NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover.

BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.

Experimental conditions and protein markers for redifferentiation of human coronary artery smooth muscle cells.

A phenotype switch from contractile type to proliferative type of arterial smooth muscle cells is known as dedifferentiation, but to the best of our knowledge, little is known about redifferentiation of coronary artery smooth muscle cells. The purpose of the present study was to determine in vitro culture conditions for inducing redifferentiation of coronary artery smooth muscle cells. In addition, the present study aimed to determine protein markers for detection of redifferentiated arterial smooth muscle cells. Human coronary artery smooth muscle cells (HCASMCs) were cultured in the presence or absence of growth factors, including epidermal growth factor, fibroblast growth factor-B and insulin. Protein expression and migration activity of HCASMCs were evaluated using western blotting and migration assay, respectively. In HCASMCs 5 days after 100% confluency, expression levels of α-smooth muscle actin (α-SMA), calponin, caldesmon and SM22α were significantly increased, while expression levels of proliferation cell nuclear antigen (PCNA) and S100A4 and migration activity were significantly decreased, compared with the corresponding levels just after reaching 100% confluency, indicating that redifferentiation occurred. Redifferentiation was also induced in a low-density culture of HCASMCs in the medium without growth factors. When the culture medium for confluent cells was replaced daily with fresh medium, the expression levels of α-SMA, caldesmon, SM22α, PCNA and S100A4 and migration activity were not significantly different but the calponin expression was significantly increased compared with the levels in dedifferentiated cells just after reaching 100% confluency. Thus, redifferentiation was induced in HCASMCs by deprivation of growth factors from culture medium. The results suggested that α-SMA, caldesmon and SM22α, but not calponin, are markers of redifferentiation of HCASMCs.

Ninjurin1 Deletion in NG2-Positive Pericytes Prevents Microvessel Maturation and Delays Wound Healing.

The formation of mature vasculature through angiogenesis is essential for adequate wound healing, such that blood-borne cells, nutrients, and oxygen can be delivered to the remodeling skin area. Neovessel maturation is highly dependent on the coordinated functions of vascular endothelial cells and perivascular cells, namely pericytes (PCs). However, the underlying mechanism for vascular maturation has not been completely elucidated, and its role in wound healing remains unclear. In this study, we investigated the role of Ninjurin-1 (Ninj1), a new molecule mediating vascular maturation, in wound healing using an inducible PC-specific Ninj1 deletion mouse model. Ninj1 expression increased temporarily in NG2-positive PCs in response to skin injury. When tamoxifen treatment induced a decreased Ninj1 expression in PCs, the neovessels in the regenerating wound margins were structurally and functionally immature, but the total number of microvessels was unaltered. This phenotypic change is associated with a reduction in PC-associated microvessels. Wound healing was significantly delayed in the NG2-specific Ninj1 deletion mouse model. Finally, we showed that Ninj1 is a crucial molecule that mediates vascular maturation in injured skin tissue through the interaction of vascular endothelial cells and PCs, thereby inducing adequate and prompt wound healing.

VEGF-Independent Angiogenic Factors: Beyond VEGF/VEGFR2 Signaling.

Tumors induce angiogenesis to acquire oxygen and nutrition from their adjacent microenvironment. Tumor angiogenesis has been believed to be induced primarily by the secretion of vascular endothelial growth factor-A (VEGF-A) from various tumors. VEGF-A binds to VEGF receptor 2 (VEGFR2), resulting in subsequent activation of cellular substances regulating cell proliferation, survival, and angiogenesis. Antiangiogenic therapies targeting the VEGF-A/VEGFR2 axis, including bevacizumab and ramucirumab, humanized monoclonal antibodies against VEGF-A and VEGFR2, respectively, have been proposed as a promising strategy aimed at preventing tumor growth, invasion, and metastasis. Phase III clinical trials using bevacizumab and ramucirumab have shown that not all tumor patients benefit from such antiangiogenic agents, and that some patients who initially benefit subsequently become less responsive to these antibodies, suggesting the possible existence of VEGF-independent angiogenic factors. In this review, we focus on VEGF-independent and VEGFR2-dependent tumor angiogenesis, as well as VEGFR2-independent tumor angiogenesis. Additionally, we discuss VEGF-independent angiogenic factors which have been reported in previous studies. Various molecular targeting drugs are currently being evaluated as potential antitumor therapies. We expect that precision medicine will permit the development of innovative antiangiogenic therapies targeting individual angiogenic factors selected on the basis of the genetic screening of tumors.

Contribution of platelet-derived microRNAs to serum microRNAs in healthy men.

