Functional Role of DUSP6 in the EGFR-mediated Progression of Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) over-expression is commonly observed in advanced head and neck squamous cell carcinoma (HNSCC) and is correlated with poor patient outcomes. However, the role of dual-specificity phosphatase 6 (DUSP6) in EGFR-associated HNSCC progression remains poorly understood. This study aimed to investigate the correlation between DUSP6 expression and EGFR signaling in malignant HNSCC tissues. MATERIALS AND METHODS: Data mining and in vitro assays were employed to assess DUSP6 expression levels in HNSCC tissues compared to normal tissues. Additionally, the correlation between DUSP6 and EGFR expression was examined. Functional assays were conducted to investigate the modulation of DUSP6 expression by EGFR signaling and its involvement in EGF-induced cell migration and anoikis resistance. RESULTS: Our analysis revealed a significant elevation in DUSP6 expression in HNSCC tissues compared to normal tissues and a strong correlation between DUSP6 and EGFR expression. EGFR signaling modulated DUSP6 expression in a dose- and time-dependent manner, primarily through the extracellular signal-regulated kinase (ERK) pathway. Knockdown experiments demonstrated the functional role of DUSP6 in EGF-induced cell migration and anoikis resistance. CONCLUSION: The findings of this study elucidate the intricate signaling networks governing DUSP6 expression and its interplay with EGFR signaling in HNSCC. Moreover, the results provide insights into the potential role of DUSP6 as a therapeutic target and highlight the importance of personalized treatment strategies in HNSCC management.

Anti-Cancer Effects and Tumor Marker Role of Glutathione S-Transferase Mu 5 in Human Bladder Cancer.

Our previous study demonstrated that the glutathione S-transferase Mu 5 (GSTM5) gene is highly CpG-methylated in bladder cancer cells and that demethylation by 5-aza-dC activates GSTM5 gene expression. The aim of the present study was to investigate the role of GSTM5 in bladder cancer. The levels of GSTM5 gene expression and DNA methylation were analyzed in patients with bladder cancer, and functional studies of GSTM5 were conducted using GSTM5 overexpression in cultured bladder cancer cells. Clinical analysis revealed that the GSTM5 mRNA expression was lower in bladder cancer tissues than in normal tissues and that the level of GSTM5 DNA methylation was higher in bladder cancer tissues than in normal urine pellets. Overexpression of GSTM5 decreased cell proliferation, migration and colony formation capacity. Glutathione (GSH) assay results indicated that cellular GSH concentration was decreased by GSTM5 expression and that GSH supplementation reversed the decrease in proliferation and migration of cells overexpressing GSTM5. By contrast, a GSH synthesis inhibitor significantly decreased 5637 cell GSH levels, survival and migration. Furthermore, GSTM5 overexpression inhibited the adhesion of cells to the extracellular matrix protein fibronectin. To elucidate the effect of GSTM5 on anticancer drugs used to treat bladder cancer, cellular viability was compared between cells with or without GSTM5 overexpression. GSTM5-overexpressed cells showed no significant change in the cytotoxicity of cisplatin or mitomycin C in 5637, RT4 and BFTC 905 cells. Though a degree of resistance to doxorubicin was noted in 5637 cells overexpressing GSTM5, no such resistance was observed in RT4 and BFTC 905 cells. In summary, GSTM5 plays a tumor suppressor role in bladder cancer cells without significantly affecting chemoresistance to cisplatin and mitomycin C, and the cellular GSH levels highlight a key mechanism underlying the cancer inhibition effect of GSTM5. These findings suggest that low gene expression and high DNA methylation levels of GSTM5 may act as tumor markers for bladder cancer.

Hinokitiol induces cell death and inhibits epidermal growth factor-induced cell migration and signaling pathways in human cervical adenocarcinoma.

