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ABSTRACT: 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. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.
Journal of Cellular Biochemistry 10/2012; · 2.87 Impact Factor
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ABSTRACT: 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-β 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.
The Journal of nutritional biochemistry 08/2012; · 4.29 Impact Factor
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ABSTRACT: Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1-5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE(-/-) mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.
Apoptosis 09/2011; 17(1):25-36. · 4.07 Impact Factor
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ABSTRACT: Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. DeltaG91 is a previously discovered congenital mutation in betaA3-crystallin that impairs protein solubility. On the other hand, the deamidation of beta-crystallin is a significant feature in aged and cataractous lenses. Several deamidation sites were also identified in betaA3-crystallin. The present study is to compare the functional consequence of DeltaG91 mutation and the deamidation of betaA3-crystallin in terms of folding properties and protein-protein interaction.
Protein secondary structure and hydrophobic properties were investigated by in silica analysis of the wild type and mutants sequences. Full-length betaA3-crystallin was cloned into a mammalian two-hybrid system in order to investigate protein-protein interactions. Deletion and deamidation were introduced by site-directed mutagenesis protocols. Both the Q85 and Q180 deamidation sites were substituted with glutamic acid residues to mimic deamidation. Different combinations of plasmid constructs were transfected in HeLa cells, and changes of protein-protein interactions were analyzed by the luciferase assay.
Bioinformatics prediction suggested that DeltaG91 mutation alters both the predicted secondary structure and hydrophobic character of betaA3-crystallin, while deamidation only exhibits minimal effects. Mammalian two-hybrid results indicated that both DeltaG91 mutation and Q85/Q180 deamidation could significantly decrease the interaction of the betaA3-crystallin homodimer.
Our results provided evidence that both mutations involved in congenital cataracts and deamidation in aged lenses commonly altered protein-protein interaction between human lens betaA3-crystallins, which may lead to protein insolubilization and contribute to cataracts.
Molecular vision 01/2010; 16:438-44. · 2.20 Impact Factor
