Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells.

Prox1 is a master regulator for the development of lymphatic vasculature and the induction of lymphangiogenesis. In this study, we identified Prox1 as a new target for small ubiquitin-like modifier 1 (SUMO-1). Lysine 556 (K556) was found to be the major sumoylation site for Prox1 in vitro and in vivo. Mutation of this site (from lysine to arginine K556R) reduced DNA binding and the transcriptional activity of Prox1. Overexpression of Prox1 in EA.hy926 endothelial cells induced expression of lymphatic endothelial cell-specific genes including vascular endothelial growth factor receptor 3 (VEGFR3), fibroblast growth factor receptor 3 (FGFR3) and p57 while expression of K556R mutant Prox1 had little effect. The induction of VEGFR3 by Prox1 in EA.hy926 endothelial cells was an indication of their response to VEGF-C-induced lymphangiogenic signals, including the enhancement of proliferation, sprouting and tube formation and the inhibition of apoptosis. This effect is SUMO-dependent because ectopic expression of SUMO-specific protease 2 (SENP2) effectively reduced Prox1 sumoylation and Prox1-induced VEGFR3 expression. In addition, K556R mutant Prox1 could not induce lymphatic phenotypes. Taken together, our results indicate that Prox1 is a target for SUMO-1 and suggest that sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells.

Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif.

BACKGROUND: Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. RESULTS: High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. CONCLUSIONS: Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

A novel sialyltransferase inhibitor AL10 suppresses invasion and metastasis of lung cancer cells by inhibiting integrin-mediated signaling.

Aberrant sialylation catalyzed by sialyltransferases (STs) is frequently found in cancer cells and is associated with increased cancer metastasis. However, ST inhibitors developed till now are not applicable for clinical use because of their poor cell permeability. In this study, a novel ST inhibitor AL10 derived from the lead compound lithocholic acid identified in our previous study is synthesized and the anti-cancer effect of this compound is studied. AL10 is cell-permeable and effectively attenuates total sialylation on cell surface. This inhibitor shows no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of alpha-2,3-ST-overexpressing A549 and CL1.5 human lung cells. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of various integrin molecules and attenuated activation of the integrin downstream signaling mediator focal adhesion kinase. More importantly, AL10 significantly suppresses experimental lung metastasis in vivo without affecting liver and kidney function of experimental animals as determined by serum biochemical assays. Taken together, AL10 is the first ST inhibitor, which exhibits potent anti-metastatic activity in vivo and may be useful for clinical cancer treatment.

Using laser-induced acoustic desorption/electrospray ionization mass spectrometry to characterize small organic and large biological compounds in the solid state and in solution under ambient conditions.

We have coupled laser-induced acoustic desorption (LIAD) with electrospray ionization (ESI) mass spectrometry (LIAD/ESI/MS) to characterize molecules in the solid state and in solution under ambient conditions. To perform an LIAD/ESI analysis, the sample droplet is deposited on the surface of a thin aluminum foil by a micropipette; the rear side of the foil with the sample spot is then irradiated with a pulse from a Nd:YAG IR laser. The resulting shockwave and heat cause the sample on the rear side to change from the condensed phase to the gas phase. The desorbed species then move upward to enter an ESI plume to react with charged solvent species (methanol- and water-related ions and droplets), forming singly or multiply charged analyte ions. A quadrupole/time-of-flight (Q-TOF) mass analyzer attached to the LIAD/ESI source detects the analyte ions to obtain an ESI-like mass spectrum. Both small organic and large biological compounds (including amino acids, peptides, and proteins) were successfully ionized and detected by the LIAD/ESI/MS system. Although native and denatured myoglobin ions were both detected from a liquid sample solution, only the denatured myoglobin ions were detected from a dried sample.

