So Young Eun

Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells

Yeast-based functional screening of a human glioblastoma cDNA library identified ras-related nuclear protein (Ran) as a novel suppressor of Bcl-2-associated X protein (Bax), a pro-apoptotic member of the Bcl-2 family of proteins. Yeast cells that expressed human Ran were resistant to Bax-induced cell death. In U373MG glioblastoma cells, stable overexpression of Ran significantly attenuated apoptotic cell death induced by the chemotherapeutic agent paclitaxel. FACS analysis demonstrated that Ran is involved in paclitaxel-induced cell cycle arrest. Stable overexpression of Ran also markedly inhibited the phosphorylation of Bcl-2 by paclitaxel, and inhibited the translocation of Bax, the release of cytochrome c and activation of caspase-3. Paclitaxel-induced phosphorylation of c-JUN N-terminal kinase (JNK), but not p38, extracellular signal-regulated kinase and Akt, was markedly suppressed in U373MG cells that stably expressed Ran. These results suggest that Ran suppresses paclitaxel-induced cell death through the downregulation of JNK-mediated signal pathways.

PPARδ promotes wound healing by up-regulating TGF-β1-dependent or -independent expression of extracellular matrix proteins

Although the peroxisome proliferator‐activated receptor (PPAR) δ has been implicated in the wound healing process, its exact role and mechanism of action have not been fully elucidated. Our previous findings showed that PPARδ induces the expression of the transforming growth factor (TGF)‐β1, which has been implicated in the deposit of extracellular matrix proteins. Here, we demonstrate that administration of GW501516, a specific PPARδ ligand, significantly promoted wound closure in the experimental mouse and had a profound effect on the expression of collagen types I and III, alpha‐smooth muscle actin, pSmad3 and TGF‐β1, which play a pivotal role in wound healing processes. Activation of PPARδ increased migration of human epidermal keratinocytes and dermal fibroblasts in in vitro scrape‐wounding assays. Addition of a specific ALK5 receptor inhibitor SB431542 significantly suppressed GW501516‐induced migration of human keratinocytes and fibroblasts. In these cells, activated PPARδ also induced the expression of collagen types I and III and fibronectin in a TGF‐β1‐dependent or ‐independent manner. The effect of PPARδ on the expression of type III collagen was dually regulated by the direct binding of PPARδ and Smad3 to a direct repeat‐1 site and a Smad‐binding element, respectively, of the type III gene promoter. Taken together, these results demonstrated that PPARδ plays an important role in skin wound healing in vivo and that it functions by accelerating extracellular matrix‐mediated cellular interactions in a process mediated by the TGF‐β1/Smad3 signaling‐dependent or ‐ independent pathway.

IL-1β enhances vascular smooth muscle cell proliferation and migration via P2Y2 receptor-mediated RAGE expression and HMGB1 release

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessel walls, and their proliferation and migration play important roles in the development of atherosclerosis. Recently, it has been reported that IL-1β mediates the inflammatory response through the upregulation of the P2Y2 receptor (P2Y2R). Thus, we examined the role of P2Y2R in IL-1β-mediated proliferation and migration of VSMCs and the underlying molecular mechanisms. VSMCs were pretreated with IL-1β for 24h to upregulate P2Y2R expression. The cells were then stimulated with UTP or ATP for the indicated times, and cell proliferation and migration and the related signaling pathways were examined. The equipotent P2Y2R agonists ATP and UTP enhanced proliferation, RAGE expression and HMGB1 secretion in IL-1β-pretreated VSMCs. Additionally, pretreatment with IL-1β enhanced UTP-mediated VSMC migration and MMP-2 release, but these effects were not observed in the P2Y2R-siRNA- or RAGE-siRNA-transfected VSMCs. Next, the signaling molecules involved in P2Y2R-mediated cell proliferation and migration were determined. The ERK, AKT, PKC, Rac-1 and ROCK2 pathways were involved in UTP-induced cell proliferation and migration, MMP-2 and HMGB1 secretion and RAGE expression in the IL-1β-pretreated VSMCs. UTP induced the phosphorylation of ERK, AKT and PKC and the translocation of Rac-1 and ROCK2 from cytosol to membrane as well as stress fiber formation, which were markedly increased in the IL-1β-pretreated VSMCs but not in the P2Y2R-siRNA-transfected VSMCs. These results demonstrate that pro-inflammatory cytokines associated with atherosclerosis, such as IL-1β, can accelerate the process of atherosclerosis through the upregulation of P2Y2R.

