CD44 connects autophagy decline and ageing in the vascular endothelium.

The decline of endothelial autophagy is closely related to vascular senescence and disease, although the molecular mechanisms connecting these outcomes in vascular endothelial cells (VECs) remain unclear. Here, we identify a crucial role for CD44, a multifunctional adhesion molecule, in controlling autophagy and ageing in VECs. The CD44 intercellular domain (CD44ICD) negatively regulates autophagy by reducing PIK3R4 and PIK3C3 levels and disrupting STAT3-dependent PtdIns3K complexes. CD44 and its homologue clec-31 are increased in ageing vascular endothelium and Caenorhabditis elegans, respectively, suggesting that an age-dependent increase in CD44 induces autophagy decline and ageing phenotypes. Accordingly, CD44 knockdown ameliorates age-associated phenotypes in VECs. The endothelium-specific CD44ICD knock-in mouse is shorter-lived, with VECs exhibiting obvious premature ageing characteristics associated with decreased basal autophagy. Autophagy activation suppresses the premature ageing of human and mouse VECs overexpressing CD44ICD, function conserved in the CD44 homologue clec-31 in C. elegans. Our work describes a mechanism coordinated by CD44 function bridging autophagy decline and ageing.

Biocontrol potential and action mechanism of Bacillus amyloliquefaciens DB2 on Bipolaris sorokiniana.

Introduction: Bipolaris sorokiniana is the popular pathogenic fungi fungus which lead to common root rot and leaf spot on wheat. Generally, chemical fungicides are used to control diseases. However, the environmental pollution resulting from fungicides should not be ignored. It is important to study the mode of antagonistic action between biocontrol microbes and plant pathogens to design efficient biocontrol strategies. Results: An antagonistic bacterium DB2 was isolated and identified as Bacillus amyloliquefaciens. The inhibition rate of cell-free culture filtrate (CF, 20%, v/v) of DB2 against B. sorokiniana reached 92.67%. Light microscopy and scanning electron microscopy (SEM) showed that the CF significantly altered the mycelial morphology of B. sorokiniana and disrupted cellular integrity. Fluorescence microscopy showed that culture filtrate destroyed mycelial cell membrane integrity, decreased the mitochondrial transmembrane potential, induced reactive oxygen species (ROS) accumulation, and nuclear damage which caused cell death in B. sorokiniana. Moreover, the strain exhibited considerable production of protease and amylase, and showed a significant siderophore and indole-3-acetic acid (IAA) production. In the detached leaves and potted plants control assay, B. amyloliquefacien DB2 had remarkable inhibition activity against B. sorokiniana and the pot control efficacy was 75.22%. Furthermore, DB2 suspension had a significant promotion for wheat seedlings growth. Conclusion: B. amyloliquefaciens DB2 can be taken as a potential biocontrol agent to inhibit B. sorokiniana on wheat and promote wheat growth.

Structure-based drug design of potential inhibitors of FBXW8, the substrate recognition component of Cullin-RING ligase 7.

FBXW8 plays an irreplaceable role in the substrate recognition of ubiquitin-dependent proteolysis, which further regulates cell cycle progression and signal transduction. However, the abnormal expression of FBXW8 triggers malignancy, inflammation, and autophagy irregulation. FBXW8 is considered as an effective therapeutic target for Cullin-RING ligase 7 (CRL7)-related cancers. Still, the lack of selective inhibitors hinders further therapeutic development and limits the exploration of its biological mechanism. This study constructed an integrated protocol that combines pharmacophore modeling, structure-based virtual screening, and Molecular Dynamic Simulation. It was then used as a screening query to identify hit compounds targeted at the substrate recognition site of FBXW8 from a large-scale compound database including 120 million compounds. Then, ten lead compounds were retrieved by using molecular docking analysis and ADMET prediction. Finally, MD simulations were performed to validate the binding stability of selected drug candidates. The result indicated that three newly obtained compounds, namely ZINC96179876, ZINC72174069, and ZINC97730272, might be potent FBXW8 inhibitors against CRL7-related cancers such as endometrial cancer.

