-7/adriamycin (MCF-7/adr), a human breast MDR cancer cell line. Puerarin treatment significantly inhibited MDR1 expression, MDR1 mRNA and MDR1 promoter activity in MCF-7/adr cells. The suppression of MDR1 was accompanied by partial recovery of intracellular drug accumulation, leading to increased toxicity of adriamycin and fluorescence of rhodamine 123, indicating that puerarin reversed the MDR phenotype by inhibiting the drug efflux function of MDR1. Moreover, nuclear factor kappa-B activity and IkappaB degradation were inhibited by puerarin. Puerarin stimulated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase and glycogen synthase kinase-3beta phosphorylation, but puerarin decreased cAMP-responsive element-binding protein phosphorylation. The puerarin-induced suppression of MDR1 expression was reduced by AMPK inhibitor (compound C). Furthermore, both MDR1 protein expression and the transcriptional activity of cAMP-responsive element (CRE) were inhibited by puerarin and protein kinase A/CRE inhibitor (H89). Taken together, our results suggested that puerarin down-regulated MDR1 expression via nuclear factor kappa-B and CRE transcriptional activity-dependent up-regulation of AMPK in MCF-7/adr cells.

Kahweol blocks STAT3 phosphorylation and induces apoptosis in human lung adenocarcinoma A549 cells

Kahweol, the coffee-specific diterpene, has been reported to have anti-carcinogenic properties. Animal data support such a chemopreventive effect of coffee. However, the precise underlying protective mechanisms are poorly understood. In this study, the apoptotic effect of kahweol in human lung adenocarcinoma A549 cells was investigated. In cell viability assays and cell proliferation assays, kahweol exhibited anti-proliferative and pro-apoptotic effects on A549 cells in a time- and dose-dependent manner. Kahweol considerably inhibited the expression of Bcl-2 but increased that of Bax; it also stimulated the cleavage of caspase-3 and PARP (poly ADP-ribose polymerase). In addition, kahweol-induced apoptosis was confirmed by TUNEL assays. Furthermore, kahweol inhibited dose-dependent phosphorylation of signal transducer and activator of transcription 3 (STAT3). An overexpression in STAT3 led to resistance to kahweol-induced apoptosis, suggesting that STAT3 was a critical target of kahweol. These findings suggest that kahweol inhibited A549 cell growth and induced apoptosis via down-regulation of STAT3 signaling pathway.

Metallothionein-III induces HIF-1α-mediated VEGF expression in brain endothelial cells

Metallothionein-III (MT-III), a metal-binding protein, is associated with resistance to neuronal injury. However, the underlying mechanism for its effects remains unclear. We therefore examined whether MT-III can induce VEGF expression and promote neuroprotective effects in brain endothelial bEND.3 cells. MT-III significantly induced VEGF mRNA and protein expression in bEND.3 cells in a dose- and time-dependent manner. Furthermore, MT-III treatment increased the stability of hypoxia-inducible factor 1alpha (HIF-1alpha) and stimulated transcription of a reporter gene under control of the VEGF promoter. MT-III also increased the accumulation of HIF-1alpha in nuclei and increased HIF-1alpha-binding to the VEGF promoter. MT-III increased PI3K/Akt and ERK1/2 phosphorylation according to Western blot analysis. However, pretreatment with PD98059 and LY294002 (ERK1/2 and Akt inhibitors) inhibited MT-III-induced stimulation of HIF-1alpha protein expression and VEGF production. These results suggest that MT-III upregulates VEGF production in brain endothelial cells by a HIF-1alpha-dependent mechanism.