P2Y2R activation by nucleotides promotes skin wound‐healing process

P2Y2R has been shown to be upregulated in a variety of tissues in response to stress or injury and to mediate tissue regeneration through its ability to activate multiple signalling pathways. This study aimed to investigate the role of P2Y2R in the wound‐healing process and the mechanisms by which P2Y2R activation promotes wound healing in fibroblasts. The role of P2Y2R in skin wound healing was examined using a full‐thickness skin wound model in wildtype (WT) and P2Y2R−/− mice and an in vitro scratch wound model in control or P2Y2R siRNA‐transfected fibroblasts. WT mice showed significantly decreased wound size compared with P2Y2R−/− mice at day 14 post‐wounding, and immunohistochemical analysis showed that a proliferation marker Ki67 and extracellular matrix (ECM)‐related proteins VEGF, collagen I, fibronectin and α‐SMA were overexpressed in WT mice, which were reduced in P2Y2R−/− mice. Scratch‐wounded fibroblasts increased ATP release, which peaked at 5 min. In addition, scratch wounding increased the level of P2Y2R mRNA. Activation of P2Y2R by ATP or UTP enhanced proliferation and migration of fibroblasts in in vitro scratch wound assays and were blocked by P2Y2R siRNA. Finally, ATP or UTP also increased the levels of ECM‐related proteins through the activation of P2Y2R in fibroblasts. This study suggests that P2Y2R may be a potential therapeutic target to promote wound healing in chronic wound diseases.

Identification of cytochrome c oxidase subunit 6A1 as a suppressor of Bax-induced cell death by yeast-based functional screening.

Human cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1) was identified as a novel suppressor of Bcl-2-associated X protein (Bax)-mediated cell death using yeast-based functional screening of a mammalian cDNA library. The overexpression of COX6A1 significantly suppressed Bax- and N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis in yeast and human glioblastoma-derived U373MG cells, respectively. The generation of reactive oxygen species (ROS) in response to Bax or 4-HPR was inhibited in yeast and U373MG cells that expressed COX6A1, indicating that COX6A1 exerts a protective effect against ROS-induced cell damage. 4-HPR-induced mitochondrial translocation of Bax, release of mitochondrial cytochrome c, and activation of caspase-3 were markedly attenuated in U373MG cells that stably expressed COX6A1. Our results demonstrate that yeast-based functional screening of human genes for inhibitors of Bax-sensitivity in yeast identified a protein that not only suppresses the toxicity of Bax in yeast, but also has a potential role in protecting mammalian cells from 4-HPR-induced apoptosis.

Honokiol inhibits U87MG human glioblastoma cell invasion through endothelial cells by regulating membrane permeability and the epithelial-mesenchymal transition

Glioblastoma is one of the most lethal and prevalent malignant human brain tumors, with aggressive proliferation and highly invasive properties. There is still no effective cure for patients with glioblastoma. Honokiol, derived from Magnolia officinalis, can cross the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), making it a strong candidate for an effective drug for the treatment of brain tumors, including glioblastoma. In our previous study, we demonstrated that honokiol effectively induced apoptotic cell death in glioblastoma. Metastasis poses the largest problem to cancer treatment and is the primary cause of death in cancer patients. Thus, in this study, we investigated the effect of honokiol on the cell invasion process of U87MG human glioblastoma cells through brain microvascular endothelial cells (BMECs) and its possible mechanisms. Honokiol dose-dependently inhibited TNF-α-induced VCAM-1 expression in BMECs and adhesion of U87MG to BMECs. Moreover, honokiol effectively blocked U87MG invasion through BMEC-Matrigel-coated transwell membranes. Increased phosphorylation of VE-cadherin and membrane permeability by TNF-α were suppressed by honokiol in BMECs. Furthermore, we investigated the effect of honokiol on the epithelial-mesenchymal transition (EMT) in U87MG cells. Honokiol reduced the expression levels of Snail, N-cadherin and β-catenin, which are mesenchymal markers, but increased E-cadherin, an epithelial marker. In conclusion, these results suggest that honokiol inhibits metastasis by targeting the interaction between U87MG and BMECs, regulating the adhesion of U87MG to BMECs by inhibiting VCAM-1, and regulating the invasion of U87MG through BMECs by reducing membrane permeability and EMT processes of U87MG cells.