OBJECTIVE: The aim of this study was to examine the antitumor activity of hinokitiol for its clinical application in the treatment of human cervical carcinoma. MATERIALS AND METHODS: Cervical carcinoma HeLa cells were treated by different concentrations of hinokitiol. Flow cytometry was used to analyze cell cycle. Senescence-associated ß-galactosidase (SA-ß-gal) assay was used to identify senescent cells. The effects of hinokitiol on EGF-induced cell migration were determined by wound healing and transwell migration assays. Western blot was used to detect proteins involved in cell cycle progression, apoptosis, autophagy, and EGF-induced signaling pathways. RESULTS: Hinokitiol suppressed cell viability in a dose-dependent manner. Flow cytometric analysis indicated that hinokitiol treatment resulted in cell cycle arrest at G1 phase, with reduced number of cells in the G2/M phase. Western blot analysis further demonstrated that hinokitiol treatment increased the levels of p53 and p21, and concomitantly reduced the expression of cell cycle regulatory proteins, including cyclin D and cyclin E. SA-ß-gal assay showed that hinokitiol treatment significantly induced ß-galactosidase activity. In addition, treatment with hinokitiol increased the accumulation of the autophagy regulators, beclin 1 and microtubule-associated protein 1 light chain 3 (LC3-II), in a dose-dependent manner; however, it did not induce caspase-3 activation and poly ADP ribose polymerase (PARP) cleavage. In addition, epidermal growth factor-induced cell migration and c-Jun N-terminal kinase (JNK) and focal adhesion kinase (FAK) phosphorylation were significantly inhibited by hinokitiol. CONCLUSION: Our findings revealed that hinokitiol might serve as a potential therapeutic agent for cervical carcinoma therapy.

Gene expression and DNA methylation regulation of arsenic in mouse bladder tissues and in human urothelial cells.

According to a report of the International Agency for Research on Cancer, arsenic and inorganic arsenic compounds are classified into Group 1 carcinogens with regard to human health. Epidemiological studies indicate that arsenic is one of the main risk factors for the development of bladder cancer. In the present study, arsenic­altered gene expression in mouse bladder tissues and in human urothelial cells was compared. In the mouse model, sodium arsenite­induced mouse urothelial hyperplasia and intracellular inclusions were present. Following DNA array analysis, four genes with differential expression were selected for quantitative real­time PCR assay. The genes were the following: Cystathionine ß­synthase (CBS), adenosine A1 receptor (ADORA1), metastasis­associated lung adenocarcinoma transcript 1 (MALAT1) and Wnt inhibitory factor 1 (Wif1). The results indicated a significant increase in the levels of Cbs and Adora1. The analysis of the DNA CpG methylation levels of the mouse Cbs and Adora1 genes revealed no significant change. In contrast to these observations, the four genes were further analyzed in the human normal urothelial cell line SV­HUC1. The data indicated that WIF1 gene expression was decreased by sodium arsenite, whereas this was not noted for CBS, MALAT1 and ADORA1. Sodium arsenite decreased mRNA and protein expression levels of the WIF1 gene. In addition, the methylation levels of the WIF1 gene were increased. Sodium arsenite inhibited cell proliferation and promoted cell migration as demonstrated in cell functional assays. The gene status was compared in 8 human urothelial cell lines, and WIF1 mRNA expression levels were determined to be higher, whereas DNA CpG methylation levels were lower in SV­HUC1 cells compared with those noted in the other 7 bladder cancer cell lines. In summary, the data indicated that sodium arsenite decreased WIF1 gene expression and promoted cell migration. The increased methylation levels of WIF1 DNA CpG could be a potential biomarker for bladder cancer.

Chrysin-induced ERK1/2 Phosphorylation Enhances the Sensitivity of Human Hepatocellular Carcinoma Cells to Sorafenib.

BACKGROUND/AIM: Sorafenib is now standard treatment for advanced hepatocellular carcinoma (HCC). However, therapeutic efficacy is not as good as was predicted. Many efforts are being made to improve HCC sensitivity to sorafenib. Our previous study demonstrated that co-treatment with chrysin enhanced sorafenib sensitivity through inhibition of ATP-binding cassette super-family G member 2 (ABCG2). Whether there is another mechanism other than inhibition of ABCG2 underlying chrysin-mediated synergistic effect is still not completely elucidated. MATERIALS AND METHODS: Phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was examined by western blot. Cell viability was examined by crystal violet staining. The importance of ERK1/2 phosphorylation was assessed by overexpression and blockage of mitogen-activated protein kinase kinase 1 (MEK1). RESULTS: Chrysin induced sustained ERK1/2 phosphorylation of HCC cells in both time- and dose-dependent manners. Overexpression of MEK1 enhanced, whereas blockage of MEK1 led to loss of chrysin-synergized sorafenib effect, through modulating ERK1/2 phosphorylation level. CONCLUSION: These results identify another novel mechanism underlying chrysin-mediated synergistic effect on sorafenib activity in HCC cells.

Trichostatin A induces bladder cancer cell death via intrinsic apoptosis at the early phase and Sp1­survivin downregulation at the late phase of treatment.

