• Journal of Life Science
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• v.31 no.11
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• pp.987-994
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• 2021

Our previous studies have reported that antimicrobial peptides (AMPs) derived from the larvae of white-spotted flower chafer (Protaetia brevitarsis seulensis) exert anti-inflammatory and neuroprotective activities. This study explored the anti-inflammatory effects of protaetiamycine 9 (CVLKKAYFLTNLKLRG-NH₂), a novel AMP, derived from P. b. seulensis against lipopolysaccharide (LPS)-mediated inflammatory response in RAW264.7 macrophage cells. Protaetiamycine 9 (25, 50, 75, and 100 ㎍/ml) did not cause cytotoxic effects against RAW264.7 cells. The RAW264.7 cells were pre-treated with various concentrations of protaetiamycine 9 (25-100 ㎍/ml) for 1 hr and then exposed to LPS (100 ng/ml) for 24 hr. Protaetiamycine 9 treatments decreased the LPS-induced secretion of inflammatory mediators, such as nitric oxide (NO), in a dose-dependent manner. Protaetiamycine 9 (25-100 ㎍/ml) effectively downregulated the LPS-induced increase in mRNA and the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are involved in the production of inflammatory mediators. Protaetiamycine 9 also suppressed the production and gene expression of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-1β, compared to the presence of LPS alone. Furthermore, protaetiamycine 9 inhibited the degradation of inhibitory kappa B alpha (IκB-α) and the phosphorylation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. In conclusion, these results suggest that protaetiamycine 9 exhibits LPS-mediated inflammatory responses by blocking IκB-α degradation and MAPK phosphorylation.

• Journal of Life Science
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• v.28 no.2
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• pp.162-169
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• 2018

Many cytoplasmic proteins are targeted to the cytoplasmic membrane of the trans-Golgi network (TGN) via an N-terminal short helix. We previously showed that the 20 N-terminal amino acids of Aplysia phosphodiesterase 4 (ApPDE4) long form are sufficient for its targeting to the plasma membrane and the TGN. The N-terminus of the ApPDE4 long form binds to PI4P and sulfatide in vitro. Therefore, in order to decipher the roles of sulfatide in Golgi complex targeting, we examined the cellular localization of sulfatide-binding peptides. In this study, we found that enhanced green fluorescent protein (EGFP) fused to the C-terminus of modified sulfatide- and heparin-binding peptides (mHSBP-EGFP) was localized to the TGN. On the other hand, its mutant, in which tryptophan was replaced with an alanine, leading to the impairment of heparin and sulfatide binding, was localized to cytosol. We also found that the TGN targeting of mHSBP-EGFP is impaired by the treatment of antimycin A, phenylarsine oxide (PAO), and adenosine but not a high concentration of wortmannin. These results suggest that PAO and adenosine-sensitive kinases, including phosphatidylinositol 4-kinase II, may play key roles in the recruitment of mHSBP-EGFP.

• KSBB Journal
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• v.20 no.1 s.90
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• pp.21-25
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• 2005

Paraquat, a widely used herbicide, has been suggested as a potential risk factor for Parkinson's disease. Heme oxygenase-1(HO-1), a marker for oxidative stress and endoplasmic reticulum(ER) stress, is known to catalyze heme to biliverdin, carbon monoxide and free iron in response to various stimuli. Here we show that paraquat activates HO-1 expression in a time-and dose-dependent manner in substantia nigra(SN) dopaminergic neuronal cells. Activation of Ho-1 by paraquat was regulated primarily at the level of gene transcription. Deletion analysis of the promoter and the 5' distal enhancers, E1 and E2, of the HO-1 gene revealed that the E2 enhancer is a potent inducer of the paraquat-dependent Ho-1 gene expression in dopamninergic neuronal cells. Mutational analysis of the E2 enhacer further demonstrated that the transcription factor activator protein-1(AP-1) plays an important role in mediating paraquat-induced HO-1 gene transcription. Moreover, using specific inhibitors of the mitogen-activated protein kinases(MAPKs), we investigated the role of paraquat and MAPKs for HO-1 gene regulation in dopaminergic cells. The c-Jun N-terminal kinase(JNK) inhibitor SP600125 significantly suppressed the expression of HO-1 by paraquat. All these results demonstrate that induction of HO-1 by paraquat requies the activation of the AP-1 and JNK pathway.

• International Journal of Oral Biology
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• v.34 no.2
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• pp.115-121
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• 2009

Bone morphogenetic protein (BMP) 2 is a potent osteogenic factor. Although both Smad1/5 and mitogenactivated protein kinases (MAPKs) are activated by BMP2, the hierarchical relationship between them is unclear. In this study, we examined if BMP2-stimulated MAPK activation is regulated by Smad1/5 or vice versa. When C2C12 cells were treated with BMP2, the activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-N-terminal kinase was evident within 5 min. The knockdown of both Smad1 and Smad5 by small interfering RNA did not affect the activation of these MAPKs. In addition, neither the overexpression of Smad1 nor Smad5 induced ERK activation. When ERK activation was induced by constitutively active MEK1 expression, the protein level and activation of Smad1 increased. Furthermore, the inhibition of constitutively active BMP receptor type IB-induced ERK activation significantly suppressed Smad1 activation. These results indicate that Smad1/5 activation is not necessary for BMP2-induced MAPK activation and also that ERK positively regulates Smad1 activation.