Identification of Compound CB-2 as a Novel Late-Stage Autophagy Inhibitor Exhibits Inhibitory Potency against A549 Cells.

Autophagy has been recognized as a stress tolerance mechanism that maintains cell viability, which contributes to tumor progression, dormancy, and treatment resistance. The inhibition of autophagy in cancer has the potential to improve the therapeutic efficacy. It is therefore of great significance to search for new autophagy inhibitors. In the present study, after screening a series of curcumin derivatives synthesized in our laboratory, (E)-3-((E)-4-chlorobenzylidene)-5-((5-methoxy-1H-indol-3-yl)methylene)-1-methylpiperidin-4-one (CB-2) was selected as a candidate for further study. We found that CB-2 increased the LC3B-II and SQSTM1 levels associated with the accumulation of autophagosomes in non-small cell lung cancer (NSCLC) A549 cells. The increased level of LC3B-II induced by CB-2 was neither eliminated when autophagy initiation was suppressed by wortmannin nor further increased when autophagosome degradation was inhibited by chloroquine (CQ). CB-2 enhanced the accumulation of LC3B-II under starvation conditions. Further studies revealed that CB-2 did not affect the levels of the key proteins involved in autophagy induction but significantly blocked the fusion of autophagosomes with lysosomes. High-dose CB-2 induced the apoptosis and necrosis of A549 cells, while a lower dose of CB-2 mainly impaired the migrative capacity of A549 cells, which only slightly induced cell apoptosis. CB-2 increased the levels of mitochondrial-derived reactive oxygen species (ROS) while decreasing the mitochondrial membrane potential (MMP). Scavenging ROS via N-acetylcysteine (NAC) reversed CB-2-induced autophagy inhibition and its inhibitory effect against A549 cells. In conclusion, CB-2 serves as a new late-stage autophagy inhibitor, which has a strong inhibitory potency against A549 cells.

Polyethylenimine-Modified Mesoporous Silica Nanoparticles Induce a Survival Mechanism in Vascular Endothelial Cells via Microvesicle-Mediated Autophagosome Release.

Surface-modified mesoporous silica nanoparticles (MSNs) have attracted more and more attention as promising materials for biomolecule delivery. However, the lack of detailed evaluation relevant to the potential cytotoxicity of these MSNs is still a major obstacle for their applications. Unlike the bare MSNs and amino- or liposome-modified MSNs, we found that polyethylenimine-modified MSNs (MSNs-PEI) had no obvious toxicity to human umbilical vein endothelial cells (HUVECs) at the concentrations up to 100 µg/mL. However, MSNs-PEI induced autophagosomes accumulation by blocking their fusion with lysosomes, an essential mechanism for the cytotoxicity of many nanoparticles (NPs). Thus, we predicted that an alternative pathway for autophagosome clearance exists in HUVECs to relieve autophagic stress induced by MSNs-PEI. We found that MSNs-PEI prevented STX17 loading onto autophagosomes instead of influencing lysosomal pH or proteolytic activity. MSNs-PEI induced the structural alternation of the cytoskeleton but did not cause endoplasmic reticulum stress. The accumulated autophagosomes were released to the extracellular space via microvesicles (MVs) when the autophagic degradation was blocked by MSNs-PEI. More importantly, blockade of either autophagosome formation or release caused the accumulation of damaged mitochondria and excessive ROS production in the MSNs-PEI-treated HUVECs, which in turn led to cell death. Thus, we propose here that the MV-mediated autophagosome release, a compensation mechanism, allows the vascular endothelial cell survival when the degradation of autophagosomes is blocked by MSNs-PEI. Accordingly, promoting the release of accumulated autophagosomes may be a protective strategy against the endothelial toxicity of NPs.

Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer.

