• Asian Pacific Journal of Cancer Prevention
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• v.15 no.15
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• pp.6391-6395
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• 2014

Overexpression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases 1/2 (Erk1/2) are associated with tumorigenesis and cancer progression and may upregulate AQP expression. In this study, we demonstrated that EGF (epidermal growth factor) induces SiHa cells migration and AQP8 expression. Wound healing results showed that cell migration was increased by 2.79-1.50-fold at 24h and 48h after EGF treatment. AQP8 expression was significantly increased (3.33-fold) at 48h after EGF treatment in SiHa cells. An EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration and AQP8 expression was decreased from 1.59-fold (EGF-treated) to 0.43-fold (PD153035-treated) in SiHa. Furthermore, the MEK (MAPK (mitogen-activated protein kinase)/Erk (extracellular signal regulated kinase)/Erk inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 1.21-fold (EGF-treated) to 0.43-fold (U0126-treated). Immunofluorescence microscopy further confirmed the results. Collectively, our findings show that EGF induces AQP8 expression and cell migration in human cervical cancer SiHa cells via the EGFR/Erk1/2 signal transduction pathway.

• Molecules and Cells
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• v.25 no.4
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• pp.479-486
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• 2008

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with $10{\mu}M$ PD98059 prevented myogenin expression in response to a low dose of SB203580 ($3{\mu}M$) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.

• Molecules and Cells
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• v.39 no.10
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• pp.742-749
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• 2016

The cancer chemo-preventive effects of equol have been demonstrated for a wide variety of experimental tumours. In a previous study, we found that equol inhibited proliferation and induced apoptotic death of human gastric cancer MGC-803 cells. However, the mechanisms underlying equol-mediated apoptosis have not been well understood. In the present study, the dual AO (acridine orange)/EB (ethidium bromide) fluorescent assay, the comet assay, MTS, western blotting and flow cytometric assays were performed to further investigate the pro-apoptotic effect of equol and its associated mechanisms in MGC-803 cells. The results demonstrated that equol induced an apoptotic nuclear morphology revealed by AO/EB staining, the presence of a comet tail, the cleavage of caspase-3 and PARP and the depletion of cIAP1, indicating its pro-apoptotic effect. In addition, equol-induced apoptosis involves the mitochondria-dependent cell-death pathway, evidenced by the depolarization of the mitochondrial membrane potential, the cleavage of caspase-9 and the depletion of Bcl-xL and full-length Bid. Moreover, treating MGC-803 cells with equol induced the sustained activation of extracellular signal-regulated kinase (ERK), and inhibiting ERK by U0126, a MEK/ERK pathway inhibitor, significantly attenuated the equol-induced cell apoptosis. These results suggest that equol induces mitochondria-dependent apoptosis in human gastric cancer MGC-803 cells via the sustained activation of the ERK1/2 pathway. Therefore, equol may be a novel candidate for the chemoprevention and therapy of gastric cancer.

• Biomolecules & Therapeutics
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• v.21 no.3
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• pp.196-203
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• 2013

Recent accumulating studies have reported that hypoxic preconditioning during ex vivo expansion enhanced the self-renewal or differentiation of various stem cells and provide an important strategy for the adequate modulation of oxygen in culture conditions, which might increase the functional bioactivity of these cells for cardiac regeneration. In this study, we proposed a novel priming protocol to increase the functional bioactivity of cardiac progenitor cells (CPCs) for the treatment of cardiac regeneration. Firstly, patient-derived c-$kit^+$ CPCs isolated from the atrium of human hearts by enzymatic digestion and secondly, pivotal target molecules identified their differentiation into specific cell lineages. We observed that hCPCs, in response to hypoxia, strongly activated ERK phosphorylation in ex vivo culture conditioning. Interestingly, pre-treatment with an ERK inhibitor, U0126, significantly enhanced cellular proliferation and tubular formation capacities of CPCs. Furthermore, we observed that hCPCs efficiently maintained the expression of the c-kit, a typical stem cell marker of CPCs, under both hypoxic conditioning and ERK inhibition. We also show that hCPCs, after preconditioning of both hypoxic and ERK inhibition, are capable of differentiating into smooth muscle cells (SMCs) and cardiomyocytes (CMs), but not endothelial cells (ECs), as demonstrated by the strong expression of ${\alpha}$-SMA, Nkx2.5, and cTnT, respectively. From our results, we conclude that the functional bioactivity of patient-derived hCPCs and their ability to differentiate into SMCs and CMs can be efficiently increased under specifically defined culture conditions such as short-term hypoxic preconditioning and ERK inhibition.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.268-276
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• 2017

