Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signalregulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. [Cancer Res 2009;69(2):518-25]

Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.Tumor suppressor protein p53 keeps us free of cancer when it is functional. Mice lacking p53 (p53 Ϫ/Ϫ ) spontaneously develop numerous neoplasms within 6 months (1). Mutated p53 genes are seen in more than 50% of all human cancers, making them the most frequently observed genetic derangement in human cancer (2). At a molecular level, the ability of p53 to eliminate cancerous cells relies on its ability to induce apoptosis, through either the transcriptional activation of proapoptotic genes such as Noxa (3), PUMA 4 (4), and Bax (5) or the direct transcription-independent activation of Bax on mitochondria (6). Growing cancers manage to keep p53 in check either by mutating the p53 gene itself (7-9) or by expressing p53 inhibitors such as Mdm2 (9, 10). The function of fortilin, a ubiquitous, highly conserved, 172-amino acid polypeptide also known as "translationally controlled tumor protein," or TCTP, remained unknown (11,12). Investigation in our laboratory and others showed that fortilin possesses potent anti-apoptotic activity (13-15). Fortilin is overexpressed in human cancers (16,17), the depletion of which is associated with spontaneous death of cancerous cells (13, 18). Higher levels of fortilin are associated with more malignant cancer phenotypes (14). Although heterozygous fortilin-deficient mice (fortilin ϩ/Ϫ ) were normal in appearance and fertile, homozygous fortilin-deficient (fortilin Ϫ/Ϫ ) mice were embryonically lethal around 3.5 days postcoitus due to massive apoptosis, as reported by our laboratory and others (19 -21).The mechanism by which fortilin functions as an anti-apoptotic molecule has been under robust investigation. First, based on the fact that fortilin physically interacts with myeloid cell leukemia protein-1 (MCL1), an anti-apoptotic Bcl-2 family member, it was suggested that fortilin stabilizes and exerts its anti-apoptotic activity through MCL1 (22). However, fortilin is capable of protecting cells from apoptosis in the...

The endoplasmic reticulum, the cytoplasmic organelle that matures a massive amount of nascent secretory polypeptides, is particularly sensitive to stress. Endoplasmic reticulum stress causes unfolded proteins to populate the organelle, eliciting the unfolded protein response. During the unfolded protein response, GRP78—an endoplasmic reticulum master stress regulator—detaches from three endoplasmic reticulum stress sensors (IRE1α, PERK, and ATF6) and allows them to activate the apoptotic signaling pathway. Fortilin, a pro-survival molecule, is known to inhibit apoptosis by binding and inhibiting p53, but its role in endoplasmic reticulum stress-induced apoptosis remains unknown. Here, we report that fortilin directly interacts with the cytoplasmic domain of IRE1α, inhibits both kinase and endoribonuclease (RNase) activities of the stress sensor, and protects cells against apoptotic cell death at both cellular and whole animal levels. Our data support a role of fortilin in the unfolded protein response and its potential participation in human diseases caused by unfolded protein response.

The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.

Atherosclerosis, a deadly disease insufficiently addressed by cholesterol-lowering drugs, needs new therapeutic strategies. Fortilin, a 172-amino acid multifunctional polypeptide, binds p53 and blocks its transcriptional activation of Bax, thereby exerting potent antiapoptotic activity. Although fortilin-overexpressing mice reportedly exhibit hypertension and accelerated atherosclerosis, it remains unknown if fortilin, not hypertension, facilitates atherosclerosis. Our objective was to test the hypothesis that fortilin in and of itself facilitates atherosclerosis by protecting macrophages against apoptosis. We generated fortilin-deficient ( fortilin+/−) mice and wild-type counterparts ( fortilin+/+) on a LDL receptor ( Ldlr)−/− apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 ( Apobec1)−/− hypercholesterolemic genetic background, incubated them for 10 mo on a normal chow diet, and assessed the degree and extent of atherosclerosis. Despite similar blood pressure and lipid profiles, fortilin+/− mice exhibited significantly less atherosclerosis in their aortae than their fortilin +/+ littermate controls. Quantitative immunostaining and flow cytometry analyses showed that the atherosclerotic lesions of fortilin+/− mice contained fewer macrophages than those of fortilin+/+ mice. In addition, there were more apoptotic cells in the intima of fortilin+/− mice than in the intima of fortilin+/+ mice. Furthermore, peritoneal macrophages from fortilin+/− mice expressed more Bax and underwent increased apoptosis, both at the baseline level and in response to oxidized LDL. Finally, hypercholesterolemic sera from Ldlr−/− Apobec1−/− mice induced fortilin in peritoneal macrophages more robustly than sera from control mice. In conclusion, fortilin, induced in the proatherosclerotic microenvironment in macrophages, protects macrophages against Bax-induced apoptosis, allows them to propagate, and accelerates atherosclerosis. Anti-fortilin therapy thus may represent a promising next generation antiatherosclerotic therapeutic strategy.