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle's balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Knockdown of KLF5 suppresses hypoxia-induced resistance to cisplatin in NSCLC cells by regulating HIF-1α-dependent glycolysis through inactivation of the PI3K/Akt/mTOR pathway.

BACKGROUND: Hypoxia-mediated chemoresistance has been regarded as an important obstacle in the development of cancer treatment. Knockdown of krüppel-like factor 5 (KLF5) was reported to inhibit hypoxia-induced cell survival and promote cell apoptosis in non-small cell lung cancer (NSCLC) cells via direct regulation of hypoxia inducible factor-1α (HIF-1α) expression. However, the roles of KLF5 in the development of hypoxia-induced cisplatin (DDP) resistance and its underlying mechanism in NSCLC cells remain to be further elucidated. METHODS: Western blot was performed to determine the protein levels of KLF5, P-glycoprotein (P-gp) and HIF-1α in treated NSCLC cells. Cell survival was examined by MTT assay. The effect of KLF5 knockdown on hypoxia-induced glycolysis was assessed by measuring glucose consumption and lactate production. The effect of KLF5 knockdown on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway was analyzed by western blot. RESULTS: Hypoxia upregulated the expression of KLF5 in NSCLC cells. KLF5 knockdown suppressed hypoxia-induced DDP resistance in NSCLC cells, as demonstrated by the increased cytotoxic effects of DDP and reduced P-gp expression in NSCLC cells in hypoxia. Moreover, KLF5 knockdown inhibited hypoxia-induced HIF-1α expression and glycolysis, and KLF5 knockdown suppressed hypoxia-induced DDP resistance by inhibiting HIF-1α-dependent glycolysis in NSCLC cells. Furthermore, KLF5 knockdown suppressed hypoxia-induced activation of the PI3K/Akt/mTOR pathway in NSCLC cells and KLF5 overexpression promoted hypoxia-induced DDP resistance in NSCLC cells through activation of the PI3K/Akt/mTOR pathway. CONCLUSIONS: KLF5 knockdown could suppress hypoxia-induced DDP resistance, and its mechanism may be due to the inhibition of HIF-1α-dependent glycolysis via inactivation of the PI3K/Akt/mTOR pathway.

Up-regulation of the active form of small GTPase Rab13 promotes macroautophagy in vascular endothelial cells.

The importance of macroautophagy (hereafter referred to as autophagy) in vascular endothelial cell (VEC) biology and dysfunction is increasingly recognized, but the molecular mechanisms of autophagy in VECs in the presence of serum are still poorly understood. Previously, we identified pterostilbene as a potent autophagy inducer of VECs in the presence of serum. In this study, we used pterostilbene as a tool to induce VEC autophagy and identified the differentially expressed genes using high-throughput DAN microarray. The small GTPase Ras-related protein in brain 13 (Rab13) was found to be the most significantly up-regulated gene in pterostilbene-treated human umbilical VECs (HUVECs). Knockdown of Rab13 blocked pterostilbene-induced mTOR inhibition and autophagy, whereas overexpression of the GTP-containing active form of Rab13 induced mTOR inhibition and autophagy in HUVECs. Using a combination of immunofluorescence and co-immunoprecipitation (co-IP) assays, we demonstrated that pterostilbene or up-regulation of the active form of Rab13 promoted the interaction between Rab13 and growth factor receptor-bound protein 2 (Grb2). Knockdown of Grb2 suppressed pterostilbene or up-regulation of the active form of Rab13-induced autophagy. Further mechanistic studies revealed that Rab13 activated the downstream AMP-activated protein kinase (AMPK) and blocked mammalian target of rapamycin (mTOR) signaling by its functional interaction with Grb2 to regulate autophagy in HUVECs. Our study firmly establishes Rab13 as a novel regulator of autophagy in VECs under nutrient-enriched conditions.

Gene expression profiling of DMU-212-induced apoptosis and anti-angiogenesis in vascular endothelial cells.