Background: UV-B-exposed keratinocytes secrete various paracrine factors. Among these factors, basic fibroblast growth factor (bFGF) stimulates the proliferation of melanocytes. Ginsenosides, the major active compounds of ginseng, are known to have broad pharmacological effects. In this study, we examined the antiproliferative effects of ginsenosides on bFGF-induced melanocyte proliferation. Methods: We investigated the inhibitory effects of Korean Red Ginseng and ginsenosides from Panax ginseng on bFGF-induced proliferation of melan-a melanocytes. Results: When melan-a melanocytes were treated with UV-B-irradiated SP-1 keratinocytes media, cell proliferation increased. This increased proliferation of melanocytes decreased with a neutralizing anti-bFGF antibody. To elucidate the effects of ginsenosides on melanocyte proliferation induced by bFGF, we tested 15 types of ginsenoside compounds. Among them, Rh3, Rh1, F1, and CK demonstrated antiproliferative effects on bFGF-induced melanocyte proliferation after 72 h of treatment. bFGF stimulated cell proliferation via extracellular signal-regulated kinase (ERK) activation in various cell types. Western blot analysis found bFGF-induced ERK phosphorylation in melan-a. Treatment with Rh3 inhibited bFGF-induced maximum ERK phosphorylation and F1-delayed maximum ERK phosphorylation, whereas Rh1 and CK had no detectable effects. In addition, cotreatment with Rh3 and F1 significantly suppressed bFGF-induced ERK phosphorylation. Western blot analysis found that bFGF increased microphthalmia-associated transcription factor (MITF) protein levels in melan-a. Treatment with Rh3 or F1 had no detectable effects, whereas cotreatment with Rh3 and F1 inhibited bFGF-induced MITF expression levels more strongly than a single treatment. Conclusion: In summary, we found that ginsenosides Rh3 and F1 have a synergistic antiproliferative effect on bFGF-induced melan-a melanocyte proliferation via the inhibition of ERK-mediated upregulation of MITF.

• Molecules and Cells
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• v.41 no.5
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• pp.436-443
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• 2018

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

• Biomolecules & Therapeutics
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• v.25 no.2
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• pp.186-193
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• 2017

Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, has been reported to have a radiosensitization effect on tumors. However, its effects on human glioma and the specific molecular mechanisms of these effects remain unknown. In this study, we demonstrated that Tet has a radiosensitization effect on human glioma cells. It has been hypothesized that Tet has a radiosensitization effect on glioma cells by affecting the glioma cell cycle and DNA repair mechanism and that ERK mediates these activities. Therefore, we conducted detailed analyses of the effects of Tet on the cell cycle by performing flow cytometric analysis and on DNA repair by detecting the expression of phosphorylated H2AX by immunofluorescence. We used western blot analysis to investigate the role of ERK in the effect of Tet on the cell cycle and DNA repair. The results revealed that Tet exerts its radiosensitization effect on glioma cells by inhibiting proliferation and decreasing the expression of phosphorylated ERK and its downstream proteins. In summary, our data indicate that ERK is involved in Tet-induced radiosensitization of glioma cells via inhibition of glioma cell proliferation or of the cell cycle at G0/G1 phase.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.90-90
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• 2019

Hibiscus syriacus exhibited promising potential as a new source of food and colorants containing various anthocyanins. However, the function of anthocyanins from H. syriacus has not been investigated. In the current study, we evaluated whether anthocyanins from the H. syriacus varieties Pulsae and Paektanshim (PS and PTS) inhibit melanin biogenesis. B16F10 cells and zebrafish larvae were exposed to PS and PTS in the presence or absence of ${\alpha}$-melanocyte-stimulating hormone (${\alpha}$-MSH), and melanin contents accompanied by its regulating genes and proteins were analyzed. PS and PTS moderately downregulated mushroom tyrosinase activity in vitro, but significantly decreased extracellular and intracellular melanin production in B16F10 cells, and inhibited ${\alpha}$-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. PS and PTS also attenuated pigmentation in ${\alpha}$-MSH-stimulated zebrafish larvae. Furthermore, PS and PTS activated the phosphorylation of extracellular signal-regulated kinase (ERK), whereas PD98059, a specific ERK inhibitor, completely reversed PS- and PTS-mediated anti-melanogenic activity in B16F10 cells and zebrafish larvae, which indicates that PS- and PTS-mediated anti-melanogenic activity is due to ERK activation. Moreover, chromatography data showed that PS and PTS possessed 17 identical anthocyanins as a negative regulator of ERK. These findings suggested that anthocyanins from PS and PTS inhibited melanogenesis in vitro and in vivo by activating the ERK signaling pathway.

• Archives of Pharmacal Research
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• v.26 no.9
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• pp.739-746
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• 2003

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.6
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• pp.593-601
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• 2021

Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H₂S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H₂S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H₂S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H₂S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-β-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H₂S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H₂S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H₂S and its downstream targets could be novel molecular targets for regulating primary cilia.