Platelets are a major source of microRNAs (miRNAs) in blood. Relationships between circulating platelet-derived miRNAs were investigated to elucidate their significance as biomarkers. Total miRNAs in serum were analyzed using the 3D-Gene miRNA Oligo chip. Among 22 miRNAs that are included in platelets and play functional roles, sufficient miRNA levels for comparison were detected for 11 miRNAs (let-7b-5p, miR-16-5p, miR-17-5p, miR-24-3p, miR-107, miR-126-3p, miR-150-3p, miR-191-5p, miR-197-3p, miR-223-3p, and miR-326). Among 55 pairs prepared by these miRNAs, relatively strong correlations (Spearman's correlation coefficient >0.8) were shown between miRNAs of 7 pairs including let-7b-5p and miR-16-5p, let-7b-5p and miR-17-5p, let-7b-5p and miR-107, miR-16-5p and miR-17-5p, miR-16-5p and miR-107, miR-17-5p and miR-107, and miR-107 and miR-126-3p. In principal component analysis, the first principal component consisted of let-7b-5p, miR-16-5p, miR-17-5p, miR-107, miR-126-3p, and miR-191-5p. These six miRNAs may be useful biomarkers that reflect platelet condition and function.

Blood levels of microRNAs associated with ischemic heart disease differ between Austrians and Japanese: a pilot study.

Mortality from ischemic heart disease (IHD) is significantly lower in Japan than in Western countries. The purpose of this study was to investigate differences in circulating microRNA (miRNA) levels related to IHD in Austrians and Japanese. Participants were middle-aged healthy male Austrians (n = 20) and Japanese (n = 20). Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Twenty-one miRNAs, previously reported as associated with IHD, were compared between Austrians and Japanese. The expression levels of miR-106a-5p, miR-135a-3p, miR-150-3p, miR-16-5p, miR-17-5p. miR-191-5p, miR-320b, miR-451a, miR-486-5p, miR-663b, and miR-92a-3p were significantly higher, while the miR-2861 expression level was significantly lower in Austrians as compared to Japanese. Both in Austrians and Japanese, there were significant positive correlations between serum expression levels of each pair of the above miRNAs except for miR-2861. The expression level of miR-2861 showed significant positive correlations with the expression levels of miR-106a-5p, miR-150-3p, miR-17-5p, miR-486-5p, miR-663b and miR-92a-3p in Austrians but not in Japanese. In pathway analysis, proinflammatory cytokine production in foam cells and collagen synthesis in vascular smooth muscle cells were associated with differentially expressed miRNAs. Difference in miRNA levels may contribute to lower cardiovascular risk in Japan than in Western countries.

HDGF enhances VEGF­dependent angiogenesis and FGF­2 is a VEGF­independent angiogenic factor in non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancer­related deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factor­A (VEGF­A) which plays a pivotal role in tumour angiogenesis. Anti­angiogenic therapies including VEGF­A neutralisation have significantly improved the response rates, progression­free survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGF­independent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC­1 using serum­free culture, to which only EBC­1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC­1 cells. Among the identified proteins, interleukin­8 (IL­8), macrophage migration inhibitory factor (MIF), galectin­1, midkine (MK), IL­18, galectin­3, VEGF­A, hepatoma­derived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGF­A and HDGF function as angiogenic factors in EBC­1 cells. To confirm whether VEGF­A and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured three­dimensionally. Tube formation, neutralisation and RNA interference assays using the three­dimensional (3D) culture supernatant showed that VEGF­A and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC­1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brain­derived neurotrophic factor (BDNF), fibroblast growth factor­2 (FGF­2) and FGF­5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF­2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGF­dependent angiogenesis and that FGF­2 is a VEGF­independent angiogenic factor in human NSCLC cells.

Relationships among erythrocyte-derived microRNAs in serum of healthy donors.

BACKGROUND: Circulating microRNAs (miRNAs) have recently been proposed to be biomarkers for various diseases including cancer and cardiovascular disease. Erythrocytes are a major source of miRNAs in blood. However, it remains unknown how miRNA levels in serum are influenced by miRNAs in erythrocytes. In this study, we investigated the relationships among serum levels of miRNAs that are contained in erythrocytes. METHODS: Participants were middle-aged healthy Japanese men. Total miRNAs in serum from each participant were analyzed using the 3D-Gene miRNA Oligo chip. Relationships among the levels of eleven miRNAs (miR-103a-3p, -144-3p, -15a-5p, -16--5p, -26a-5p, -423-5p, -451a, -484, -486-5p, -92a-3p, and -93-5p) that have been reported to exist in erythrocytes were investigated by using correlation analysis. RESULTS: Among 55 pairs prepared by the above 11 miRNAs, there were significant correlations between miRNA levels of 31 pairs. In principal component analysis, 4 major erythrocyte-derived miRNAs, miR-16-5p, -451a, -486-5p and -92a-3p, were included in the first principal component. There were strong correlations between miR-16-5p and -451a levels (Spearman's rank correlation coefficient: 0.920) and between miR-486-5p and -92a-3p levels (Spearman's rank correlation coefficient: 0.863). CONCLUSION: There are significant associations among serum levels of erythrocyte-derived miRNAs, and these associations should be taken into account when considering the miRNAs as disease biomarkers.