Histone deacetylase (HDAC) inhibitors have been widely shown to result in cancer cell death. The present study investigated the mechanisms underlying the antitumor effects of the phytochemical trichostatin A (TSA), a classic pan-HDAC inhibitor, in 5,637 urinary bladder cancer cells. It was found that TSA caused cell cycle arrest at the G2/M and G1 phase accompanied by reduced expression of cyclin D1 and upregulated induction of p21. In addition, TSA induced morphological changes, reduced cell viability and apoptotic cell death in 5,637 cells through caspase-3 activation followed by PARP cleavage. The loss of mitochondrial membrane potential (MMP) indicated that TSA induced apoptosis in 5,637 cells through the intrinsic mitochondrial pathway. TSA significantly suppressed Akt activity at 12 h after treatment, suggesting that the apoptosis in the early phase was mediated by Akt inhibition. In addition, the protein level of transcription factor Sp1 was decreased at 24 h after TSA treatment, which likely led to the downregulation of survivin gene expression, and then contributed to the antitumor activity of TSA. Taken together, the present study delineated that TSA-induced growth inhibition and apoptosis in 5,637 cells was associated with pAKT inhibition and MMP loss at the early phase, followed by downregulation of Sp1 and survivin at the late phase of treatment.

Alkaloids from Pandanus amaryllifolius: Isolation and Their Plausible Biosynthetic Formation.

Pandanus amaryllifolius Roxb. (Pandanaceae) is used as a flavor and in folk medicine in Southeast Asia. The ethanolic crude extract of the aerial parts of P. amaryllifolius exhibited antioxidant, antibiofilm, and anti-inflammatory activities in previous studies. In the current investigation, the purification of the ethanolic extract yielded nine new compounds, including N-acetylnorpandamarilactonines A (1) and B (2); pandalizines A (3) and B (4); pandanmenyamine (5); pandamarilactones 2 (6) and 3 (7), and 5(E)-pandamarilactonine-32 (8); and pandalactonine (9). The isolated alkaloids, with either a γ-alkylidene-α,ß-unsaturated-γ-lactone or γ-alkylidene-α,ß-unsaturated-γ-lactam system, can be classified into five skeletons including norpandamarilactonine, indolizinone, pandanamine, pandamarilactone, and pandamarilactonine. A plausible biosynthetic route toward 1-5, 7, and 9 is proposed.

Activation of MAPK pathways and downstream transcription factors in 2-aminobiphenyl-induced apoptosis.

2-Aminobiphenyls (2-ABP) induces oxidative DNA damage and leads to apoptosis. The precise signaling pathways of inducing apoptosis in vitro are still unknown. This study provides insight into the relationship between 2-ABP-induced apoptosis and the activation of MAPK and downstream transcription factors using pharmacological inhibitors of ERK, p38, and JNK pathways. Results showed that 2-ABP induced the activation of ERK and JNK but not p38. The ERK/JNK pathways downstream transcription factors, c-Jun and ATF-2, were also activated by 2-ABP. The inhibitory effects of ERK inhibitor, U0126, on 2-ABP-induced caspase-3 activity were not detected. However, JNK inhibitor, SP600125, significantly attenuated the caspase-3 activity induced by 2-ABP. The expression of the transcription factors c-Jun and ATF-2 were decreased in 2-ABP treated cells in the presence of ERK/JNK inhibitors, suggesting that the expression of ERK/JNK pathways leads to the downstream activation of c-Jun and ATF-2. N-acetylcysteine, an ROS scavenger, inhibited 2-ABP-induced activation of ERK and JNK, the cell death and caspase-3 activity, which suggested that oxidative stress plays a crucial role in apoptosis through activation of caspase-3 in a ROS/JNK-dependent signaling cascade.

Hepatitis B virus X upregulates HuR protein level to stabilize HER2 expression in hepatocellular carcinoma cells.

Hepatitis B virus- (HBV-) associated hepatocellular carcinoma (HCC) is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx) has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR) family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2) is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC.

Molecular mechanisms of 3,3'-dichlorobenzidine-mediated toxicity in HepG2 cells.

