Developing Natural Products-inspired Prooxidative Antiangiogenic Agents And Exploring Their Action Mechanisms

Angiogenesis,forming new blood vessels by budding from the existing vasculature,plays an important role in the development of diseases such as tumors.Vascular density can be used as an important prognostic indicator for tumor metastasis potential.Anti-angiogenic therapy has become one of the effective strategies to control the growth and metastasis of solid tumor.Compared with normal cells,cancer cells exhibit increased levels of reactive oxygen species(ROS)to maintain their malignant phenotypes and are more vulnerable to further production of ROS by using prooxidants.However,relatively little is known about designing natural product-inspired angiogenesis inhibitor from the point of view of prooxidants.In this dissertation,natural products including resveratrol,[6]-shogaol,curcumin and their analogs were selected to explore their structural basis and mechanisms as angiogenesis inhibitors with the aim of giving useful information on how to design prooxidative angiogenesis inhibitor.The main research contents are as follows:(1)As part of our research project in understanding why dietary polyphenols with the catechol skeleton tend to exhibit cancer chemopreventive activity,a resveratrol analog(3,4-DHS)was selected as a model of dietary catechols to probe their antiangiogenic effects and mechanisms.We examined the antiangiogenic effects of 3,4-DHS on angiogenesis-related endothelial cell functions,including migration,invasion and tube formation,and in vivo angiogenesis on a chick chorioallantoic membrane assay.The potential molecular mechanisms for the suppression of cell migration by 3,4-DHS were analyzed using various specific inhibitors.3,4-DHS was identified as a potent angiogenesis inhibitor by constructing an efficient catalytic redox cycle with intracellular copper ions and NAD(P)H quinone oxidoreductase I(NQO1)to generate reactive oxygen species and thereby down-regulate matrix metalloproteinase-9.This work provides further evidence that dietary catechols manifest antiangiogenic activity by virtue of their copper-dependent prooxidative instead of antioxidative role,and useful information for designing polyphenol-inspired angiogenesis inhibitors.(2)Inspired from the above work,we chosen a catechol-type diphenylbutadiene((1E,3E)-3,4-dihydroxy-diphenylbuta-1,3-diene,3,4-DHB)by elongating the conjugated links of 3,4-DHS but preserving its catechol moiety,to improve its reactivity with copper and lipophilicity facilitating penetration into the cell membranes.As expected,3,4-DHB become more proficient at constructing a redox cycle with intracellular copper ions and NQO1 to generate ROS and inhibit angiogenesis than 3,4-DHS.(3)Three active constituents of ginger including[6]-paradol(6P),[6]-shogaol(6S)and[6]-dehydroshogaol(6-DHS)differentiated by various numbers of the Michael acceptor units were selected to explore the dependence of angiogenesis inhibition on the Michael acceptor structure.It was found that among the tested molecules[6]-DHS with a double Michael acceptor structure exhibited the highest anti-angiogenic activity by virtue of its ability to generate ROS.Taking 6S and its analogs as a case,this works illustrates the feasibility of developing angiogenesis inhibitor based on the Michael receptor structure.(4)Curcumin has been identified as a potent anti-angiogenic,anti-invasive and anti-tumor metastatic agents,but it is unstable under physiological conditions due to the presence of the active methylene group.We designed its analogs 2a in which the labile methylene group was omitted by Knoevenagel condensation.Compared with the parent curcumin,2a is a more potent angiogenesis inhibitor by virtue of its Michael acceptor-dependent ROS generation and inhibiting PMA-induced MMP-9expression and phosphorylation of AKT protein kinase,thereby inhibiting angiogenesis.(5)Piperlongumine(PL),a natural alkaloid isolated from the long pepper,works as a potent prooxidative anticancer agent.However,its anticancer mechanisms under hypoxic environment remain unclear to date.Here we used cobalt chloride(Co Cl₂)to simulate hypoxia in human liver cancer cells(HepG2)to probe anticancer mechanisms of PL under hypoxic environment.It was found that PL exhibited improved cytotoxicity under hypoxic environment.Mechanistic investigation revealed that PL mediated apoptosis of HepG2 cells by generating ROS and up-regulating the expression of p-P38 and hypoxia-inducible factor HIF-1?.Additionally,it also down-regulated the expression of VEGF induced by Co Cl₂,indicating that inhibition against solid tumor growth by PL may be related to its angiogenesis inhibition.