Avenanthramide C suppresses hypoxia-induced cyclooxygenase-2 expression through sirtuin1 activation in non-small-cell lung cancer cells

Summary

Avenanthramide C (AVC), found mainly in oats, mediates anti-inflammatory activities by reducing the anti-inflammatory cytokine levels. This study investigated the effects of AVC on hypoxia-induced cyclooxygenase-2 (COX-2) expression in A549 cells. AVC suppressed the hypoxia-induced increase in COX-2 protein levels and promoter activity. We also observed that the effects of AVC were reversed by a SIRT1 inhibitor, indicating that the inhibitory effects of AVC on hypoxia-induced COX-2 expression are mediated by SIRT1. Therefore, AVC inhibits the hypoxic induction of COX-2 expression via SIRT1 activation. Our results suggest that AVC could be beneficial for preventing lung inflammation under hypoxia.     

Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells

Background  

Stanniocalcin-1(STC-1) is up-regulated in several cancers including gastric cancer. Evidences suggest that STC-1 is associated with carcinogenesis and angiogenic process. However, it is unclear on the exact role for STC-1 in inducing angiogenesis and tumorigeneisis.     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.     

EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.     

Epigallocatechin-3-gallate induces cell apoptosis of human chondrosarcoma cells through apoptosis signal-regulating kinase 1 pathway

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.     

IGFBP7 regulates sepsis-induced acute kidney injury through ERK1/2 signaling

IGFBP7 as an early biomarker has been used to identify patients at risk of developing acute kidney injury (AKI). Nevertheless, its role in AKI remains obscure. The aim of our study is to determine the role and mechanism of IGFBP7 in lipopolysaccharide (LPS)-induced HK-2 cells in vitro and on sepsis-induced AKI by cecal ligation and puncture (CLP) in vivo. Here, we identified that IGFBP7 expression was increased in patients with AKI and HK-2 cells with LPS (1, 2, and 5 μg/mL) induction. HK-2 cells with LPS induction showed cell cycle arrest at G1-G0 phases and cell apoptosis and activated ERK1/2 parallel with the changes in the proteins belonging to the ERK1/2 pathway, including Cyclin D1, P21, Bax, and Bcl-2, which were inhibited by the IGFBP7 knockdown. Moreover, IGFBP7 overexpression significantly induced cell cycle arrest at G1-G0 phases and cell apoptosis of HK-2 cells, which were inhibited by PD98509, an ERK1/2 signaling inhibitor. IGFBP7 knockdown effectively alleviated the severity of the renal injury, evidenced by decreases in the urinary levels of creatinine, blood urea nitrogen, and albumin, cell apoptosis, and activation of ERK1/2 signaling in CLP mice. Taken together, our findings indicate that IGFBP7 regulates sepsis-induced AKI through ERK1/2 signaling.     

Rap2b promotes proliferation,migration, and invasion of lung cancer cells

Rap2b, a member of the guanosine triphosphate-binding proteins, is widely up-regulated in many types of tumors. However, the functional role of Rap2b in tumorigenesis of lung cancer remains to be fully elucidated. In this study, we investigated the effect of Rap2b on the lung cancer malignant phenotype, such as cell proliferation and metastasis. We found that Rap2b could promote the abilities of lung cancer cell wound healing, migration, and invasion via increasing matrix metalloproteinase-2 enzyme activity. Furthermore, Rap2b overexpression could increase the phosphorylation level of extracellular signal-regulated protein kinases 1/2. In conclusion, our results suggested that Rap2b may be a potential therapeutic target for lung cancer.     

Long noncoding RNA NEAT1-modulated miR-506 regulates gastric cancer development through targeting STAT3

Accumulating evidence has indicated that long noncoding RNA NEAT1 exerts critical roles in cancers. So far, the detailed biological role and mechanisms of NEAT1, which are responsible for human gastric cancer (GC), are still largely unknown. Here, we observed that NEAT1 and STAT3 expressions were significantly upregulated in human GC cells including BGC823, SGC-7901, AGS, MGC803, and MKN28 cells compared with normal gastric epithelial cells GES-1, while miR-506 was downregulated. We inhibited NEAT1 and observed that NEAT1 inhibition was able to repress the growth, migration, and invasion of GC cells. Conversely, overexpression of NEAT1 exhibited an increased ability of GC progression in BGC823 and SGC-7901 cells. Bioinformatics analysis, dual luciferase reporter assays, RIP assays, and RNA pull-down tests validated the negative binding correlation between NEAT1 and miR-506. In addition, it was found that miR-506 can modulate the expression of NEAT1 in vitro. STAT3 was predicted as a messenger RNA (mRNA) target of miR-506, and miR-506 mimics can suppress STAT3 mRNA expression. Subsequently, it was observed that downregulation of NEAT1 can restrain GC development by decreasing STAT3, which can be reversed by miR-506 inhibitors. Therefore, it was hypothesized in our study that NEAT1 can be recognized as a competing endogenous RNA to modulate STAT3 by sponging miR-506 in GC. In conclusion, we implied that NEAT1 can serve as an important biomarker in GC diagnosis and treatment.     

Prostasin regulates PD-L1 expression in human lung cancer cells

The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial–mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of the present study was to investigate if prostasin regulates PD-L1 expression. We established sublines overexpressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-γ (IFNγ) is further enhanced in cells overexpressing the wildtype membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.     

EGCG synergizes the therapeutic effect of irinotecan through enhanced DNA damage in human colorectal cancer cells

Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin-3-gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 μmol L⁻) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.     

