GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

BCR-ABL tyrosine kinase inhibition induces metabolic vulnerability by preventing the integrated stress response in K562 cells.

The integrated stress response (ISR) is a cellular process that is characterized by activation of eukaryotic initiation factor (eIF)2α kinases and subsequent induction of activating transcription factor (ATF)4. The ISR plays an important role in protecting cells from tumor-related metabolic stresses, such as nutrient deprivation and perturbed proteostasis. Here, we showed that disruption of the ISR, together with increased cellular stress vulnerability, was produced by pharmacological inhibition of BCR-ABL, the oncogenic driver in chronic myeloid leukemia (CML). Treatment of CML-derived K562 cells with BCR-ABL tyrosine kinase inhibitors, including imatinib, dasatinib, nilotinib and ponatinib, prevented activation of eIF2α kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control nonderepressible 2, and downstream ATF4 induction during metabolic stress. Prevention of ATF4 induction likely occurred as a result of the combinatorial suppression of the eIF2α kinase and phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathways. In addition, we found that pharmacological inhibition of PERK mimicked BCR-ABL inhibition to enhance apoptosis induction under stress conditions. These findings indicate that the ISR is under the control of BCR-ABL and may foster adaptation to tumorigenic stresses in CML cells.

BRAF-mutated cells activate GCN2-mediated integrated stress response as a cytoprotective mechanism in response to vemurafenib.

In BRAF-mutated melanoma cells, the BRAF inhibitor, vemurafenib, induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) and subsequent induction of activating transcription factor 4 (ATF4), the central regulation node of the integrated stress response (ISR). While the ISR supports cellular adaptation to various stresses, the role of vemurafenib-triggered ISR has not been fully characterized. Here, we showed that in response to vemurafenib, BRAF-mutated melanoma and colorectal cancer cells rapidly induced the ISR as a cytoprotective mechanism through activation of general control nonderepressible 2 (GCN2), an eIF2α kinase sensing amino acid levels. The vemurafenib-triggered ISR, an event independent of downstream MEK inhibition, was specifically prevented by silencing GCN2, but not other eIF2α kinases, including protein kinase-like endoplasmic reticulum kinase, which transmits endoplasmic reticulum (ER) stress. Consistently, the ER stress gatekeeper, GRP78, was not induced by vemurafenib. Interestingly, ATF4 silencing by siRNA rendered BRAF-mutated melanoma cells sensitive to vemurafenib. Thus, the GCN2-mediated ISR can promote cellular adaptation to vemurafenib-induced stress, providing an insight into the development of drug resistance.

Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.

Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation.

The unfolded protein response (UPR) is an adaptive stress response pathway that is essential for cancer cell survival under endoplasmic reticulum stress such as during glucose starvation. In this study, we identified spautin-1, an autophagy inhibitor that suppresses ubiquitin-specific peptidase 10 (USP10) and USP13, as a novel UPR inhibitor under glucose starvation conditions. Spautin-1 prevented the induction of UPR-associated proteins, including glucose-regulated protein 78, activating transcription factor 4, and a splicing variant of x-box-binding protein-1, and showed preferential cytotoxicity in glucose-starved cancer cells. However, USP10 and USP13 silencing and treatment with other autophagy inhibitors failed to result in UPR inhibition and preferential cytotoxicity during glucose starvation. Using transcriptome and chemosensitivity-based COMPARE analyses, we identified a similarity between spautin-1 and mitochondrial complex I inhibitors and found that spautin-1 suppressed the activity of complex I extracted from isolated mitochondria. Our results indicated that spautin-1 may represent an attractive mitochondria-targeted seed compound that inhibits the UPR and cancer cell survival during glucose starvation.

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 ID(50) value being 10µg (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions.

Antiangiogenic effects of indole-3-carbinol and 3,3'-diindolylmethane are associated with their differential regulation of ERK1/2 and Akt in tube-forming HUVEC.

