Toshiro Ohta

Resveratrol and quercetin inhibit angiogenesis in vitro

Resveratrol and quercetin are polyphenolic compounds found in grapes, red wine and other food products. In this study, we examined the effect of resveratrol and quercetin on the inhibition of angiogenesis in vitro. Resveratrol and quercetin inhibited the growth of bovine aorta endothelial (BAE) cells in a concentration-dependent manner (6-100 microM).The migration of BAE was obviously inhibited by resveratrol and weakly inhibited by quercetin. When the lengths of all tubes constructed in the 3-D culture system with or without resveratrol or quercetin were measured, resveratrol was found to inhibit the tube formation of BAE cells in a concentration-dependent manner (6-100 microM). On the other hand, quercetin at concentrations above 100 microM significantly inhibited the tube formation of vascular endothelial cells. From these results, we suggest that resveratrol and quercetin may prove useful in the development of useful therapeutic agents or preventive food factors for tumor angiogenesis.

Tea catechins inhibit angiogenesis in vitro, measured by human endothelial cell growth, migration and tube formation, through inhibition of VEGF receptor binding

We have investigated whether tea catechins (EC, ECg, EGC, EGCg) have any inhibitory effects on angiogenesis and which step they affect during the process. The effects of catechins were tested on in vitro models of angiogenesis, namely, growth, migration and tube formation of human umbilical vein endothelial cells. All four catechins inhibited angiogenesis in vitro in the three different bioassays with concentrations ranging from 1.56 to 100 microM. Among the four catechins tested, epigallocatechin gallate (EGCg) was the most effective in inhibiting angiogenesis in all three assays. When these four catechins were tested on VEGF binding assay, only EGCg inhibited the binding of VEGF, a major angiogenesis inducing factor, to endothelial cells in a concentration dependent manner. These results indicate that while all four tea catechins inhibit the process of angiogenesis, EGCg alone can reduce the binding of VEGF to its receptors and thus affects the downstream signaling.

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

Correlation between antiangiogenic activity and antioxidant activity of various components from propolis

Propolis possesses various physiological activities. In this study, we examined the antiangiogenic and antioxidant activities of various components from propolis: acacetin, apigenin, artepillin C, caffeic acid phenethyl ester, chrysin, p-coumaric acid, galangin, kaempferol, pinocembrin, and quercetin. The effects of these components were tested on in vitro models of angiogenesis, tube formation and growth of human umbilical vein endothelial cells (HUVECs). Furthermore, these components were evaluated for their antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferric reducing/antioxidant power (FRAP) assays. Two propolis components, caffeic acid phenethyl ester, and quercetin, possessed strong inhibitory effects on tube formation and on endothelial cell proliferation and, coincidentally, showed strong antioxidant activity. Artepillin C, galangin, and kaempferol also possessed strong antiangiogenic and antioxidant activities to a slightly less degree. In contrast, acacetin, apigenin, and pinocembrin possessed a considerable degree of antiangiogenic activities, although they showed very low antioxidant activities. From these results, we propose that components from propolis such as artepillin C, caffeic acid phenethyl ester, galangin, kaempferol, and quercetin might represent a new class of dietary-derived antioxidative compounds with antiangiogenic activities. These propolis components may have the potential to be developed into pharmaceutical drugs for the treatment of angiogenesis-dependent human diseases such as tumors.

Artepillin C (ARC) in Brazilian Green Propolis Selectively Blocks Oncogenic PAK1 Signaling and Suppresses the Growth of NF Tumors in Mice

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)‐based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)‐based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)‐associated tumors require the kinase PAK1 for their growth, and CAPE‐based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE‐based and ARC‐based propolis extracts are natural anti‐PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30. Copyright

Arsenic trioxide induces apoptosis through JNK and ERK in human mesothelioma cells