The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: the suppression of tumor cell growth and angiogenesis.

Type I-interferon (IFN) is considered to exert antitumor effects through the inhibition of cancer cell proliferation and angiogenesis. Based on the species-specific biological activity of IFN, we evaluated each antitumor mechanism separately. We further examined the antitumor effects of type I-IFN combined with sorafenib. Human IFN (hIFN) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) Hep3B cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Although mouse IFN (mIFN) did not inhibit the proliferation of Hep3B cells in vitro, mIFN, as well as hIFN, showed significant antitumor effects in mouse Hep3B cell-xenograft model. Furthermore, mIFN treatment amplified the antitumor effects of sorafenib in vivo with the suppression of angiogenesis. The DNA chip analysis showed that the mIFN treatment promoted the antitumor signal pathways of sorafenib, including anti-angiogenic effects. Unlike the effects observed in in vitro experiments, mIFN showed an antitumor effect in the mouse Hep3B cell-xenograft model, suggesting a role of the anti-angiogenic activity in the in vivo tumoricidal effects of type I-IFN. In addition, our findings suggested the clinical utility of combination therapy with type І-IFN and sorafenib for HCC.

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure.

The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of m/z 1467, 2380 and 2662 changed significantly after drinking. The m/z 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the m/z 2380 peptide was identified to be a fragment of complement C4. The intensities of the m/z 2380 and m/z 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the m/z 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The m/z 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.

Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Early detection of cytomegalovirus-specific cytotoxic T lymphocytes against cytomegalovirus antigenemia in human leukocyte antigen haploidentical hematopoietic stem cell transplantation.

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation (haplo-SCT) is associated with a high incidence of cytomegalovirus (CMV) infection, probably originating from the delayed reconstitution of CMV-specific T cell immunity. There have been few reports on the presence of CMV-specific cytotoxic T lymphocytes (CMV-CTLs) after haplo-SCT. We have studied CMV-specific immune reconstitution by measuring the absolute number of CMV-CTLs using a flow cytometry method with HLA-A2-restricted NLVPMVATV peptide dextramers. We examined the association between reconstitution patterns of CMV-CTLs and the duration of CMV antigenemia in 15 patients who underwent first allogeneic SCT from HLA-haploidentical-related donors with HLA-A2. In seven and eight patients, CMV antigenemia consecutively resolved for more than 4 weeks (the CMV antigenemia 'resolved' group) and intermittently persisted (the CMV antigenemia 'persistent' group) during a 100-day observation period, respectively. The group of the seven patients, in whom levels of CMV antigenemia were reduced to zero, had a significantly lower maximum level of CMV antigenemia than the CMV antigenemia persistent group. In contrast, the CMV antigenemia persistent group had a significantly higher maximum level of CMV-CTLs, but the levels took longer to peak. Despite no difference in general lymphocyte recovery between the two groups, the CMV antigenemia resolved group had significantly higher median CMV-CTL counts than the CMV antigenemia persistent group at 6 weeks after onset of CMV infection. Flow cytometry analysis of CMV-CTLs is a convenient method of monitoring reconstitution of CMV-specific lymphocyte immunity following haplo-SCT.

Recombinant human soluble thrombomodulin attenuates FK506-induced endothelial dysfunction through prevention of Akt inactivation.

Thrombomodulin (TM), a transmembrane glycoprotein on vascular endothelial cells, is a naturally occurring anticoagulant. Recombinant human soluble TM (rTM), composed of the extracellular domain of TM, also shows anti-coagulant and anti-inflammatory activity, but the effects of rTM on microangiopathy remain unclear. We reported that FK506 induced endothelial dysfunction through inactivation of Akt and extracellular-regulated kinase 1/2 using a three-dimensional culture blood vessel model. In the present study, we examined the effects of rTM on FK506-induced endothelial dysfunction. We found that rTM suppressed FK506-induced endothelial cell death, but not the breakdown of capillary-like tube structures. rTM prevented FK506-induced inactivation of Akt, but not of extracellular-regulated kinase 1/2. Akt inhibition by LY294002 abrogated the preventive effect of rTM on FK506-induced Akt inactivation and the suppressive effect of rTM on FK506-induced cell death. These results suggest that rTM attenuates FK506-induced endothelial dysfunction through prevention of Akt inactivation.

FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.