CONTEXT: trans-3,4,5,4'-Tetramethoxystilbene (DMU-212), an derivative of resveratrol, shows strong antiproliferative activities against many cancer cells. In our previous study, we demonstrated that DMU-212 possesses potent proapoptosis and antiangiogenesis effects on vascular endothelial cells (VECs), which made it a promising agent for the treatment of angiogenesis-related diseases. OBJECTIVE: We studied the gene expression profile of DMU-212-treated VECs to gain further insight into the mechanisms by which DMU-212 exerts its potent pro-apoptosis and antiangiogenesis effects. MATERIALS AND METHODS: The potential changes in the gene expression of VECs incubated with DMU-212 were identified and analyzed using the Affymetrix HG-U133 Plus 1.0 array. In addition, the gene expression profile was validated by quantitative real-time PCR (qRT-PCR) analysis for seven of those altered genes. RESULTS AND CONCLUSION: DMU-212 was found to regulate a diverse range of genes, including cytokines (IL8, selectin E, MPZL2, EGR1, CCL20, ITGB8, CXCL1, VCAM1, KITLG, and AREG), transport proteins (TRPC4, SLC41A2, SLC17A5, and CREB5), metabolism (CYP1B1, CYP1A1, PDK4, CSNK1G1, MVK, TCEB3C, and CDKN3), enzymes (RAB23, SPHK1, CHSY3, PLAU, PLA2G4C, and MMP10), and genes involved in signal transduction (TMEM217, DUSP8, and SPRY4), chromosome organization (HIST1H2BH and GEM), cell migration and angiogenesis (ERRFI1, HBEGF, and NEDD9), and apoptosis (TNFSF15, TNFRSF9, CD274, BCL2L11, BIRC3, TNFAIP3, and TIFA), as well as other genes with unknown function (PGM5P2, SNORD1142, LOC151760, KRTAP5-2, C1orf110, SNORA14A, MIR31, C2CD4B, SCARNA4, C2orf66, SC4MOL, LOC644714, and LOC283392). This is the first application of microarray technique to investigate and analyze the profile of genes regulated by DMU-212 in VECs. Our results lead to an increased understanding of the signaling pathways involved in DMU-212-induced apoptosis and antiangiogenesis.

Magnetic ferroferric oxide nanoparticles induce vascular endothelial cell dysfunction and inflammation by disturbing autophagy.

Despite the considerable use of magnetic ferroferric oxide nanoparticles (Fe3O4NPs) worldwide, their safety is still an important topic of debate. In the present study, we detected the toxicity and biological behavior of bare-Fe3O4NPs (B-Fe3O4NPs) on human umbilical vascular endothelial cells (HUVECs). Our results showed that B-Fe3O4NPs did not induce cell death within 24h even at concentrations up to 400 µg/ml. The level of nitric oxide (NO) and the activity of endothelial NO synthase (eNOS) were decreased after exposure to B-Fe3O4NPs, whereas the levels of proinflammatory cytokines were elevated. Importantly, B-Fe3O4NPs increased the accumulation of autophagosomes and LC3-II in HUVECs through both autophagy induction and the blockade of autophagy flux. The levels of Beclin 1 and VPS34, but not phosphorylated mTOR, were increased in the B-Fe3O4NP-treated HUVECs. Suppression of autophagy induction or stimulation of autophagy flux, at least partially, attenuated the B-Fe3O4NP-induced HUVEC dysfunction. Additionally, enhanced autophagic activity might be linked to the B-Fe3O4NP-induced production of proinflammatory cytokines. Taken together, these results demonstrated that B-Fe3O4NPs disturb the process of autophagy in HUVECs, and eventually lead to endothelial dysfunction and inflammation.

Novel role for TRPC4 in regulation of macroautophagy by a small molecule in vascular endothelial cells.