Malignant mesothelioma is an aggressive tumor of serosal surfaces, which is refractory to current treatment options. Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia, and also to inhibit proliferation of several solid tumors including hepatoma, esophageal, and gastric cancer in vitro. Here we found that As2O3 inhibited cell viability of a mesothelioma cell line, NCI‐H2052. As2O3 induced apoptosis of NCI‐H2052 cells, which was accompanied by activation of c‐Jun NH2‐terminal kinase (JNK)1/2, extracellular signal‐regulated kinase (ERK)1/2, and caspase‐3. zVAD‐fmk, a broad‐spectrum caspase inhibitor, inhibited As2O3‐induced apoptosis and activation of caspase‐3, but not that of JNK1/2 and ERK1/2. Small interfering RNAs (siRNAs) targeting JNK1/2 suppressed As2O3‐induced caspase‐3 activation and apoptosis, indicating that JNK1/2 regulate As2O3‐induced apoptosis though caspase cascade. Furthermore, JNK1 siRNA abrogated As2O3‐induced JNK2 phosphorylation and JNK2 siRNA abrogated As2O3‐induced JNK1 phosphorylation, suggesting that JNK1 and JNK2 interact with each other. Moreover, JNK1 siRNA, but not JNK2 siRNA, abrogated As2O3‐induced ERK1/2 phosphorylation. JNK2 siRNA together with PD98059, a specific MAPK/ERK kinase inhibitor, suppressed As2O3‐induced apoptosis more significantly than JNK2 siRNA alone. These results indicated that As2O3 induces apoptosis of NCI‐H2052 cells mainly through JNK1/2 activation, and that ERK1/2 is involved in As2O3‐induced apoptosis when JNK1/2 are inactivated. J. Cell. Physiol. 226: 762–768, 2011.

Brazilian Propolis Suppresses Angiogenesis by Inducing Apoptosis in Tube-Forming Endothelial Cells through Inactivation of Survival Signal ERK1/2

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.

Nobiletin, a citrus polymethoxyflavonoid, suppresses multiple angiogenesis-related endothelial cell functions and angiogenesis in vivo

Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration‐dependent inhibitory effects on multiple functions of angiogenesis‐related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro‐matrix metalloproteinase‐2 (proMMP‐2) production and MMP‐2 mRNA expression in ECGS‐stimulated HUVEC. Nobiletin also downregulated cell‐associated plasminogen activator (PA) activity and urokinase‐type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis‐related signaling molecules, such as extracellular signal‐regulated kinase 1/2 and c‐Jun N‐terminal kinase, and transcriptional factors (c‐Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID50 value being 10 μg (24.9 nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions. (Cancer Sci 2010; 101: 2462–2469)

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia‐induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary‐like tubes, was used to analyze hypoxia‐induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase‐3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia‐induced apoptosis and tube breakdown by using zVAD‐fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD‐fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD‐fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase‐3. Inhibition of p38 suppressed activation of caspase‐3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so‐called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD‐fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase‐3 is activated, a p38‐regulated caspase‐independent pathway is crucial for the execution of hypoxia‐induced apoptosis and tube breakdown. J. Cell. Physiol. 211: 673–681, 2007.

Resveratrol derivative-rich melinjo (Gnetum gnemon L.) seed extract suppresses multiple angiogenesis-related endothelial cell functions and tumor angiogenesis.

Angiogenesis is a promising target for cancer prevention and treatment. This study aimed to determine the antiangiogenic effects of melinjo (Gnetum gnemon L.) seed extract and its resveratrol derivative components, such as gnetin C (GC), gnetin L (GL), gnemonoside A (GMA), gnemonoside C (GMC), and gnemonoside D (GMD). An ethanol extract of melinjo seeds (EEMS) and the two gnetins markedly inhibited the proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor and basic fibroblast growth factor. The inhibitory effects of GC and GL were much stronger than those of resveratrol. GMC and GMD inhibited only proliferation, whereas GMA had almost no effect on the two endothelial cell functions. The EEMS and GC also reduced the cell viability of tube-forming HUVEC, with accompanying ERK1/2 inactivation, and suppressed the migration of HUVEC. Furthermore, dietary intake of EEMS significantly inhibited tumor angiogenesis in a mouse dorsal air sac assay. In conclusion, we found that the EEMS and its resveratrol derivatives, particularly GC, suppress multiple angiogenesis-related endothelial cell functions and/or tumor angiogenesis, indicating that the melinjo seeds and the natural resveratrol derivatives may be useful for cancer prevention and treatment.