3,3'-Dichlorobenzidine (DCB) (CAS 91-94-1), a synthetic, chlorinated, primary aromatic amine, is typically used as an intermediate in the manufacturing of pigments for printing inks, textiles, paints, and plastics. In this study, we found that DCB could significantly inhibit the cell viability of HepG2 cells in a concentration-dependent manner. Flow cytometry revealed that DCB induced G2/M-phase arrest and apoptosis in HepG2 cells. DCB treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψm ) and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that DCB-induced apoptosis was accompanied by the down-regulation of Bcl-2/Bax ratio. These results suggested that DCB led to cytotoxicity involving activation of mitochondrial-dependent apoptosis through Bax/Bcl-2 pathways in HepG2 cells. Furthermore, HepG2 cells treated with DCB showed significant DNA damage as supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by concentration- and time-dependent increase in histone H2AX phosphorylation (γ-H2AX). Two-dimensional-difference gel electrophoresis (2D-DIGE), combined with mass spectrometry (MS), was used to unveil the differences in protein expression between cells exposed to 25 µM or 100 µM of DCB for 24 hr and the control cells. Twenty-seven differentially expressed proteins involved in DNA repair, unfolded protein response, metabolism, cell signaling, and apoptosis were identified. Among these, 14-3-3 theta, CGI-46, and heat-shock 70 protein 4 were confirmed using Western blot assay. Taken together, these data suggest that DCB is capable of inducing DNA damage and some cellular stress responses in HepG2 cells, thus eventually leading to cell death by apoptosis.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient epithelial cells are less tolerant to infection by Staphylococcus aureus.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. The most common clinical manifestations in patients with G6PD deficiency are neonatal jaundice and acute hemolytic anemia. The effects of microbial infection in patients with G6PD deficiency primarily relate to the hemolytic anemia caused by Plasmodium or viral infections and the subsequent medication that is required. We are interested in studying the impact of bacterial infection in G6PD-deficient cells. G6PD knock down A549 lung carcinoma cells, together with the common pathogen Staphylococcus aureus, were employed in our cell infection model. Here, we demonstrate that a lower cell viability was observed among G6PD-deficient cells when compared to scramble controls upon bacterial infection using the MTT assay. A significant increase in the intracellular ROS was detected among S. aureus-infected G6PD-deficient cells by observing dichlorofluorescein (DCF) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. The impairment of ROS removal is predicted to enhance apoptotic activity in G6PD-deficient cells, and this enhanced apoptosis was observed by annexin V/PI staining under a confocal fluorescence microscope and quantified by flow cytometry. A higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by Western blotting analysis of G6PD-deficient cells following bacterial infection. In conclusion, we propose that bacterial infection, perhaps the secreted S. aureus α-hemolysin in this case, promotes the accumulation of intracellular ROS in G6PD-deficient cells. This would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells.

Differential regulation and activity against oxidative stress of Dps proteins in Bacillus cereus.

In this study, the sequence similarity, structure, ferroxidase activity and efficacy in antagonizing oxidative stress of three Dps-like proteins, Dps1, Dps2 and Dps3, encoded by Bacillus cereus were comparatively analyzed. The three Dps-like proteins are homologous to other bacterial Dps proteins that exhibit ferroxidase activity. Both Dps1 and Dps2 have a typical Dps spherical structure, but Dps3 has a unique filamentous structure. Several dps mutant strains were generated to investigate the functional role of dps genes in cell protection. The dps1 null strain was the most labile to oxidative stress in the stationary phase, and the loss of dps2 resulted in greater sensitivity to peroxide exposure compared with the other mutant strains in the log phase. Interestingly, after simultaneous deletion of dps1 and dps2, the survival rate was dramatically reduced by approximately 5 log in the stationary phase. Immunoblotting analysis demonstrated that Dps1 and Dps2 in the wild-type strain were induced by oxidative stress, and Dps3 responded to general stress in the log phase. Constitutively high expression of Dps2 in a perR null mutant and PerR-specific binding of the promoter region of dps2 confirmed Dps2 as a member of the PerR regulon. In addition, the expression of Dps1 and Dps2, absent any stress, was initiated in the log phase and was abundant in the stationary phase, suggesting that the expression of Dps1 and Dps2 was dependent on the bacterial growth stage. In summary, the three Dps proteins conferred cellular protection, particularly from oxidative stress, and were differentially regulated in response to varied stress conditions.

BCRP/ABCG2 inhibition sensitizes hepatocellular carcinoma cells to sorafenib.

The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine kinase inhibitors (TKIs) to attenuate their efficacy. This study aimed to investigate the role of BCRP/ABCG2 in the sensitivity of HCC to sorafenib. Our data showed that BCRP/ABCG2 mediated the efflux of sorafenib. Co-treatment with a BCRP/ABCG2 inhibitor greatly augmented the cytotoxicity of sorafenib in HCC cells. Similar results were also achieved by the competitive inhibitor of BCRP/ABCG2, gefitinib, in combination with sorafenib. These results suggest not only that BCRP/ABCG2 is a potential predictor for the sorafenib sensitivity in HCC, but also that blockage of BCRP/ABCG2 may be a potential strategy to increase the response of HCC cells to sorafenib.