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.     

Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义

目的探讨Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义。方法收集武汉大学人民医院病理科2006-2009年非小细胞肺癌存档蜡块40例,采用免疫组织化学S-P法检测40例非小细胞肺癌及癌旁组织中Bmi-1和EZH2基因的表达水平,并采用HPIAS-1000高清晰度彩色病理图文报告管理系统对Bmi-1和EZH2基因的表达进行定量分析,用SPSS11.5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 1.Bmi-1在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织Bm i-1的表达明显高于癌旁组织,差异有显著性(P<0.05)。2.EZH2基因的表达EZH2基因在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织中EZH2基因的表达明显高于癌旁组织(P<0.05)。结论 EZH2和Bmi-1基因对肺癌的生长有着不同的调节作用,在肺癌的发生、发展中发挥重要的作用。     

Targeting EP4 downstream c‐Jun through ERK1/2‐mediated reduction of DNMT1 reveals novel mechanism of solamargine‐inhibited growth of lung cancer cells

Lung cancer is the most common cancer and the leading cause of cancer deaths worldwide. We previously showed that solamargine, one natural phytochemicals from traditional plants, inhibited the growth of lung cancer cells through inhibition of prostaglandin E2 (PGE₂) receptor EP4. However, the potential downstream effectors of EP4 involving in the anti‐lung cancer effects of solamargine still remained to be determined. In this study, we further verified that solamargine inhibited growth of non‐small‐cell lung cancer (NSCLC) cells in multiple cell lines. Mechanistically, solamargine increased phosphorylation of ERK1/2. Moreover, solamargine inhibited the protein expression of DNA methyltransferase 1 (DNMT1) and c‐Jun, which were abrogated in cells treated with MEK/ERK1/2 inhibitor (PD98059) and transfected with exogenously expressed DNMT1 gene, respectively. Interestingly, overexpressed DNMT1 gene antagonized the effect of solamargine on c‐Jun protein expression. Intriguingly, overexpressed c‐Jun blocked solamargine‐inhibited lung cancer cell growth, and feedback resisted the solamargine‐induced phosphorylation of ERK1/2. A nude mouse xenograft model implanted with lung cancer cells in vivo confirmed the results in vitro. Collectively, our results show that solamargine inhibits the growth of human lung cancer cells through reduction of EP4 protein expression, followed by increasing ERK1/2 phosphorylation. This results in decrease in DNMT1 and c‐Jun protein expressions. The inter‐correlations between EP4, DNMT1 and c‐Jun and feedback regulation of ERK1/2 by c‐Jun contribute to the overall responses of solamargine in this process. This study uncovers an additional novel mechanism by which solamargine inhibits growth of human lung cancer cells.     

Quercetin inhibits IL-1 beta-induced ICAM-1 expression in pulmonary epithelial cell line A549 through the MAPK pathways

Quercetin is a herbal flavonoid derived from various foods of plant origin and widely used as a major constituent of nutritional supplements. Quercetin has been shown to have anti-inflammatory properties and can play a role in anti-inflammatory procedure. Intercellular adhesion molecule-1 (ICAM-1) is one of the important pro-inflammatory factors, especially in early phage of inflammation. However, the mechanisms regulating ICAM-1 expression by quercetin in human A549 cells were still unclear. In this study, the inhibitory effect of quercetin on ICAM-1 expression by interleukin-1 beta (IL-1 beta)-stimulated A549 cells was investigated, and the roles of mitogen-activated protein kinases (MAPK) pathways were explored. Quercetin attenuated IL-1 beta-induced expression of ICAM-1 mRNA and protein in a dose-dependent manner. The experiment suggested that quercetin actively inhibited inhibitory protein of nuclear factor-kappa B (I kappa B) degradation, and nuclear factor-kappa B (NF-kappa B) activity. The c-fos and c-jun, components of activator protein-1 (AP-1), were mediated by MAPK pathways. ERK and p38 were involved in the c-fos mRNA expression, and JNK was involved in the c-jun mRNA expression. The inhibitory effect of quercetin on ICAM-1 expression was mediated by the sequential attenuation of the c-fos and c-jun mRNA expressions. These inhibitory effects were partially inhibited by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a specific inhibitors of extracellular signal-regulated kinase (ERK), and SP600125, a specific inhibitor of c-Jun-N-terminal kinase (JNK). Taken together, these results suggest that quercetin negatively modulating ICAM-1 partly dependent on MAPK pathways. Binwu Ying and Ting Yang have contributed equally to this work.     

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.     

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway

Both estrogen and leptin play an important role in the regulation of physiological processes of endochondral bone formation in linear growth. Estrogen receptors (ERα and ERβ) are known as members of the superfamily of nuclear steroid hormone receptors and are detected in all zones of growth plate chondrocytes. They can be regulated in a ligand-independent manner. Whether leptin regulates ERs in the growth plate is still not clear. To explore this issue, chondrogenic ATDC5 cells were used in the present study. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting. We found that both ERα and ERβ were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERβ mRNA and protein levels 48 h after leptin stimulation (P<0.05) at day 14. The up-regulation of ERα and ERβ mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively). Furthermore, we confirmed that leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. Finally, a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs. These data demonstrate, for the first time, that leptin regulates the expression of ERs in growth plate chondrocytes via ERK signaling pathway, thereby suggesting a crosstalk between leptin and estrogen receptors in the regulation of bone formation.