Resveratrol protects cardiomyocytes from oxidative stress through SIRT1 and mitochondrial biogenesis signaling pathways

Reactive oxygen species (ROS) is generated by oxidative stress and plays an important role in various cardiac pathologies. The SIRT1 signaling pathway and mitochondrial biogenesis play essential roles in mediating the production of ROS. SIRT1 activated by resveratrol protects cardiomyocytes from oxidative stress, but the exact mechanisms by which SIRT1 prevents oxidative stress, and its relationship with mitochondrial biogenesis, remain unclear. In this study, it was observed that after stimulation with 50 μM H₂O₂ for 6 h, H9C2 cells produced excessive ROS and downregulated SIRT1. The mitochondrial protein NDUFA13 was also downregulated by ROS mediated by SIRT1. Resveratrol induced the expression of SIRT1 and mitochondrial genes NDUFA1, NDUFA2, NDUFA13 and Mn-SOD. However, the production of these genes was reversed by SIRT1 inhibitor nicotinamide. These results suggest that resveratrol inhibits ROS generation in cardiomyocytes via SIRT1 and mitochondrial biogenesis signaling pathways.     

Association of Gap-43 (neuromodulin) with microtubule-associated protein MAP-2 in neuronal cells

Gap-43 (B-50, neuromodulin) is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, and regeneration. Its activities are regulated by its dynamic interactions with various neuronal proteins, including actin and brain spectrin. Recently we have shown that Gap-43 co-localizes with an axonal protein DPYSL-3 in primary cortical neurons. In the present study we provide evidence that Gap-43 co-localizes and potentially interacts with microtubule-associated protein MAP-2 in adult and fetal rat brain, as well as in primary neuronal cultures. Our studies suggest that this interaction may be developmentally regulated.     

Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.     

Demethoxycurcumin induces Bcl-2 mediated G2/M arrest and apoptosis in human glioma U87 cells

Docking analysis of curcumin (C1), demethoxycurcumin (C2) and bisdemethoxycurcumin (C3) with Bcl-2 illustrated that among the three curcuminoids, C2 binds more efficiently into its putative active site. C1, C2 and C3 were purified from turmeric rhizomes to demonstrate the molecular mechanism of their anticancer activity on human glioma U87 cells. Human glioma U87 cells treated with curcuminoids resulted in activation of Bcl-2 mediated G2 checkpoint, which was associated with the induction of G2/M arrest and apoptosis. The binding of C1, C2 and C3 with Bcl-2 protein was confirmed with circular dichroism (CD) spectroscopy. Present work revealed that C2 induced Bcl-2 mediated G2/M arrest and apoptosis most effectively.     

Effects of tau phosphorylation on proteasome activity

Dysfunction of proteasome contributes to the accumulation of the abnormally hyperphosphorylated tau in Alzheimer's disease. However, whether tau hyperphosphorylation and accumulation affect the activity of proteasome is elusive. Here we found that a moderate tau phosphorylation activated the trypsin-like activity of proteasome, whereas further phosphorylation of tau inhibited the activity of the protease in HEK293 cells stably expressing tau441. Furthermore, tau hyperphosphorylation could partially reverse lactacystin-induced inhibition of proteasome. These results suggest that phosphorylation of tau plays a dual role in modulating the activity of proteasome.     

Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy

Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson’s disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P < 0.001), decreased cell apoptosis (by 54.38%, P < 0.001), increased SOD and GSH (by 120.53% and 90.46%, P < 0.01), reduced accumulation of α-synuclein (by 35.93%, P < 0.001) and ROS generation (by 33.99%, P < 0.001), preserved MMP (33.93 ± 3.62%, vs. 15.10 ± 0.71% of JC-1 monomer, P < 0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P < 0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P < 0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.     

Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Although many cancer cells are sensitive to TRAIL-induced apoptosis, chronic myeloid leukemia (CML) develops resistance to TRAIL. In this study, we investigated whether apicidin, a novel histone deacetylase inhibitor, could overcome the TRAIL resistance in CML-derived K562 cells. Compared to treatment with apicidin or TRAIL alone, cotreatment with apicidin and TRAIL-induced apoptosis synergistically in K562 cells. This combination led to activation of caspase-8 and Bcl-2 interacting domain (Bid), resulting in the cytosolic accumulation of cytochrome c from mitochondria as well as an activation of caspase-3. Treatment with apicidin resulted in down-regulation of Bcr-Abl and inhibition of its downstream target, PI3K/AKT-NF-κB pathway. In addition, apicidin decreased the level of NF-κB-dependent Bcl-x_L, leading to caspase activation and Bid cleavage. These results suggest that apicidin may sensitize K562 cells to TRAIL-induced apoptosis through caspase-dependent mitochondrial pathway by regulating expression of Bcr-Abl and its related anti-apoptotic proteins. Therefore, the present study suggests that combination of apicidin and TRAIL may be an effective strategy for treating TRAIL-resistant Bcr-Abl expressing CML cells.     

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.     