Calcineurin inhibitors such as cyclosporin A (CsA) and FK506 have been used in solid organ and hematopoietic stem cell transplantations to suppress immune function. However, these immunosuppresants are associated with severe endothelial dysfunction. We investigated whether CsA and FK506 induce endothelial dysfunction using a three-dimensional culture blood vessel model, in which human umbilical vein endothelial cells form and maintain capillary-like tube and lumen structures. We found that FK506, but not CsA, induced breakdown of the tube structures and endothelial cell death. FK506 inhibited calcineurin activity, but FK506-induced tube breakdown and cell death was not suppressed by RNA interference targeting calcineurin Aα. FK506 also induced caspase activation, but caspase inhibition by zVAD(OMe)-fmk failed to suppress FK506-induced tube breakdown and cell death. FK506 induced attenuation of Akt and extracellular-regulated kinase 1/2 (ERK1/2). Furthermore, Akt inhibition by LY294002 or ERK1/2 inhibition by PD98059 induced tube breakdown and cell death. Present results suggest that FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.

Serum thioredoxin-1 as a diagnostic marker for malignant peritoneal mesothelioma.

BACKGROUND: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive malignant tumor of mesothelial origin that shows a limited response to cytoreductive surgery along with intraperitoneal chemotherapy. Therefore, diagnosing DMPM early is very important. Reactive oxygen species play an important role in asbestos toxicity, which is associated with the pathogenesis of DMPM growth. Thioredoxin-1 (TRX) is a small redox-active protein that demonstrates antioxidative activity associated with tumor growth. Here, we investigated the serum levels of TRX in patients with DMPM and compared them with those of a population that had been exposed to asbestos but did not have DMPM. STUDY: The serum concentrations of TRX were measured in 15 DMPM patients and 34 individuals with benign asbestos-related diseases. RESULTS: We demonstrated that the patients with DMPM had significantly higher serum levels of TRX than the population that had been exposed to asbestos but did not have DMPM. CONCLUSIONS: Our data suggest that serum TRX concentration is a useful serum marker for DMPM.

Different mechanisms causing loss of mismatched human leukocyte antigens in relapsing t(6;11)(q27;q23) acute myeloid leukemia after haploidentical transplantation.

Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA-mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA-A unshared between recipients and donors in relapsing leukemic cells after HLA-haploidentical transplantation. However, high-resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T-cell immune surveillance after HLA-haploidentical transplantation. Most importantly, down-regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.

Deficiency of Fyn protein is prerequisite for apoptosis induced by Src family kinase inhibitors in human mesothelioma cells.

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes. Src family kinases (SFKs) have a pivotal role in cell adhesion, proliferation, survival and apoptosis. Here, we examined the effect of SFK inhibitors in NCI-H2052, ACC-MESO-4 and NCI-H28 cells, mesothelioma cell lines and Met5A, a human non-malignant mesothelial cell line. We found that PP2, a selective SFK inhibitor, inhibited SFK activity and induced apoptosis mediated by caspase-8 in NCI-H28 but not Met5A, NCI-H2052 and ACC-MESO-4 cells. Src, Yes, Fyn and Lyn protein, which are members of the SFK, were expressed in these cell lines, whereas NCI-H28 cells were deficient in Fyn protein. Small interfering RNA (siRNA) targeting Fyn facilitated PP2-induced apoptosis mediated by caspase-8 in NCI-H2052 and ACC-MESO-4 cells. PP2 reduced Lyn protein levels and suppressed SFK activity in all mesothelioma cell lines. Lyn siRNA induced caspase-8 activation and apoptosis in NCI-H28 cells but not in NCI-H2052 and ACC-MESO-4 cells. However, double RNA interference knockdown of Fyn and Lyn induced apoptosis accompanied by caspase-8 activation in NCI-H2052 and ACC-MESO-4 cells. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, also inhibited SFK activity and induced reduction of Lyn protein levels, caspase-8 activation and apoptosis in NCI-H28 cells but not in other cell lines. Present study suggests that SFK inhibitors induce caspase-8-dependent apoptosis caused by reduction of Lyn protein in Fyn-deficient mesothelioma cells.

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.

Is serum thioredoxin-1 a useful clinical marker for malignant pleural mesothelioma?

Malignant pleural mesothelioma (MPM), an asbestos-related aggressive malignant tumor of mesothelial origin, shows limited response to therapy and overall survival remains very poor. Reactive oxygen species play an important role in asbestos toxicity. Here, we found that the patients with MPM had significantly higher serum levels of thioredoxin-1 (TRX) than control population. The patients with advanced-stage MPM showed higher levels of TRX than those with early-stage MPM. The difference in overall survival between the groups with lower and higher serum TRX levels was significant. Our data suggest that serum TRX concentration could be a useful clinical marker for MPM.

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.