We previously reported that indole-3-carbinol (I3C), found in cruciferous vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying molecular mechanisms still remain unclear. Antiangiogenic effects of its major metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In this study, we investigated the effects of these indoles on angiogenesis and tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by affecting angiogenic signal transduction in human umbilical vein endothelial cells (HUVEC). We found that I3C and DIM at 25 micromol/L significantly inhibited tube formation and only DIM induced a significant increase in apoptosis in tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At the molecular level, I3C and DIM markedly inactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was significantly greater than that of I3C. DIM treatment also resulted in activation of the caspase pathway and inactivation of Akt, whereas I3C did not affect them. These results indicate that I3C and DIM had a differential potential in the regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt was enough to inhibit tube formation and induce caspase-dependent apoptosis in tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is responsible for its stronger antiangiogenic effects than those of I3C.

Taurine's health influence on Japanese high school girls.

BACKGROUND: The prevalence of metabolic syndrome (MS) in children and adolescents has been increasing at an alarming rate. MS risks during childhood and adolescence adversely affect health conditions in later life. Thus, the characterization of their MS risks is a critical research field. The aims of this study are to survey the health status of Japanese adolescent females, a poorly characterized population, and to investigate the potential relationship between their MS risks and dietary factors like potassium (K) and taurine. METHODS: Anthropometric characteristics of 243 healthy school girls aged 13 to 18 years were measured. Serum levels of triglycerides, total cholesterol and high-density lipoprotein (HDL), and plasma levels of glucose and insulin were analyzed in fasting blood samples. We assessed overweight, disturbed lipid prolife, higher blood pressure (hBP) and higher plasma glucose (hGlc) levels as indicators of MS risks. The relationships between MS risks and urinary K or taurine excretion were investigated by dividing into higher and lower groups at medians of their urinary excretions. RESULTS: Half of junior high school (JHS) and one-quarter of senior high school (SHS) girls had at least one MS risk. The quite common risk was hGlc, the rates being 21% in JHS girls and 14% in SHS. The prevalence of being overweight and obesity were only small portions, the rate being 0% and 0% in JHS girls, and 10% and 1% in SHS, respectively. Substantial differences in the prevalence of hBP were observed between JHS (22%) and SHS (4%) girls. Furthermore, higher urinary K excretion group showed a significant decrease in triglyceride level (P = 0.03) and increase in HDL level (P = 0.003) compared with the lower. Also, the higher urinary taurine excretion group exhibited a significant reduction in triglyceride level (P = 0.04) compared with the lower. CONCLUSIONS: These results indicate that control of plasma glucose level rather than body weight is a crucial task in Japanese pubertal girls, and that a dietary habit rich in K and taurine could improve their lipid profile. Nutritional education based on these findings would help to prevent the future development of MS in Japanese female adolescents.

Taurine in health and diseases: consistent evidence from experimental and epidemiological studies.

Taurine (T) was first noted as beneficial for stroke and cardiovascular diseases (CVD) prevention in genetic rat models, stroke-prone spontaneously hypertensive rats (SHRSP). The preventive mechanisms of T were ascribed to sympathetic modulation for reducing blood pressure (BP) and anti-inflammatory action. Recent epidemiological surveys revealed the involvement of inflammatory mediators in the pathogenesis of stroke and also atherosclerosis for which T was proven to be effective experimentally. Arterio-lipidosis prone rats, a substrain of SHRSP selectively bred for higher reactive hypercholesterolemia, quickly develop not only arterial fat deposition but also fatty liver which could be attenuated by dietary T supplementation. CARDIAC (CVD and Alimentary Comparison) Study was a WHO-coordinated multi-center epidemiological survey on diets and CVD risks and mortalities in 61 populations. Twenty-four-hour urinary (24U) T was inversely related significantly with coronary heart disease mortality. Higher 24U-T excreters had significantly lower body mass index, systolic and diastolic BP, total cholesterol (T-Cho), and atherogenic index (AI: T-Cho/high density lipoprotein-cholesterol) than lower T excreters. T effects on CVD risks were intensified in individuals whose 24U-T and -magnesium (M) excretions were higher. Furthermore, higher Na excreters with higher heart rate whose BP were significantly higher than those with lower heart rate were divided into two groups by the mean of 24U-T, high and low T excreters. Since the former showed significantly lower BP than the latter, T may beneficially affect salt-sensitive BP rise. Included among the typical 61 populations, were Guiyang, China or St. John's, Newfoundland, Canada where in which the means of both 24U-T and -M were high or low, respectively. The former and the latter had low and high CVD risks, respectively. Australian Aboriginals living at the coastal area in Victoria were supposed to eat T- and M-rich bush and sea foods and be free from CVD 200 years ago, but they presently have nearly the highest CVD risks indicating that T- and/or M-containing seafood, vegetables, fruits, nuts, milk, etc, similar to prehistoric hunters' and gatherers' food should be good for CVD prevention. The preventive effects of T, good for health and longevity, first noted experimentally, were also proven epidemiologically in humans.