Background Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. Methods & Results In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin+/− mice have the ability to survive and exhibit normal growth, while fortilin−/− mice are embryonically lethal around the 3.5 days post-coitum (dpc). Cultured blastocysts from fortilin+/− embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin−/− embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin+/− embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin’s message severely compromises the formation of neural tissue, including the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. Conclusion We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. General Significance Abnormalities in the fortilin gene may be associated with early pregnancy loss.

BackgroundThe aim of this study was to evaluate the efficiency of a simple prototype device for training respiratory muscles in lung function, respiratory muscle strength, walking capacity, quality of life (QOL), dyspnea, and oxidative stress in patients with COPD.MethodsThirty COPD patients with moderate severity of the disease were randomized into three groups: control (n=10, 6 males and 4 females), standard training (n=10, 4 males and 6 females), and prototype device (n=10, 5 males and 5 females). Respiratory muscle strength (maximal inspiratory pressure [PImax] and maximal expiratory pressure [PEmax]), lung function (forced vital capacity [FVC], percentage of FVC, forced expiratory volume in 1 second [FEV1], percentage of FEV1 [FEV1%], and FEV1/FVC), 6-minute walking distance (6MWD), QOL, and oxidative stress markers (total antioxidant capacity [TAC]), glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) were evaluated before and after 6 weeks of training. Moreover, dyspnea scores were assessed before; during week 2, 4, and 6 of training; and at rest after training.ResultsAll parameters between the groups had no statistical difference before training, and no statistical change in the control group after week 6. FVC, FEV1/FVC, PImax, PEmax, QOL, MDA, and NO showed significant changes after 6 weeks of training with either the standard or prototype device, compared to pre-training. FEV1, FEV1%, 6MWD, TAC, and GSH data did not change statistically. Furthermore, the results of significant changes in all parameters were not statistically different between training groups using the standard and prototype device. The peak dyspnea scores increased significantly in week 4 and 6 when applying the standard or prototype device, and then lowered significantly at rest after 6 weeks of training, compared to pre-training.ConclusionThis study proposes that a simple prototype device can be used clinically in COPD patients as a standard device to train respiratory muscles, improving lung function and QOL, as well as involving MDA and NO levels.

Methamphetamine (METH) is known as a toxin for neuronal and glial cells. Previous studies have found that METH-induced glial cell death and inflammation is mediated by oxidative stress. However, the exact mechanisms of the inflammatory response remain unclear. Therefore, we hypothesized that the activation of nuclear factor-κB (NF-κB) signaling, a key mediator of inflammation, and the inhibition of nuclear factor erythroid 2-related factor-2 (Nrf2) signaling, a regulator of the antioxidant response, would be significant events occurring in response to METH-induced inflammation in a rat glioma cell line (C6 cells). Our results show that METH increased the production of nitric oxide (NO) and up-regulated the expression of its main regulatory protein, inducible nitric oxide synthase (iNOS). METH also induced NF-κB activation by increasing inhibitory κBα (IκBα) degradation and translocation of the NF-κB (p65) subunit into the nucleus. Additionally, METH inhibited the activation of the Nrf2 pathway by decreasing the translocation of Nrf2 into the nucleus and also by suppressing the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO-1), and glutamate-cysteine ligase catalytic subunit (γ-GCLC), resulting in the suppression of superoxide dismutase (SOD) activity. Pretreatment with melatonin effectively promoted Nrf2 activation and reversed the METH-induced NF-κB response. Melatonin increased the expression of HO-1, NQO-1, and γ-GCLC, resulting in increased SOD activity. In addition, melatonin also decreased IκBα degradation, translocation of the p65 subunit, and expression of iNOS, resulting in decreased NO production. Taken together, our results indicate that melatonin diminishes the proinflammatory mediator in METH-stimulated C6 cells by inhibiting NF-κB activation and inducing Nrf2-mediated HO-1, NQO-1, and γ-GCLC expression.