Interplay of RsbM and RsbK controls the σ(B) activity of Bacillus cereus.

The alternative transcription factor σ(B) of Bacillus cereus controls the expression of a number of genes that respond to environmental stress. Four proteins encoded in the sigB gene cluster, including RsbV, RsbW, RsbY (RsbU) and RsbK, are known to be essential in the σ(B)-mediated stress response. In the context of stress, the hybrid sensor kinase RsbK is thought to phosphorylate the response regulator RsbY, a PP2C serine phosphatase, leading to the dephosphorylation of the phosphorylated RsbV. The unphosphorylated RsbV then sequesters the σ(B) antagonist, RsbW, ultimately liberating σ(B). The gene arrangement reveals an open reading frame, bc1007, flanked immediately downstream by rsbK within the sigB gene cluster. However, little is known about the function of bc1007. In this study, the deletion of bc1007 resulted in high constitutive σ(B) expression independent of environmental stimuli, indicating that bc1007 plays a role in σ(B) regulation. A bacterial two-hybrid analysis demonstrated that BC1007 interacts directly with RsbK, and autoradiographic studies revealed a specific C(14)-methyl transfer from the radiolabelled S-adenosylmethionine to RsbK when RsbK was incubated with purified BC1007. Our data suggest that BC1007 (RsbM) negatively regulates σ(B) activity by methylating RsbK. Additionally, mutagenic substitution was employed to modify 12 predicted methylation residues in RsbK. Certain RsbK mutants were able to rescue σ(B) activation in a rsbK-deleted bacterial strain, but RsbK(E439A) failed to activate σ(B), and RsbK(E446A) only moderately induced σ(B). These results suggest that Glu439 is the preferred methylation site and that Glu446 is potentially a minor methylation site. Gene arrays of the rsbK orthologues and the neighbouring rsbM orthologues are found in a wide range of bacteria. The regulation of sigma factors through metylation of RsbK-like sensor kinases appears to be widespread in the microbial world.

Cyclooxygenase-2 expression is up-regulated by 2-aminobiphenyl in a ROS and MAPK-dependent signaling pathway in a bladder cancer cell line.

Overexposure to biphenyl amine compounds, which are found in smoke and azo-dyes, is linked to the occurrence of bladder cancer. However, the molecular mechanisms of biphenyl amine compound-induced bladder cancer are still unclear. Many studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) in neoplastic lesions is associated with carcinogenesis. In this study, we have demonstrated that 2-aminobiphenyl (2-ABP) up-regulated the expression of COX-2 in a dose- and time-dependent manner in TSGH-8301 bladder cancer cells. This 2-ABP-induced COX-2 expression was attenuated by ROS scavenger NAC and NADPH oxidase inhibitors apocynin and DPI. The p22phox subunit of NADPH oxidase, but not p67, and Nox2 was up-regulated by 2-ABP. Knocking down p22phox by siRNA significantly reduced 2-ABP-induced COX-2 expression. Furthermore, 2-ABP also activated the ERK/JNK-AP1 pathways, and this effect was also abolished by NADPH oxidase inhibitors. Blocking the ERK/JNK-AP1 signaling pathways by pharmacological inhibitors attenuated 2-ABP-induced COX-2 expression. Overexpression of the upstream ERK activator MEK1 significantly and consistently increased 2-ABP-mediated COX-2 expression. Transfection of a dominant negative c-Jun mutant, TAM-67, blocked 2-ABP-mediated COX-2 expression, demonstrating that c-Jun was responsible for the transcriptional activation. Taken together, these results demonstrate that 2-ABP induces the carcinogenic factor COX-2 and that this induction is mediated through NADPH oxidase-derived ROS-dependent JNK/ERK-AP-1 pathways.

Induction of DNA damage signaling genes in benzidine-treated HepG2 cells.

We examined genotoxicity and DNA damage response in HepG2 cells following exposure to benzidine. Using the Comet assay, we showed that benzidine (50-200 µM) induces DNA damage in HepG2 cells. DNA damage signaling pathway-based PCR arrays were used to investigate expression changes in genes involved in cell-cycle arrest, apoptosis, and DNA repair and showed upregulation of 23 genes and downregulation of one gene in benzidine-treated cells. Induction of G2/M arrest and apoptosis was confirmed at the protein level. Real-time PCR and Western blots were used to demonstrate the expression of select DNA repair-associated genes from the PCR array. Upregulation of the p53 protein in benzidine-treated cells suggests the induction of the p53 DNA damage signaling pathway. Collectively, DNA damage response genes induced by benzidine indicate recruitment complex molecular machinery involved in DNA repair, cell-cycle arrest, and potentially, activation of the apoptosis.