High susceptibility of obese hypertensive SHRSP.Z-Lepr(fa) /IzmDmcr rats to lipid deposition in the mesenteric artery.

1. Atherosclerosis is commonly observed in obesity. Obese atherosclerosis-prone animals may be a promising tool for understanding the pathophysiology of obesity-associated atherosclerosis. However, most rat strains are resistant to atherosclerosis. The aim of the present study was to assess the susceptibility of two obese hypertensive rat models, namely SHRSP.Z-Lepr(fa) /IzmDmcr rats (SHRSP-fatty) and SHR.Cg-Lepr(cp) /NDmcr rats (SHR-cp), to arterial lipid deposition, an initial stage of atherosclerosis, by comparing these strains with non-obese stroke-prone spontaneously hypertensive rats (SHRSP). 2. Eight-week-old male SHRSP, SHRSP-fatty and SHR-cp were fed a high-fat and high-cholesterol diet containing 20% palm oil, 5% cholesterol and 2% cholic acid for 5weeks. Bodyweight, blood pressure and fasting serum levels of total cholesterol and triglycerides were measured in 12-week-old rats. Oil red O staining was used to visualize lipid deposition in the mesenteric artery. 3. The bodyweight of 12-week-old SHRSP-fatty and SHR-cp was higher than that of SHRSP (P<0.005). Systolic blood pressure in SHRSP and SHRSP-fatty was higher than in SHR-cp (P<0.005). Serum total cholesterol and triglyceride levels were elevated in SHRSP-fatty (P<0.005) and SHR-cp (P<0.05) compared with levels in SHRSP. Lipid deposition in the mesenteric artery was significantly greater in SHRSP-fatty than in SHRSP (37.7±4.9 vs 13.1±2.8%, respectively; P<0.005), but markedly reduced in SHR-cp (1.8±0.4%; P<0.05). 4. The results of the present study indicate that SHRSP-fatty are highly susceptible to arterial lipid deposition, whereas SHR-cp are resistant. Thus, SHRSP-fatty may be a useful obese rat model in which to investigate atherosclerotic processes.

Disrupting ATF4 Expression Mechanisms Provides an Effective Strategy for BRAF-Targeted Melanoma Therapy.

BRAF V600 mutation influences cellular signaling pathways for melanoma development. However, the role of oncogenic BRAF in adaptive stress response pathways is not fully understood. Here, we show that oncogenic BRAF plays an essential role in the induction of ATF4 following the activation of general control non-derepressible 2 (GCN2) kinase during nutrient stress and BRAF-targeted, therapeutic stress. Under GCN2 activation, BRAF ensures ATF4 induction by utilizing mTOR and eIF4B as downstream regulators. In contrast to the MEK-ERK pathway, this signaling pathway remains temporarily active even during treatment with BRAF inhibitors, thereby enabling the transient induction of ATF4. We also identify a chemical compound that prevents BRAF inhibitor-induced activation of the GCN2-ATF4 pathway and produces synergistic cell killing with BRAF inhibitors. Our findings establish a collaborative relationship between oncogenic BRAF and the GCN2-ATF4 signaling pathway, which may provide a novel therapeutic approach to target the adaptive stress response.

Identification of nobiletin, a polymethoxyflavonoid, as an enhancer of adiponectin secretion.

