α-Tomatine Suppresses Invasion and Migration of Human Non-Small Cell Lung Cancer NCI-H460 Cells Through Inactivating FAK/PI3K/Akt Signaling Pathway and Reducing Binding Activity of NF-κB

α-Tomatine, isolated from Lycopersicon esculentum Linn., is a naturally occurring steroidal glycoalkaloid in immature green tomatoes. Some reports demonstrated that α-tomatine had various anticarcinogenic properties. The purpose of this study is to investigate the anti-metastatic effect of α-tomatine in NCI-H460 human non-small cell lung cancer cells. First, the results showed that α-tomatine significantly suppressed the abilities of the adhesion, invasion, and migration of NCI-H460 cells under non-cytotoxic concentrations. Molecular data also showed α-tomatine could inhibit the activation of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K)/Akt signal involve in the downregulation the enzyme activities, protein and messenger RNA levels of matrix metalloproteinase-7 (MMP-7). Next, α-tomatine also strongly inhibited the degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding ability of NF-κB by α-tomatine treatment was further observed. Furthermore, α-tomatine significantly decreased the levels of phospho-Akt and MMP-7 in Akt1-cDNA-transfected cells concomitantly with a marked reduction on cell invasion and migration. Presented results indicated α-tomatine might be further application for treating cancer metastasis.     

CXCL12–CXCR7 Signaling Activates ERK and Akt Pathways in Human Choriocarcinoma Cells

Abstract

CXCL12 acts as a physiological ligand for the chemokine receptor CXCR7. Chemokine receptor expression by human trophoblast and other placental cells have important implications for understanding the regulation of placental growth and development. We had previously reported the differential expression of CXCR7 in different stages of the human placenta suggesting its possible role in regulation of placental growth and development. In this study, we determined the expression of CXCR7 in human choriocarcinoma JAR cells at the mRNA level and protein level and the downstream signaling pathway mediated by CXCL12–CXCR7 interaction. We observed that binding of CXCL12 to CXCR7 activates the ERK and Akt cell-survival pathways in JAR cells. Inhibition of the ERK and Akt pathways using specific inhibitors (Wortmanin & PD98509) led to the activation of the p38 pathway. Our findings suggest a possible role of CXCR7 in activating the cell survival pathways ERK and Akt in human choriocarcinoma JAR cells.     

Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

Background

Pathological angiogenesis plays an essential role in tumor aggressiveness and leads to unfavorable prognosis. The aim of this study is to detect the potential role of Retinoblastoma binding protein 2 (RBP2) in the tumor angiogenesis of non-small cell lung cancer (NSCLC).

Methods

Immunohistochemical staining was used to detect the expression of RBP2, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and CD34. Two pairs of siRNA sequences and pcDNA3-HA-RBP2 were used to down-regulate and up-regulate RBP2 expression in H1975 and SK-MES-1 cells. An endothelial cell tube formation assay, VEGF enzyme-linked immunosorbent assay, real-time PCR and western blotting were performed to detect the potential mechanisms mediated by RBP2 in tumor angiogenesis.

Results

Of the 102 stage I NSCLC specimens analyzed, high RBP2 protein expression is closely associated with tumor size (P = 0.030), high HIF-1α expression (P = 0.028), high VEGF expression (P = 0.048), increased tumor angiogenesis (P = 0.033) and poor prognosis (P = 0.037); high MVD was associated with high HIF-1α expression (P = 0.034), high VEGF expression (P = 0.001) and poor prognosis (P = 0.040). Multivariate analysis indicated that RBP2 had an independent influence on the survival of patients with stage I NSCLC (P = 0.044). By modulating the expression of RBP2, our findings suggested that RBP2 protein depletion decreased HUVECs tube formation by down-regulating VEGF in a conditioned medium. RBP2 stimulated the up-regulation of VEGF, which was dependent on HIF-1α, and activated the HIF-1α via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Moreover, VEGF increased the activation of Akt regulated by RBP2.

Conclusions

The RBP2 protein may stimulate HIF-1α expression via the activation of the PI3K/Akt signaling pathway under normoxia and then stimulate VEGF expression. These findings indicate that RBP2 may play a critical role in tumor angiogenesis and serve as an attractive therapeutic target against tumor aggressiveness for early-stage NSCLC patients.     

Cell Type- and Isotype-Specific Expression and Regulation of β-Tubulins in Primary Olfactory Ensheathing Cells and Schwann Cells In Vitro

Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on β-tubulin isotype expression in primary adult canine glia as a translational large animal model. β-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed βII–V isotype mRNA. Interestingly, βIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to βV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of βIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of βIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (βIII) and isotype-specific regulation (βIII, βV) of β-tubulin isotypes in OECs and SCs. While differential expression of βIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong βIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Salsolinol,an Endogenous Neurotoxin,Activates JNK and NF-κB Signaling Pathways in Human Neuroblastoma Cells

Salsolinol, an endogenous neurotoxin, is known to be involved in the pathogenesis of Parkinson’s disease (PD). In the present study, we have investigated the effects of salsolinol on the activation of two different signaling pathways that involve c-Jun N-terminal kinase (JNK), and nuclear factor-κB, (NF-κB) in human dopaminergic neuroblastoma SH-SY5Y cells. Salsolinol treatment caused upregulation in the levels of c-Jun and phosphorylated c-Jun. It also caused degradation of IκBα and translocated the active NF-κB into the nucleus. The binding activity of NF-κB to DNA was enhanced by salsolinol in a concentration dependent manner. Furthermore, salsolinol decreased the levels of the anti-apoptotic protein Bcl-2, and increased pro-apoptotic protein Bax, while enhancing the release of cytochrome-c from mitochondria. Mitochondrial complex-I activity was significantly decreased and reactive oxygen species (ROS) were increased in salsolinol treated cells. These results partly suggest that salsolinol-induced JNK and NF-κB signaling pathways may be involved in induction of apoptosis in human dopaminergic neurons, as seen in Parkinson’s disease.     

Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src,NADPH Oxidase,PI3K,and Akt in Human Colon Cancer Cells

Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47^phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.     

VEGF,HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.     

Overexpression of RNF146 in Non-Small Cell Lung Cancer Enhances Proliferation and Invasion of Tumors through the Wnt/β-catenin Signaling Pathway

Studies have suggested a possible correlation between the newly identified E3 ubiquitin ligase ring finger protein 146 (RNF146) and tumor development. However, until now, studies on RNF146 have been restricted to poly(ADP-ribosyl)ation and ubiquitin ligation, whereas the role of RNF146 in tumor biology has rarely been reported. In the present study, the role of RNF146 in non-small cell lung cancer (NSCLC) was investigated. The results showed that the expression of RNF146 was increased in clinical lung cancer samples and cell lines. RNF146 expression correlated with tumor size, differentiation level, lymphatic metastasis, pTNM staging, and prognosis of patients in stage I. RNF146 expression was negatively correlated with Axin expression but positively correlated with the nuclear expression of β-catenin in NSCLC tissues. RNF146 downregulated the expression of Axin in lung cancer cell lines and induced the expression and nuclear distribution of β-catenin. Overexpression of RNF146 in NSCLC cell lines increased the levels of cyclinD1, cyclinE, and CDK4, promoted cell cycle G₀/G₁-S transitions, and regulated cell proliferation. Overexpression of RNF146 led to upregulated levels of matrix metalloproteinases 2 and 7 and enhanced lung cancer cell invasiveness, events that were mediated by the classical Wnt/β-catenin signaling pathway. In summary, the data in the present study indicate that RNF146 regulated the development and progression of NSCLC by enhancing cell growth, invasion, and survival, suggesting that RNF146 may be a potential treatment target in NSCLC.     

Zinc Chloride Inhibits Human Lens Epithelial Cell Migration and Proliferation Involved in TGF-β1 and TNF-α Signaling Pathways in HLE B-3 Cells

Zinc is one of the most abundant essential elements in the human body, which is an essential, coenzyme-like component of many enzymes, and is indispensable to their functions. However, high levels of zinc ions can lead to cell damage. In the present study, we explored the effects of high concentrations of zinc chloride (ZnCl₂) on lens epithelial cell proliferation and migration and further investigated the effects of different concentrations of ZnCl₂ on caspase-9 and caspase-12, transforming growth factor-beta 1 (TGF-β1), and tumor necrosis factor-alpha (TNF-α). We found that ZnCl₂ could inhibit human lens epithelial (HLE) B-3 cell migration and induce apoptosis/necrosis. In addition, ZnCl₂ can efficiently decrease the expressions of caspase-9 and caspase-12, increase the expression of TNF-α at both gene and protein levels, and thus induces cell death. Taken together, our results indicate that ZnCl₂ can inhibit HLE B-3 cell migration and proliferation by decreasing the expression of TGF-β1 and increasing the expression of TNF-α and finally lead to HLE B-3 cell death.     

Galectin Expression Profiling Identifies Galectin-1 and Galectin-9Δ5 as Prognostic Factors in Stage I/II Non-Small Cell Lung Cancer

Approximately 30–40% of the patients with early stage non-small cell lung cancer (NSCLC) will present with recurrent disease within two years of resection. Here, we performed extensive galectin expression profiling in a retrospective study using frozen and paraffin embedded tumor tissues from 87 stage I/II NSCLC patients. Our data show that galectin mRNA expression in NSCLC is confined to galectin-1, -3, -4, -7, -8, and -9. Next to stage, univariable Cox regression analysis identified galectin-1, galectin-9FL and galectin-9Δ5 as possible prognostic markers. Kaplan-Meier survival estimates revealed that overall survival was significantly shorter in patients that express galectin-1 above median levels, i.e., 23.0 (2.9–43.1) vs. 59.9 (47.7–72.1) months (p = 0.020) as well as in patients that express galectin-9Δ5 or galectin-9FL below the median, resp. 59.9 (41.9–75.9) vs. 32.8 (8.7–56.9) months (p = 0.014) or 23.2 (−0.4–46.8) vs. 58.9 (42.9–74.9) months (p = 0.042). All three galectins were also prognostic for disease free survival. Multivariable Cox regression analysis showed that for OS, the most significant prognostic model included stage, age, gal-1 and gal-9Δ5 while the model for DFS included stage, age and gal-9Δ5. In conclusion, the current study confirms the prognostic value of galectin-1 and identifies galectin-9Δ5 as novel potential prognostic markers in early stage NSCLC. These findings could help to identify early stage NSCLC patients that might benefit most from adjuvant chemotherapy.     

TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways

Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.