Non-steroidal anti-inflammatory drugs suppress the ERK signaling pathway via block of Ras/c-Raf interaction and activation of MAP kinase phosphatases.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit cancer cell growth, induce apoptosis and decrease tumor metastasis. We have previously reported that a NSAID NS398 repressed the expression of matrix metalloproteinase-2 (MMP-2) via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the underlying mechanism of this inhibition. In vitro kinase assay indicated that NS398 could not directly inhibit c-Raf, MEK1 and ERK enzymatic activity. We found that NS398 increased the inhibitory phosphorylation of Ser259 in c-Raf, attenuated membrane recruitment of c-Raf and inhibited Ras/c-Raf interaction to attenuate activation of this kinase. This is a general effect for NSAIDs because sulindac sulfide, aspirin and indomethacin also inhibited the binding of c-Raf to Ras. Immunofluorescent staining verified that NS398 reduced the serum-induced membrane recruitment of c-Raf in cells. However, overexpression of constitutively active c-Raf only partly reversed NS398-induced inhibition of MMP-2 expression. Interestingly, we found that NS398 up-regulated the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and MKP-3. Block of MKP activity by sodium orthovanadate also partly counteracted the inhibitory effect of NS398. Overexpression of constitutively active c-Raf and treatment of sodium orthovanadate together completely reversed the inhibition of MMP-2 by NS398. Taken together, we conclude that NS398 and other NSAIDs act via inhibition of Ras/c-Raf interaction and up-regulation of MKPs to suppress the ERK-mediated signaling.

Overexpression of Jab1 in hepatocellular carcinoma and its inhibition by peroxisome proliferator-activated receptor{gamma} ligands in vitro and in vivo.

PURPOSE: Jun activation domain-binding protein 1 (Jab1) is the fifth subunit of the COP9 signalosome and exhibits oncogenic activity. We investigated Jab1 expression in hepatocellular carcinoma (HCC) tissues and cell lines and tested the effect of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on Jab1 expression. EXPERIMENTAL DESIGN: Jab1 expression in HCC tissues and cell lines was studied by real-time reverse transcription-PCR, immunohistochemical staining, and Western blotting. Promoter activity and chromatin immunoprecipitation assays were done to address the inhibition of Jab1 promoter by PPARgamma ligands. RNA interference was used to clarify PPARgamma ligand-induced inhibition of Jab1. Anticancer and Jab1-suppressing activity of PPARgamma ligands was tested in nude mice. RESULTS: Jab1 was detected in the nucleus and cytoplasm of HCC tissues and 37% (37 of 99) of tissues exhibited Jab1 overexpression. Jab1 expression correlated with sex and hepatitis C virus infection, whereas it was negatively associated with hepatitis B virus infection. Additionally, Jab1 was overexpressed in HCC cell lines. PPARgamma ligands troglitazone and rosiglitazone down-regulated Jab1 expression in HCC cells, and troglitazone directly suppressed Jab1 promoter activity by inhibiting Sp1- and Tcf4-mediated transcription. This suppression was mediated via both PPARgamma-dependent and PPARgamma-independent mechanisms. Ectopic expression of Jab1 counteracted troglitazone-induced growth inhibition. Animal studies verified that intratumor or i.p. injection of troglitazone attenuated HCC growth and reduced Jab1 expression in tumor tissues. CONCLUSIONS: Our results indicate that Jab1 is overexpressed in HCC and PPARgamma ligands may suppress Jab1 to inhibit the proliferation of HCC cells.

Electrospray-assisted laser desorption/ionization mass spectrometry for continuously monitoring the states of ongoing chemical reactions in organic or aqueous solution under ambient conditions.