Identification of ADP-ribosylation factor 4 as a suppressor of N-(4-hydroxyphenyl)retinamide-induced cell death

Yeast-based functional screening for inhibitors of Bcl-2-associated X protein (Bax)-induced cell death in yeast identified ADP-ribosylation factor 4 (ARF4) as a novel anti-apoptotic gene in human glioblastoma-derived U373MG cells. Yeast or U373MG cells that overexpressed ARF4 exhibited reduced reactive oxygen species (ROS) generation in response to Bax or N-(4-hydroxyphenyl)retinamide (4-HPR), respectively, which suggests that ROS play a role in the inhibition of cell death by ARF4. The 4-HPR-mediated phosphorylation of c-JUN N-terminal kinase, p38, and extracellular signal-regulated kinase was markedly suppressed in U373MG cells that stably expressed ARF4. Stable ARF4 transfectants were also refractory to 4-HPR-induced mitochondrial translocation of Bax, release of mitochondrial cytochrome c, and activation of caspase-3. Our results suggest that ARF4 participates in the regulation of glioblastoma apoptosis through the inhibition of stress-mediated apoptotic signals.

P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells

Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

Mepivacaine-induced contraction involves phosphorylation of extracellular signal-regulated kinase through activation of the lipoxygenase pathway in isolated rat aortic smooth muscle

Mepivacaine is an aminoamide local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. This study investigated the arachidonic acid metabolic pathways involved in mepivacaine-induced contraction, and elucidated the associated cellular mechanism with a particular focus on extracellular signal-regulated kinase (ERK) in endothelium-denuded rat aorta. Isolated rat thoracic aortic rings were suspended for isometric tension recording. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, indomethacin, NS-398, SC-560, fluconazole, PD 98059, and verapamil. Mepivacaine-induced ERK phosphorylation, 5-lipoxygenase (5-LOX) expression, and cyclooxygenase (COX)-2 expression in rat aortic smooth muscle cells were detected by Western blot analysis in the presence or absence of inhibitors. Mepivacaine produced tonic contraction in isolated endothelium-denuded rat aorta. Quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, NS-398, PD 98059, and verapamil attenuated mepivacaine-induced contraction in a concentration-dependent manner. However, fluconazole had no effect on mepivacaine-induced contraction. PD 98059, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, and indomethacin attenuated mepivacaine-induced ERK phosphorylation. Mepivacaine upregulated 5-LOX and COX-2 expression. These results suggest that mepivacaine-induced contraction involves ERK activation, which is primarily mediated by the 5-LOX pathway and in part by the COX-2 pathway.

Farnesyl diphosphate synthase attenuates paclitaxel-induced apoptotic cell death in human glioblastoma U87MG cells.

Increased expression of farnesyl diphosphate synthase (FPPS) by stable transfection appeared to attenuate paclitaxel-induced apoptotic cell death in human glioblastoma U87MG cells. The present results suggest that the apoptotic functions of p53 and c-Jun N-terminal kinase (JNK) are affected by FPPS. Farnesyl diphosphate, a catalytic product of FPPS, also attenuated mentioned paclitaxel-induced apoptotic cell death. As expected, the FPPS inhibitor, pamidronate, enhanced paclitaxel-induced apoptotic cell death. The present results suggest that FPPS plays an important role in apoptotic cell death of cancer cells by blocking the JNK signaling cascade and activating mevalonate metabolism in paclitaxel-treated glioblastoma cells.