Adiponectin, an adipocyte-derived protein with insulin-sensitizing, anti-diabetic and anti-atherogenic activities, is known to be induced during adipocyte differentiation. Nobiletin, a citrus polymethoxy flavonoid, was found to induce the differentiation of ST-13 preadipocytes into mature adipocytes and enhance the production of adiponectin protein at a concentration of 10 microM.

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.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Propolis suppresses tumor angiogenesis by inducing apoptosis in tube-forming endothelial cells.

We have reported that propolis suppresses tumor-induced angiogenesis in vivo and in vitro, but antiangiogenic mechanism of propolis at cellular level remains unclear. In this study, we observed that propolis not only inhibited tube formation but also induced apoptosis of endothelial cells. These results suggest that propolis exerts its antiangiogenic effects at least in part through induction of apoptosis.

Indole-3-carbinol suppresses tumor-induced angiogenesis by inhibiting tube formation and inducing apoptosis.

Indole-3-carbinol (I3C) has been reported to exert anticancer activity in vivo. However, its anticancer mechanism has not been fully elucidated. In this study, we demonstrate that I3C suppressed tumor-induced angiogenesis and tube formation of endothelial cells. I3C also induced apoptosis in endothelial cells by activating the caspase cascade. We propose that I3C exerts its anticancer effect through the induction of endothelial apoptosis.

Suppression of tumor-induced angiogenesis by Brazilian propolis: major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation.

Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis.

Inhibition of in vivo angiogenesis by N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine.

N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, inhibits human tumor growth in the nude mice model; however, the mechanism of its action is unclear. The in vivo antitumor effect includes the inhibition of tumor cell proliferation and suppression of angiogenesis. Angiogenesis is essential for tumor growth in vivo. In this study, we examined whether 5-S-GAD inhibits tumor cell-induced angiogenesis by performing the mouse dorsal air sac assay. We found that intraperitoneal administration of 5-S-GAD inhibited the angiogenesis induced by S180 mouse sarcoma cells. Furthermore, 5-S-GAD also inhibited vascular endothelial growth factor-induced angiogenesis in the Matrigel plug assay and embryonic angiogenesis in the chick embryo chorioallantoic membrane assay. However, 5-S-GAD did not show any effect on the proliferation, migration, and tube formation of vascular endothelial cells. These results provide the first evidence that a bioactive substance derived from the flesh fly has antiangiogenic activity in vivo, although the mechanisms involved could not be explained.

Antitumor activity of melinjo (Gnetum gnemon L.) seed extract in human and murine tumor models in vitro and in a colon-26 tumor-bearing mouse model in vivo.

Melinjo (Gnetum gnemon L.) seed extract (MSE) and its active ingredient gnetin C (GC), a resveratrol dimer, have been shown to possess a broad spectrum of pharmacological activities. In this study, we investigated the antitumor activity of MSE and GC using human and murine tumor cell culture models in vitro. The antitumor activity of GC was compared with trans-resveratrol (tRV), a stilbenoid polyphenol. Our results show that MSE and GC at clinically achievable concentrations significantly inhibited the proliferation of pancreatic, prostate, breast, and colon cancer cell types (P < 0.05), without affecting normal cells. Interestingly, GC exerts enhanced antitumor activity than that of tRV (P < 0.05). MSE and GC significantly induced apoptosis in all the cancer cells, indicating MSE and GC inhibit tumor cell growth by inducing apoptosis (P < 0.001). Our findings provide evidence that MSE might induce apoptosis in cancer cells via caspase-3/7-dependent and -independent mechanisms. However, GC might trigger both early and late stage apoptosis in cancer cells, at least in part by activating caspase 3/7-dependent mechanisms. Furthermore, the antitumor efficacy of MSE observed in vitro was also validated in a widely used colon-26 tumor-bearing mouse model. Oral administration of MSE at 50 and 100 mg/kg per day significantly inhibited tumor growth, intratumoral angiogenesis, and liver metastases in BALB/c mice bearing colon-26 tumors (P < 0.05). In conclusion, our findings provide evidence that MSE and GC have potent antitumor activity. Most importantly, we provide the first evidence that MSE inhibits tumor growth, intratumoral angiogenesis, and liver metastasis in a colon-26 tumor-bearing mice.