• Biomolecules & Therapeutics
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• v.21 no.4
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• pp.258-263
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• 2013

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-${\alpha}$, and $IL1{\beta}$. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.

• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.99-105
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• 2022

This study assessed the antioxidant and anti-inflammatory activities of leaf extracts from grain amaranths (Amaranthus spp.). Among all the extracts, the ethanol extract of Amaranthus cruentus leaves (Ar) exhibited the highest antioxidant activity, including the DPPH free radical scavenging activity and ORAC. In addition, Ar strongly inhibited nitric oxide production by suppressing the MEK/ERK signaling pathway in lipopolysaccharide-stimulated RAW264 murine macrophages. HPLC analysis revealed 13 polyphenolic compounds in the leaf extracts of grain amaranth and indicated that Ar contained more rutin than the other extracts. Taken together, these results show the impact of species diversity on the phytochemical contents and bioactivities of plant extracts and suggest that the nonedible parts, such as leaves, of A. cruentus should be considered for use as crude drugs and dietary health supplements.

• Molecules and Cells
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• v.26 no.5
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• pp.468-473
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• 2008

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that $Cl^-$ channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of $Cl^-$ channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. $Cl^-$ channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated $Cl^-$ concentration, as judged by flow cytometry analysis using MQAE as a $Cl^-$-detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS ($Cl^-$ channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive $Cl^-$-channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.31-37
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• 2020

We previously identified a new heparinase inhibitor fungal metabolite, named CRM646-A, which showed inhibition of heparinase and telomerase activities in an in vitro enzyme assay and antimetastatic activity in a cell-based assay. In this study, we elucidated the mechanism by which CRM646-A rapidly induced nucleus condensation, plasma membrane disruption and morphological changes by increasing intracellular Ca²⁺ levels. Furthermore, PD98059, a mitogen-activated protein kinase (MEK) inhibitor, inhibited CRM646-A-induced nucleus condensation through ERK1/2 activation in rat 3Y1 fibroblast cells. We identified CRM646-A as a Ca²⁺ ionophore-like agent with a distinctly different chemical structure from that of previously reported Ca²⁺ ionophores. These results indicate that CRM646-A has the potential to be used as a new and effective antimetastatic drug.

• Development and Reproduction
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• v.6 no.1
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• pp.1-6
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• 2002

To elucidate the mechanism underlying the embryotropic effect of extracellular matrix(ECM) on the preimplantation development of mammalian embryos, the involvement of mitogen-activated protein kinase(MAPK) downstream the integrin signaling was examined in mouse blastocysts. Blastocysts were cultured in the presence of growth factor-reduced(GFR) Matrigel(0.5％, v/v). MAPK activity was measured by in vitro phosphorylation of myelin basic protein by the Erk1/2 antibody immunoprecipitates of embryonic extract following the Matrigei treatment. MAPK activity of the early blastocysts rapidly increased within 10 min fo1lowing the Matrigel treatment. When the embryos were cultured for 12 h in the presence of Matrigel, the MAPK activity was significantly higher than that ot the control embryos. PD098059, a MAPK kinase(MEK) inhibitor, attenuated the effect of Matrigel on the change in MAPK activity. Taken together, it suggested that the embryotropic effect of ECM proteins might be mediated by the activation of MAPK cascade.

• Herbal Formula Science
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• v.15 no.2
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• pp.127-137
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• 2007

The purpose of this study was to investigate the effect of Yong-dam-sa-gan-tang (YST) on apoptosis in HepG2 cells, First of all. to study the cytotoxic effect of methanol extract of YST on HepG2 cells, the cells were treated with various concentrations of YST and then cell viability was determined by XTT reduction method and trypan blue exclusion assay. YST reduced proliferation of HepG2 cells in a dose-dependent manner. To confirm the induction of apoptosis, HepG2 cells were treated with various concentrations of YST. The cleavage of poly AD P-ribose polymerase (P ARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of caspase-3, procaspase-8 and procaspase-8 were examined by western blot analysis. YST decreased procaspase-3, procaspase-8 and procaspase-9 levels in a dose-dependent manner and induced the clevage of PARP. YST triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome c from mitochondria to cytosol. Furthermore, YST also downregulated the anti-apoptotic Bcl-2 and upregulated the pro-apoptotic-Bax. Therefore, this result suggest that YST induced HepG2 cell death through the mitochondrial pathway. Sustained activation of the Ras/Raf/MEK/ERK cascade in cells results in a cell cycle arrest and has been implicated in the differentiation of certain cell types, in many cases acting to promote differentiation. YST decreased the activation of Ras/Raf/MEK/ERK cascade in a dose-dependent manner. These results suggest that YST is potentially useful as a chemo-therapeutic agent in HepG2.

