• Korean Journal of Plant Resources
• /
• v.28 no.3
• /
• pp.297-304
• /
• 2015

The flower buds of Sophora japonica L (SF), as a well-known traditional Chinese medicinal herb, have been used to treat bleeding-related disorders such as hematochezia, hemorrhoidal bleeding, dysfunctional uterine bleeding, and diarrhea. However, no specific anti-cancer effect and its molecular mechanism of SF have been described. Thus, we performed in vitro study to investigate if treatment of SF affects activating transcription factor 3 (ATF3) expression and ATF3-mediated apoptosis in human colorectal cancer cells. The effects of SF on cell viability and apoptosis were measured by MTT assay and Western blot analysis against cleaved poly (ADP-ribose) polymerase (PARP). ATF3 activation induced by SF was evaluated using Western blot analysis, RT-PCR and ATF3 promoter assay. SF treatment caused decrease of cell viability and increase of apoptosis in a dose-dependent manner in HCT116 and SW480 cells. Exposure of SF activated the levels of ATF3 protein and mRNA via transcriptional regulation in HCT116 and SW480 cells. Inhibition of extracellular signal-regulated kinases (ERK) 1/2 by PD98059 and p38 by SB203580 attenuated SF-induced ATF3 expression and transcriptional activation. Ectopic ATF3 overexpression accelerated SF-induced cleavage of PARP. These findings suggest that SF-mediated apoptosis may be the result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

• Archives of Pharmacal Research
• /
• v.26 no.12
• /
• pp.1055-1060
• /
• 2003

Lysophosphatidic acid (LPA) is a well-known mitogen in various cell types. However, we found that LPA inhibits melanocyte proliferation. Thus, we further investigated the possible signaling pathways involved in melanocyte growth inhibition. We first examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by LPA. The activations of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were observed in concert with the inhibition of melanocyte proliferation by LPA, whereas p38 MAP kinase and Akt were not influenced by LPA. However, the specific inhibition of the ERK or JNK pathways by PD98059 or D-JNKI1, respectively, did not restore the antiproliferative effect. We next examined changes in the expression of cell cycle related proteins. LPA decreased cyclin $D_1 and cyclin D_2$ levels but increased $p21^{WAF1/CIP1}$ (p21) and $p27^{KIP1}$ (p27) levels, which are known inhibitors of cyclin-dependent kinase. Flow cytometric analysis showed the inhibition of DNA synthesis by a reduction in the S phase and an increase in the $G_0/G_1$ phase of the cell cycle. Our results suggest that LPA induces cell cycle arrest by regulating the expressions of cell cycle related proteins.

• Molecules and Cells
• /
• v.19 no.1
• /
• pp.60-66
• /
• 2005

Low density lipoproteins (LDL) play important roles in the pathogenesis of atherosclerosis. Diabetes is associated with accelerated atherosclerosis leading to cardiovascular disease in diabetic patients. Although LDL stimulates the proliferation of arterial smooth muscle cells (SMC), the mechanisms are not fully understood. We examined the effects of native LDL and glycated LDL on the extracellular signal-regulated kinase (ERK) pathway. Addition of native and glycated LDL to rat aorta SMCs (RASMCs) stimulated ERK phosphorylation. ERK phosphorylation was not affected by exposure to the $Ca^{2+}$ chelator BAPTA-AM but inhibition of protein kinase C (PKC) with GF109203X, inhibition of Src kinase with PP1 ($5{\mu}M$) and inhibition of phospholipase C (PLC) with U73122/U73343 ($5{\mu}M$) all reduced ERK phosphorylation in response to glycated LDL. In addition, pretreatment of the RASMCs with a cell-permeable mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059, $5{\mu}M$) markedly decreased ERK phosphorylation in response to native and glycated LDL. These findings indicate that ERK phosphorylation in response to glycated LDL involves the activation of PKC, PLC, and MEK, but is independent of intracellular $Ca^{2+}$.

• Korean Journal of Plant Resources
• /
• v.29 no.3
• /
• pp.297-304
• /
• 2016

The seed of safflower (Carthamus tinctorius L) has been reported to suppress human cancer cell proliferation. However, the mechanisms by which safflower seed inhibits cancer cell proliferation have remained nuclear. In this study, the inhibitory effect of the safflower seed (SS) on the proliferation of human colorectal cancer cells and the potential mechanism of action were examined. SS inhibited markedly the proliferation of human colorectal cancer cells (HCT116, SW480, LoVo and HT-29). In addition, SS suppressed the proliferation of human breast cancer cells (MDA-MB-231 and MCF-7). SS treatment decreased cyclin D1 protein level in human colorectal cancer cells and breast cancer cells. But, SS-mediated downregulated mRNA level of cyclin D1 was not observed. Inhibition of proteasomal degradation by MG132 attenuated cyclin D1 downregulation by SS and the half-life of cyclin D1 was decreased in SS-treated cells. In addition, SS increased cyclin D1 phosphorylation at threonine-286 and a point mutation of threonine-286 to alanine attenuated SS-mediated cyclin D1 degradation. Inhibition of ERK1/2 by PD98059 suppressed cyclin D1 phosphorylation and downregulation of cyclin D1 by SS. In conclusion, SS has anti-proliferative activity by inducing cyclin D1 proteasomal degradation through ERK1/2-dependent threonine-286 phosphorylation of cyclin D1. These findings suggest that possibly its extract could be used for treating colorectal cancer.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.4
• /
• pp.399-406
• /
• 2016

Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.4
• /
• pp.347-355
• /
• 2016