• Biomolecules & Therapeutics
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• v.25 no.5
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• pp.519-527
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• 2017

Excessive activation of microglia causes the continuous production of neurotoxic mediators, which further causes neuron degeneration. Therefore, inhibition of microglial activation is a possible target for the treatment of neurodegenerative disorders. Balanophonin, a natural neolignoid from Firmiana simplex, has been reported to have anti-inflammatory and anti-cancer effects. In this study, we aimed to evaluate the anti-neuroinflammatory effects and mechanism of balanophonin in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. BV2 microglia cells were stimulated with LPS in the presence or absence of balanophonin. The results indicated that balanophonin reduced not only the LPS-mediated TLR4 activation but also the production of inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), $Interleukin-1{\beta}$ ($IL-1{\beta}$), and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), in BV2 cells. Balanophonin also inhibited LPS-induced inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) protein expression and mitogen activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK. Interestingly, it also inhibited neuronal cell death resulting from LPS-activated microglia by regulating cleaved caspase-3 and poly ADP ribose polymerase (PARP) cleavage in N2a cells. In conclusion, our data indicated that balanophonin may delay the progression of neuronal cell death by inhibiting microglial activation.

• Biomolecules & Therapeutics
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• v.24 no.5
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• pp.501-509
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• 2016

Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin's biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35-250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/agent for cancer chemotherapy.

• Journal of Ginseng Research
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• v.40 no.2
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• pp.135-140
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• 2016

Background: Nephrotoxicity is a common side effect of medications. Panax ginseng is one of the best-known herbal medicines, and its individual constituents enhance renal function. Identification of its efficacy and mechanisms of action against drug-induced nephrotoxicity, as well as the specific constituents mediating this effect, have recently emerged as an interesting research area focusing on the kidney protective efficacy of P. ginseng. Methods: The present study investigated the kidney protective effect of fermented black ginseng (FBG) and its active component ginsenoside 20(S)-Rg3 against cisplatin (chemotherapy drug)-induced damage in pig kidney (LLC-PK1) cells. It focused on assessing the role of mitogen-activated protein kinases as important mechanistic elements in kidney protection. Results: The reduced cell viability induced by cisplatin was significantly recovered with FBG extract and ginsenoside 20(S)-Rg3 dose-dependently. The cisplatin-induced elevated protein levels of phosphorylated c-Jun N-terminal kinase (JNK), p53, and cleaved caspase-3 were decreased after cotreatment with FBG extract or ginsenoside 20(S)-Rg3. The elevated percentage of apoptotic LLC-PK1 cells induced by cisplatin treatment was significantly abrogated by cotreatment with FBG and the ginsenoside 20(S)-Rg3. Conclusion: FBG and its major ginsenoside 20(S)-Rg3, ameliorated cisplatin-induced nephrotoxicity in LLC-PK1 cells by blocking the JNKep53ecaspase-3 signaling cascade.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.1-7
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• 2011

Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 112 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.

• The Korea Journal of Herbology
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• v.28 no.5
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• pp.7-11
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• 2013

Objectives : Seungmagalgeun-tang (SMGGT) is traditional medicine widely used for inflammatory disease and flu. But SMGGT exhibits potent anti-inflammatory activity with an unknown mechanism. To elucidate the molecular mechanisms of SMGGT water extract on pharmacological and biochemical actions in inflammation, we examined the effect of SMGGT on pro-inflammatory mediators in Phorbol-12-myristate-13-acetate (PMA)+A23187-stimulated mast cells. Methods : In the present study, pro-inflammatory cytokine production was determined by performing enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and western blot analysis to measure the activation of MAPKs. Cells were treated with SMGGT 1 h prior to the addition of 50 nM of PMA and $1{\mu}M$ of A23187. Cell viability was measured by MTS assay. The investigation focused on whether SMGGT inhibited the expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and mitogen-activated protein kinases (MAPKs) in PMA+A23187-stimulated mast cells. Results : SMGGT has no cytotoxicity at examined concentration (100, 250, and $500{\mu}g/ml$). Also, gene expression of IL-6 and IL-8 in HMC-1 cells stimulated by PMA+A23187 was down regulated by SMGGT. Furthermore, SMGGT suppressed the PMA+A23187-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal Kinase(JNK). But, SMGGT could not regulate phosphorylation of p38 MAPK. Conclusions : These results suggest that SMGGT has inhibitory effects on PMA+A23187-induced IL-6 and IL-8 production. These inhibitory effects occur through blockades on the phosphorylation of ERK and JNK.

• Preventive Nutrition and Food Science
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• v.21 no.1
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• pp.38-43
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• 2016

Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that take important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Natural products are on the rise for their potential to provide remarkable health benefits. In this context, halophytes have been of interest in the nutraceutical field with reported instances of isolation of bioactive compounds. In this study, Limonium tetragonum, an edible halophyte, was studied for its ability to inhibit MMP-2 and -9 using HT1080 fibrosarcoma cells. Results showed that L. tetragonum extract was able to inhibit the enzymatic activity and mRNA expression of MMP-2 and -9 according to gelatin zymography and RT-PCR assays, respectively, but it was not able to significantly change the MMP pathway related factors such as tissue inhibitors of metalloproteinases. Also, Mitogen-activated protein kinases pathway-related protein levels and their phosphorylation were assayed. While the phosphorylated p38 levels were decreased, extracellular signal-regulated kinase and c-Jun N-terminal kinase were not affected by L. tetragonum treatment. In conclusion, it was suggested that L. tetragonum contains substances acting as MMP inhibitors on enzymatic activity rather than intracellular pathway intervention, which could be useful for further utilization of L. tetragonum as a source for anti-MMP agents.