Cloning and characterization of ß-agarase AgaYT from Flammeovirga yaeyamensis strain YT.

A bacterium with potent agar-degrading capability was isolated from the surface of a red algae, Gracilaria tenuistipitata. Based on phenotypic characteristics, 16S rDNA gene sequence and a phylogenetic analysis, this bacterium was identified and named as Flammeovirga yaeyamensis strain YT. PCR using homology-based degenerate primers was employed to clone any agarase gene belonging to GH16 family encoded in F. yaeyamensis strain YT. The resolved 1512 nucleotides revealed that the cloned gene, namely AgaYT, encodes a protein of 503 amino acids comprising a signal peptide, a glycosyl hydrolase catalytic module and a C-terminal domain with an unknown function. The recombinant protein r-AgaYT is an endo-type ß-agarase hydrolyzing agarose to yield neoagarobiose and neoagarotetraose as the main hydrolytic products. The specific activity of r-AgaYT was determined about 178.6 U mg(-1) at 40°C and pH 8.0.

Fenthion and terbufos induce DNA damage, the expression of tumor-related genes, and apoptosis in HEPG2 cells.

This study investigates the effects of fenthion and terbufos, two organophosphorous pesticides, on DNA damage, tumor-related gene expression, and apoptosis in HepG2 cells. We found that exposure to concentrations ranging from 50 to 200 µM of fenthion and terbufos for 2 hr caused significant death in HepG2 cells. Both compounds induced DNA damage in a concentration-dependent manner as measured using the alkaline comet assay. Tumor-related genes (jun, myc, and fos) and apoptosis-related genes (socs3, tnfaip3, ppp1r15a, and nr4a1) were up-regulated by both compounds. Finally, both compounds induced apoptosis. The results demonstrate that both terbufos and fenthion induce DNA damage and should be considered potentially hazardous to humans.

A novel alkaloid, aristopyridinone A and anti-inflammatory phenanthrenes isolated from Aristolochia manshuriensis.

A novel alkaloid, aristopyridinone A (1) and a new phenanthrene, aristolamide II (2), were isolated from Aristolochia manshuriensis (Guanmutong) together with eight known phenanthrenes (3-10). All structures were elucidated by spectroscopic methods. Compound 2 showed a selective inhibitory effect on elastase release by human neutrophils in response to fMLP with an IC(50) value of 4.11 µg/mL. Compound 7 exhibited significant inhibitory effects on superoxide anion generation and elastase release with IC(50) values of 0.12 and 0.20 µg/mL, respectively.

Functional role of NF-IL6beta and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase 2 gene.

NF-IL6beta regulates gene expression and plays function roles in many tissues. The EGF-regulated cyclooxygenase-2 (cox-2) expression is mediated through p38(MAPK) signaling pathway and positively correlates with NF-IL6beta expression in A431 cells. NF-IL6beta coordinated with c-Jun on cox-2 transcriptional activation by reporter and small interfering RNA assays. NF-IL6beta could directly bind to CCAAT/enhancer-binding protein (C/EBP) and cyclic AMP-response element (CRE) sites of the cox-2 promoter by in vitro-DNA binding assay. The C/EBP site was important for basal and, to a lesser extent, for EGF-regulated cox-2 transcription, while the CRE site was a more specific response to EGF inducibility of cox-2 gene. SUMO1 expression attenuated EGF- and NF-IL6beta-induced cox-2 promoter activities. NF-IL6beta was found to be sumoylated by in vivo- and in vitro-sumoylation assays, and the SUMO1-NF-IL6beta (suNF-IL6beta) lost its ability to interact with p300 in in vitro-binding assay. NF-IL6beta was also acetylated by p300, and acetylation of NF-IL6beta enhanced the cox-2 promoter activity stimulated by NF-IL6beta itself. In vivo-DNA binding assay demonstrated that EGF stimulated the recruitment of p300 and NF-IL6beta to the cox-2 promoter, yet promoted the dissociation of SUMO1-modificated proteins from the promoter. These results indicated that NF-IL6beta plays a pivotal role in the regulation of basal and EGF-induced cox-2 transcription.