Macroautophagy (autophagy) is an important factor affecting the function of vascular endothelial cells (VECs) and must be tightly regulated in these cells. However, the precise mechanisms underlying this process, particularly in the presence of serum, remain obscure. In this study, we identified trans-3,5,4'-trimethoxystilbene (TMS) as a potent small molecule inducer of autophagy in human umbilical vascular endothelial cells (HUVECs) in the presence of serum. Using high-throughput DNA microarray and siRNA transfection technologies, we demonstrated that TMS induced autophagy by up-regulating the expression of the transient receptor potential canonical channel 4 (TRPC4), an important cation channel in HUVECs. In addition, the overexpression of TRPC4 by plasmid transfection also induced autophagy. Mechanistic studies revealed that the up-regulation of TRPC4 increased the intracellular Ca²âº concentration, which, in turn, activated the Ca²âº/CaMKKß/AMPK pathway, leading to mTOR inhibition and autophagy. Our study identifies a novel role for TRPC4 in the regulation of autophagy in VECs. TMS is a useful new tool for investigating the molecular mechanism of autophagy in VECs and may serve as a potential lead compound for developing a class of autophagy inducers to treat autophagy-related diseases.

Highly Efficient Labeling of Human Lung Cancer Cells Using Cationic Poly-l-lysine-Assisted Magnetic Iron Oxide Nanoparticles.

Cell labeling with magnetic iron oxide nanoparticles (IONPs) is increasingly a routine approach in the cell-based cancer treatment. However, cell labeling with magnetic IONPs and their leading effects on the biological properties of human lung carcinoma cells remain scarcely reported. Therefore, in the present study the magnetic γ-Fe2O3 nanoparticles (MNPs) were firstly synthesized and surface-modified with cationic poly-l-lysine (PLL) to construct the PLL-MNPs, which were then used to magnetically label human A549 lung cancer cells. Cell viability and proliferation were evaluated with propidium iodide/fluorescein diacetate double staining and standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide assay, and the cytoskeleton was immunocytochemically stained. The cell cycle of the PLL-MNP-labeled A549 lung cancer cells was analyzed using flow cytometry. Apoptotic cells were fluorescently analyzed with nuclear-specific staining after the PLL-MNP labeling. The results showed that the constructed PLL-MNPs efficiently magnetically labeled A549 lung cancer cells and that, at low concentrations, labeling did not affect cellular viability, proliferation capability, cell cycle, and apoptosis. Furthermore, the cytoskeleton in the treated cells was detected intact in comparison with the untreated counterparts. However, the results also showed that at high concentration (400 µg mL-1), the PLL-MNPs would slightly impair cell viability, proliferation, cell cycle, and apoptosis and disrupt the cytoskeleton in the treated A549 lung cancer cells. Therefore, the present results indicated that the PLL-MNPs at adequate concentrations can be efficiently used for labeling A549 lung cancer cells and could be considered as a feasible approach for magnetic targeted anti-cancer drug/gene delivery, targeted diagnosis, and therapy in lung cancer treatment.

Innovative fluorescent magnetic albumin microbead-assisted cell labeling and intracellular imaging of glioblastoma cells.

Superparamagnetic nanoparticle-based polymer microbeads utilized as carriers are attractive materials widely applied in the biomedical field. However, the deficiency of toxicity, biocompatibility, and biodegradability for polymer materials often limits the application of these microbeads. In the present study, magnetic albumin microbeads (MAMbs), i.e., human serum albumin-coated γ-Fe2O3 nanoparticles, are synthesized to label human U251 glioblastoma multiforme cells. The effects of MAMbs on the biological behavior of U251 glioblastoma cells, including their proliferation, cell viability, cytoskeletal structure, cell cycle, and apoptosis rate, are investigated. Moreover, fluorescein isothiocyanate (FITC)-MAMbs are fabricated by reaction with fluorescent dye FITC used for intracellular imaging of U251 glioblastoma cells. MAMbs possess undetectable cytotoxicity and excellent biocompatibility with U251 glioblastoma cells, as demonstrated by the biological behavior and morphology of U251 cells exposed to MAMbs. Furthermore, the constructed fluorescent MAMbs allow effective intracellular imaging, as illustrated by fluorescence microscopic analysis. The fabricated fluorescent MAMbs have promising perspectives in biomedical research, especially in cell-targeted labeling and intracellular fluorescence magnetic dual-mode imaging in cancer-targeted diagnosis and therapy.