Platycodin D inhibits migration,invasion, and growth of MDA-MB-231 human breast cancer cells via suppression of EGFR-mediated Akt and MAPK pathways

Platycodin D (PD), an active triterpenoid saponin from Platycodon grandiflorum, has been known to inhibit the proliferation of a variety of cancer cells, but the effect of PD on the invasiveness of cancer cells is largely unknown. In this study, we first determined the molecular mechanism by which PD inhibits the migratory and invasive abilities of the highly metastatic MDA-MB-231 breast cancer cell line. We demonstrated that a non-cytotoxic concentration of PD markedly suppressed wound healing migration, invasion through the matrigel, and adhesion to an ECM-coated substrate in a dose-dependent manner. Moreover, PD inhibited cell invasion by reducing matrix metalloproteinase (MMP)-9 enzyme activity and mRNA expression. Western blot analysis indicated that PD potently suppressed the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) as well as blocked the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway. Furthermore, PD treatment inhibited the DNA binding activity of NF-κB, which is known to mediate the expression of epidermal growth factor receptor (EGFR), as observed by electrophoretic mobility shift assay. Specific mechanisms of action exerted by PD involved the downregulation of EGFR and the inhibition of EGF-induced activation of the EGFR, MAPK, and PI3K/Akt pathways. The in vivo studies showed that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in BALB/c nude mice. These results suggest that PD might be a potential therapeutic candidate for the treatment of breast cancer metastasis.     

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H₂O₂ stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H₂O₂, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.     

Wisp2/CCN5 up-regulated in the central nervous system of GM3-only mice facilitates neurite formation in Neuro2a cells via integrin-Akt signaling

Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function of Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin β1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H₂O₂. These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.     

Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells

Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine.     

CUB-domain containing protein 1 represses the epithelial phenotype of pancreatic cancer cells

The prognosis of pancreatic cancer is dismal due to the frequent metastasis and invasion to surrounding organs. Numerous molecules are involved in the malignant behavior of pancreatic cancer cells, but the entire process remains unclear. Several reports have suggested that CUB-domain containing protein-1 (CDCP1) is highly expressed in pancreatic cancer, but its impact on the invasive growth and the upstream regulator remain elusive. To clarify the role of CDCP1 in pancreatic cancer, we here examined the effects of CDCP1 knockdown on the cell behaviors of pancreatic cancer cells. Knockdown of CDCP1 expression in Panc-1 resulted in reduced cellular migration accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin. Knockdown of CDCP1 attenuated the spheroid formation and resistance against gemcitabine, which are some of the cancer stem cell-related phenotypes. Bone morphogenetic protein 4 (BMP4) was found to induce CDCP1 expression via the extracellular signal regulated kinase pathway, suggesting that CDCP1 has a substantial role in the BMP4-induced epithelial-mesenchymal transition. These results indicate that CDCP1 represses the epithelial phenotype of pancreatic cancer cells.     

Netrin-1 induces proliferation of Schwann cells through Unc5b receptor

Netrin and its receptors, DCC (Deleted in Colorectal Cancer) and Unc5, are proposed to be involved in the axon guidance and neuroglial migration during development. However, accumulating evidence implies that they may also participate in the cell survival and apoptosis. Here, we show that netrin-1 induces proliferation of Schwann cells. Unc5b is the sole receptor expressed in RT4 schwannoma cells and adult primary Schwann cells, and netrin-1 and Unc5b are found to be expressed in the injured sciatic nerve. It was also found that the netrin-1-induced Schwann cell proliferation was blocked by the specific inhibition of Unc5b expression with RNAi. These data suggest that netrin-1 could be an endogenous trophic factor for Schwann cells in the injured peripheral nerves.     

The aspartic acid of Fyn at 390 is critical for neuronal migration during corticogenesis

The mammalian cerebral cortex develops through the coordinated migration of postmitotic neurons. Fyn, a member of the Src tyrosine kinase family (SFKs), is involved in the neuronal migration and the absence of Fyn leads to abnormal migration. However, the molecular mechanism whereby Fyn acts on migrating neurons has remained unclear. Here, we employed two Fyn mutants (Fyn259T and FynD390A) to investigate the function of Fyn kinase domain in neuronal migration. Using in utero electroporation, we co-transfected the migrating neurons in embryonic cortex with these mutants combined with plasmid expressing GFP. Interestingly, although both of them impaired neuronal migration, FynD390A, rather than Fyn259T, induced remarkable morphology change. Our work provides in vivo and in vitro evidence that the aspartic acid of Fyn at 390 is indispensable for the radial migration, and it is required for precise cooperation with focal adhesion kinase.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.     

Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22 kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200 μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100–400 μg/ml for 24 h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP.     

Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.     

Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells

Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork that helps maintain the uniform size and shape of cells. As cells of hepatocellular carcinoma are morphologically different from healthy human hepatocytes, we hypothesized that plectin deficiency and cytoskeletal disorganization underlies this pleomorphic transformation. To test this hypothesis we induced apoptosis as the most accessible pathway for creating plectin deficiency status in vivo. We analyzed expression levels and organization of plectin and other cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after staurosporine-induced apoptosis in human Chang liver cells. The results revealed the expression of plectin and cytokeratin 18 were downregulated in hepatocellular carcinoma tissues in vivo. The expression of actin and tubulin, however, were not altered. In vitro analysis indicated that plectin and cytokeratin 18 were cleaved following staurosporine-treatment of human Chang liver cells. Time course experiments revealed that plectin was cleaved 2 h earlier than cytokeratin 18. The organization of plectin and cytokeratin 18 networks collapsed after staurosporine-treatment. Conclusively, degradation of plectin induced by staurosporine-treatment in liver cells resulted in cytoskeleton disruption and induced morphological changes in these cells by affecting the expression and organization of cytokeratin 18.