Metallothionein-III provides neuronal protection through activation of Nuclear Factor-κB via the TrkA/Phosphatidylinositol-3 kinase/Akt signaling pathway

Metallothionein (MT)-III is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. The present study investigated the mechanisms of MT-III protection of neuronal cells from hypoxia or DNA damage-induced cell death. MT-III reduced the hydrogen peroxide- or DNA damage-induced effects on neuronal cells, including the cell death, the activation of caspase-3 and -9, and the release of mitochondrial cytochrome c to the cytoplasm in a dose-dependent manner. MT-III also increased the activation of Akt, the phosphorylation and degradation of IkappaB, the nuclear translocation/accumulation and the transcriptional activity of nuclear factor-kappaB (NF-kappaB) in neuronal cells in a dose-dependent manner. The MT-III-induced antiapoptotic effects and increase in NF-kappaB activity were blocked by specific inhibitors of TrkA, phosphatidylinositol-3 kinase (PI3K), Akt, or NF-kappaB, indicating that MT-III provides neuronal protection by activating NF-kappaB through the TrkA/PI3K/Akt signaling pathway.

Chemopreventive effects of Furan-2-yl-3-pyridin-2-yl-propenone against 7,12-dimethylbenz[a]anthracene-inducible genotoxicity.

1-Furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) is an anti-inflammatory agent with a propenone moiety and chemically synthesized recently. In this study, we examined the chemopreventive effect of FPP-3 on 7,12-dimethylbenz[a]anthracene (DMBA)-induced genotoxicity in MCF-7 cells. FPP-3 reduced the formation of the DMBA-DNA adduct. DMBA-induced CYP1A1 and CYP1B1 gene expression and enzyme activity were inhibited by FPP-3. It inhibited DMBA-induced aryl hydrocarbon receptor (AhR) transactivation and DMBA-inducible nuclear localization of the AhR. Induction of detoxifying phase II genes by chemopreventive agents represents a coordinated protective response against oxidative stress and neoplastic effects of carcinogens. Transcription factor NF-E2 related factor 2 (Nrf2) regulates antioxidant response element (ARE) of phase II detoxifying and antioxidant enzymes, such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase (QR). FPP-3 increased the expression and enzymatic activity of GST and QR. Moreover, FPP-3 increased transcriptional activity of GST and QR. GST and QR induction and Nrf2 translocation by FPP-3 were blocked by the PKC inhibitor Gö6983, and the p38 inhibitor SB203580. These results reflected a partial role of PKC delta and p38 signaling in FPP-3-mediated GSTA and QR induction through nuclear translocation of Nrf2. Classically, chemopreventive agents either inhibit CYP metabolizing enzyme or induce phase II detoxifying enzymes. These results suggest that FPP-3 has a potent protective effect against DMBA-induced genotoxicity through modulating phase I and II enzymes and that it has potential as a chemopreventive agent.

Effect of troglitazone on CYP1A1 induction.

Several peroxisome proliferators enhance CYP1A1 activity, but the mechanisms involved in this enhancement remain unknown. In this study, we examined the effect of troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, on CYP1A1 gene expression and explored the mechanisms involved in these effects. Troglitazone increased gene expression of CYP1A1 mRNA and also increased CYP1A1-specific 7-ethoxyresorufin-O-deethylase (EROD) activity in a dose-dependent manner. Moreover, concomitant treatment with troglitazone and GW9662, a PPAR antagonist, markedly reduced the troglitazone-inducible EROD activity. Luciferase reporter assays using Hepa-1c1c7 cells showed a significant transactivation by troglitazone with a reporter plasmid containing a region from -1395 to +7 of the CYP1A1 gene. We found that a putative peroxisome proliferator-response element (PPRE) between -521 and -500 is located in the CYP1A1 gene promoter. Their inactivation by deletion mutagenesis suppressed the inductive effect of troglitazone on CYP1A1 promoter activation. Electrophoretic mobility shift assay revealed that troglitazone induced the activation of the PPAR-gamma to a form capable of binding specifically to the PPRE sequence of the CYP1A1 gene promoter. Furthermore, troglitazone increased the formation of the benzo[a]pyrene (BaP)-DNA adduct. Overall, our results suggest that troglitazone induces CYP1A1 enzyme activity and gene expression through PPAR-gamma activation, and may be involved in carcinogenesis.