Characterization of the microtubule-binding activity of kinesin-like calmodulin binding protein from Dunaliella salina.

Although the C-terminal motor and the N-terminal myosin-like domains of KCBP in Dunaliella salina (DsKCBP) are implicated in interaction with the microtubules, its microtubule binding property has not been addressed. It has been shown that several calmodulin isoforms suppress the microtubule binding activity of KCBP, but whether the calmodulin-like protein (CLP) has this ability remains unknown. The results of our previous study showed that there are two microtubule binding sites in DsKCBP, motor domain at the C-terminus and MyTH4-FREM at the N-terminus. In the present study, MyTH4, without the companion of FERM, was identified as the minimal domain responsible for interaction with the microtubules in the N-terminal of DsKCBP. CLP interacted with the calmodulin-binding domain of DsKCBP in the presence of Ca(2+), and inhibited the microtubule-binding activity of motor domain but not MyTH4 domain. Furthermore, MyTH4 domain in the N-terminus of DsKCBP was responsible for binding to the microtubules, and had 10-fold weaker affinity to the microtubules than the motor domain.

Pterostilbene, a natural small-molecular compound, promotes cytoprotective macroautophagy in vascular endothelial cells.

Chemical modulators of macroautophagy (herein referred to as autophagy) have aroused widespread interest among biologists and clinical physicians because of their potential for disease therapy. Pterostilbene (PT), a natural small-molecular compound, has been demonstrated to inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis in vascular endothelial cells (VECs). The aim of the present study was to investigate whether and how PT could induce VEC autophagy. PT at 0.5 or 1 µM could effectively induce autophagosome formation in human umbilical vein VECs (HUVECs). PT promoted autophagy via a rapid elevation in intracellular calcium ([Ca(2+)]i) concentration and subsequent AMP-activated protein kinase α1 subunit (AMPKα1) activation, which in turn inhibited mammalian target of rapamycin, a potent inhibitor of autophagy. PT-induced AMPKα1 activation and autophagy were refractory to the depletion of serine/threonine kinase 11 but depended on calcium/calmodulin-dependent protein kinase kinase-ß activation. Interestingly, PT stimulated cytoprotective autophagy so as to aid in the removal of accumulated toxic oxLDL and inhibit apoptosis in HUVECs. Our study provides a potent small molecule enhancer of autophagy and a novel useful tool in exploring the molecular mechanisms for crosstalk between apoptosis and autophagy. PT could serve as a potential lead compound for developing a class of autophagy regulator as autophagy-related diseases therapy.

3,4-Dimethoxystilbene, a resveratrol derivative with anti-angiogenic effect, induces both macroautophagy and apoptosis in endothelial cells.

Angiogenesis plays an important role in many pathological processes. Identification of novel anti-angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans-3,4-dimethoxystilbene (3,4-DMS) with the fullest potential to develop as an anti-angiogenic agent. In vitro and in vivo analyses suggested that 3,4-DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4-DMS exerted its anti-angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4-DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca(2+)](i)) might bridge the 3,4-DMS-induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4-DMS with inhibitor of autophagy, such as 3-methyladenine (3-MA) and autophagy-related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro-apoptotic and anti-angiogenic effects of 3,4-DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4-DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis-related diseases.

Characterization of the glucose-6-phosphate isomerase (GPI) gene from the halotolerant alga Dunaliella salina.

Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes, studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of 6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD. Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including "GEPGTNGQHSFYQLIHQG" and "VQGFIWGINSFDQWGVELGK", and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light.