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha ($TNF-{\alpha}$) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of $TNF-{\alpha}$ on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe $CM-DCFH_2-DA$. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. $TNF-{\alpha}$ induced LOX-1 expression in a dose- and time- dependent manner in endothelial cells. $TNF-{\alpha}$ induced ROS formation, phosphorylation of $NF-{\kappa}B$ p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. $NF-{\kappa}B$ inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited $TNF-{\alpha}-induced$ LOX-1 expression. CPT and NAC suppressed $TNF-{\alpha}-induced$ LOX-1 expression and phosphorylation of $NF-{\kappa}B$ p65 and ERK in rat aorta. These data suggested that $TNF-{\alpha}$ induced LOX-1 expression via ROS activated $NF-{\kappa}B/ERK$ pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

• Environmental Analysis Health and Toxicology
• /
• v.20 no.1
• /
• pp.75-85
• /
• 2005

This study was aimed to know the effect of cadmium chloride (CdCl₂) on glucose transport in L6 myotube and its action mechanism. CdCl₂ increased the 2-deoxy- (l-3H)-D-glucose (2-DOG) uptake 1.9 and 2.4 fold at 10 and 25 μM respectively. To investigate the stimulating-mechanism of glucose transport induced by CdCl₂, the wortmannin and PD98059 were used as PI3K (phosphatidylinositol 3-kinase) inhibitor and MAPK inhibitor respectively, which did not affect 2-DOG uptake. This fact suggests that CdCl₂ induced 2-DOG uptake may not be concerned to the insulin signalling pathway. Whereas nifedipine, a calcium channel blocker, and trifluoperazine, a calmodulin inhibitor, were found to inhibit the 2-DOG uptake stimulted by CdCl₂. In addition, we also measured the ROS (reactive oxygen species) production and GSH level in L6 myotube to investigate the correlation between the glucose uptake and ROS. CdCl₂(25 μM) increased ROS generation approximately 1.5 fold and changed the cellular GSH level, but GSSG/GSH ratio remained unchanged. CdCl₂ stimulated 2-DOG uptake and ROS generation were inhibited by N-acetylcystein. And BSO pretreatment, a potent inhibitor of γ-GCS, resulted in the dramatic decrease of 2-DOG uptake and also the increase of the sensitivity to cadmium cytotoxicity. The obtained results suggest that CdCl₂-stimulated glucose uptake might be based on the activation of HMP shunt as an antioxidant defense mechanism of the cells.

• Environmental Analysis Health and Toxicology
• /
• v.20 no.1
• /
• pp.87-95
• /
• 2005

Cadmium is well known as a toxic metal and has insulin mimicking effects in rat adipose tissue. This study was undertaken to investigate the effect of CdCl₂ on glucose transport and its mechanism in 3T3 - L1 adipocytes. CdCl₂ exhibits respectively 2.2 and 2.8 fold increases in the 2-deoxyglucose uptake when exposed to 10 and 25 μM of CdCl₂ for 12 hr. To investigate the stimulating mechanism of glucose transport induced by CdCl₂. Wortmannin and PD98059 were used respectively as PI3K inhibitor and MAPK inhibitor, which did not affect 2-DOG uptake. This results suggest that induced 2-deoxy-(l-3H)-D-glucose (2-DOG) uptake by CdCl₂ may not be concerned with the insulin signalling pathway. Whereas nifedipine, a calcium channel blocker inhibited the 2- DOG uptake stimulated by CdCl₂. In addition, we also measured the increased production of Reactive oxygen substances (ROS) and glutathione (GSH) level in 3T3-L1 adipocytes to investigate correlation between the glucose uptake and increased production of ROS with H2DCFDA. CdCl₂ increased production of ROS. Induced 2-DOG uptake and increased production of ROS by CdCl₂ were decreased by N-acetylcystein (NAC). And L-buthionine sulfoximine (BSO) a potent inhibitor of γ-GCS, decreased of 2-DOG uptake. Also NAC and BSO changed the cellular GSH level, but GSH/GSSG ratio remained unchanged at 10, 25 μM of CdCl₂.

• Journal of Periodontal and Implant Science
• /
• v.34 no.3
• /
• pp.489-498
• /
• 2004

Recent studies have demonstrated that human periodontal ligament cells express receptor activation of nuclear factor ${\kappa}B$ ligand (RANKL) which enhances the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The purpose of this study is to determine the effects of p38 MAPK and JNK kinase upon regulating RANKL and OPG in response to $IL-1{\beta}$(l ng/ml) in rat periodontal ligament cells. Soluble RANKL was measured by immunoassay. The effects of p38 MAPK on RANKL and OPG expression was determined by RT-PCR. The results were as follows: 1. Periodontal ligament cells which stimulated by $IL-1{\beta}$ increased soluble RANKL synthesis by dose-dependent pattern. 2. p38 MAP kinase inhibitor (SB203580) showed regulation of soluble RANKL expression by dose-dependent manners. 3. p38 MAP kinase inhibitor (SB203580) regulated the expression of RANKL, but it dose regulate the expresseion of OPG. 4. JNK (c-jun $NH_2-terminal$ kinase) inhibitor (PD98059) did not regulate mRANKL and mOPG. These results suggested that p38 MAPK play a significant role in RANKL gene expression.

• BMB Reports
• /
• v.37 no.4
• /
• pp.480-486
• /
• 2004

Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its $IC_{50}$ value was $400\;{\mu}M$. The DNA fragment became evident after incubation of the cells with $300\;{\mu}M$ cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).