• IMMUNE NETWORK
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• v.9 no.5
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• pp.192-202
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• 2009

Background: Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages. Methods: Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors. Results: Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-${\gamma}$ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-${\kappa}B$ and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-${\gamma}$ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3. Conclusion: These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

• Archives of Pharmacal Research
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• v.27 no.3
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• pp.324-333
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• 2004

To determine whether Insulin-like growth factor (IGF-I) treatment represents a potential means of enhancing the survival of cardiac muscle cells from adriamycin (ADR)-induced cell death, the present study examined the ability of IGF-I to prevent cell death. The study was performed utilising the embryonic, rat, cardiac muscle cell line, H9C2. Incubating cardiac muscle cells in the presence of adriamycin increased cell death, as determined by MTT assay and annexin V-positive cell number. The addition of 100 ng/mL IGF-I, in the presence of adriamycin, decreased apoptosis. The effect of IGF-I on phosphorylation of PI, a substrate of phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase B (AKT), was also examined in H9C2 cardiac muscle cells. IGF-I increased the phosphorylation of ERK 1 and 2 and $PKC{\;}{\zeta}{\;}kinase$. The use of inhibitors of PI 3-kinase (LY 294002), in the cell death assay, demonstrated partial abrogation of the protective effect of IGF-I. The MEK1 inhibitor-PD098059 and the PKC inhibitor-chelerythrine exhibited no effect on IGF-1-induced cell protection. In the regulatory subunit of PI3K-p85- dominant, negative plasmid-transfected cells, the IGF-1-induced protective effect was reversed. This data demonstrates that IGF-I protects cardiac muscle cells from ADR-induced cell death. Although IGF-I activates several signaling pathways that contribute to its protective effect in other cell types, only activation of PI 3-kinase contributes to this effect in H9C2 cardiac muscle cells.

• Molecules and Cells
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• v.42 no.5
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• pp.397-405
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• 2019

The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in both cancerous and noncancerous cells have been widely reported. This study aimed to evaluate the role of lncRNA GAS5 in heart failure caused by myocardial infarction. We reported that silence of lncRNA GAS5 attenuated hypoxia-triggered cell death, as cell viability was increased and apoptosis rate was decreased. This phenomenon was coupled with the down-regulated expression of p53, Bax and cleaved caspase-3, as well as the up-regulated expression of CyclinD1, CDK4 and Bcl-2. At the meantime, the expression of four heart failure-related miR-NAs was altered when lncRNA GAS5 was silenced (miR-21 and miR-142-5p were up-regulated; miR-30b and miR-93 were down-regulated). RNA immunoprecipitation assay results showed that lncRNA GAS5 worked as a molecular sponge for miR-142-5p. More interestingly, the protective actions of lncRNA GAS5 silence on hypoxia-stimulated cells were attenuated by miR-142-5p suppression. Besides, TP53INP1 was a target gene for miR-142-5p. Silence of lncRNA GAS5 promoted the activation of PI3K/AKT and MEK/ERK signaling pathways in a miR-142-5p-dependent manner. Collectively, this study demonstrated that silence of lncRNA GAS5 protected H9c2 cells against hypoxia-induced injury possibly via sponging miR-142-5p, functionally releasing TP53INP1 mRNA transcripts that are normally targeted by miR-142-5p.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.743-748
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• 2016

Background: $K^-Ras$ activation is an early event in colorectal carcinogenesis and associated mutations have been reported in about 40% of colorectal cancer patients. These mutations have always been responsible for enhancing malignancy and silencing them is associated with attenuation of tumorigenicity. Among downstream effectors are the RAF/MEK/ERK and the PI3K/Akt signaling pathways. PI3K/Akt signaling leads to reduction of apoptosis, stimulated cell growth and enhanced proliferation. Ellagic acid (EA), a naturally occurring antioxidant, has recently emerged as a promising anti-cancer agent. Purpose: To evaluate the impact of cellular genetic makeup of two colon cancer cell lines with different genetic backgrounds, HCT-116 ($K^-Ras^-/p53^+$) and Caco-2 ($K^-Ras^+/p53^-$), on response to potential anti-tumour effects of EA. In addition, the influence of $K^-Ras$ silencing in HCT-116 cells was investigated. Materials and Methods: Cellular proliferation, morphology and cell cycle analysis were carried out in addition to Western blotting for detecting total Akt and p-Akt (at Thr308 and Ser473) in the presence and absence of different concentrations of EA. Cell proliferation was also assessed in cells transfected with different concentrations of $K^-Ras$ siRNA or incubated with ellagic acid following transfection. Results: The results of the present study revealed that EA exerts anti-proliferative and dose-dependent pro-apoptotic effects. Cytostatic and cytotoxic effects were also observed. p-Akt (at Thr308 and Ser473) was downregulated. Moreover, EA treatment was found to (i) reduce $K^-Ras$ protein expression; (ii) in cells transfected with siRNA and co-treated with EA, pronounced anti-proliferative effects as well as depletion of p-Akt (at Thr308) were detected. Conclusions: Cellular genetic makeup ($K^-Ras^-/p53^-$) was not likely to impose limitations on targeting EA in treatment of colon cancer. EA had a multi-disciplinary pro-apoptotic anti-proliferative approach, having inhibited Akt phosphorylation, induced cell cycle arrest and showed an anti-proliferative potential in HCT-116 cells (expressing mutant $K^-Ras$).