Electrospray-assisted laser desorption/ionization (ELDI) combined with mass spectrometry allows chemical and biochemical compounds to be characterized directly from hydrophilic and hydrophobic organic solutions mixed with carbon powders under ambient conditions. Organic and inorganic compounds dissolved in polar or nonpolar solvent such as methanol, tetrahydrofuran, ethyl acetate, toluene, dichloromethane, or hexane can be detected using this ambient ionization technique without prior pretreatment. We have used this technique to monitor the progress in several ongoing reactions: the epoxidation of chalcone in ethanol, the chelation of ethylenediaminetetraacetic acid with copper and nickel ions in aqueous solution, the chelation of 1,10-phenanthroline with iron(II) in methanol, and the tryptic digestion of cytochrome c in aqueous solution. Liquid-ELDI analyses simply require irradiation of the surface of the sample solution with a pulsed ultraviolet laser; the laser energy is adsorbed by the carbon powder presuspended in the sample solution; the absorbed laser energy is then transferred to the surrounding solvent and to the analyte molecules in the solution, leading to their desorption; the desorbed gaseous analyte molecules are then postionized within an electrospray (ESI) plume to generate ESI-like analyte ions.

Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry.

Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.

The nonsteroidal anti-inflammatory drug NS398 reactivates SPARC expression via promoter demethylation to attenuate invasiveness of lung cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to exhibit potent anticancer effects in vitro and in vivo. One of the mechanisms by which NSAIDs suppress tumorigenesis is inhibition of angiogenesis and metastasis. In this study, we used a microarray system to study the change of expression profile of metastasis-related genes regulated by NS398, a NSAID and a cyclooxygenase-2 (COX-2) inhibitor. We found that several negative regulators of cell invasion, including secreted protein acidic and rich in cysteine (SPARC), thrombospondin 1 (TSP-1), thrombospondin 3 (TSP-3), and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) are upregulated by NS398. In addition, we demonstrated that upregulation of SPARC expression by NS398 in human lung cancer cells is mediated by promoter demethylation and associated with a decrease in DNA methyltransferase (DNMT) expression. This is the first report to show that NS398 can inhibit the expression of DNMT1 and 3b. Functional assay indicated that SPARC is a critical mediator for NS398 to inhibit cell invasion. Our results provide new insights for the understanding of the anticancer actions of NSAIDs.

A bioactive withanolide Tubocapsanolide A inhibits proliferation of human lung cancer cells via repressing Skp2 expression.

Withanolides are generally defined as C(28) steroidal lactones built on an intact or rearranged ergostane skeleton and have been shown to exhibit antiproliferative activity on various types of cancer cells. In this study, we investigated the effect of a new withanolide Tubocapsanolide A isolated from Tubocapsicum anomalum and addressed its molecular action. Tubocapsanolide A inhibited proliferation of A549, H358, and H226 human lung cancer cells via induction of G(1) growth arrest. We found that Tubocapsanolide A treatment led to up-regulation of cyclin E, p21, and p27, whereas other cyclins and cyclin-dependent kinases were not affected in A549 cells. Conversely, Skp2, the F-box protein that is implicated in the mediation of degradation of p21 and p27, was significantly down-regulated. Chromatin immunoprecipitation assay suggested that Tubocapsanolide A suppressed Skp2 expression by inhibiting the binding of Rel A to the nuclear factor-kappaB site of Skp2 gene promoter. In addition, we showed that inhibition of Skp2 is a critical step for the suppression of cell proliferation by Tubocapsanolide A because ectoexpression of Skp2 effectively reversed Tubocapsanolide A-induced p27 up-regulation and growth inhibition in human lung cancer cells. Collectively, we have identified Skp2 as a molecular target for Tubocapsanolide A and suggest that this withanolide may be useful for the prevention or treatment of cancer cells with Skp2 overexpression.

HER-2/neu transcriptionally activates Jab1 expression via the AKT/beta-catenin pathway in breast cancer cells.

Jab1 is a co-activator of activating protein-1 (AP-1) transcription factor and the fifth subunit of the constitutive photomorphogenesis 9 (COP9) signalosome, which has been shown to mediate nuclear exportation and ubiquitin-dependent degradation of the tumor suppressor p27(Kip1). Jab1 is overexpressed in several types of human cancer. However, de-regulation of Jab1 gene expression in cancer cells is largely unclear. In this study, we reported that expression of Jab1 was stimulated by HER-2/neu oncogene via transcriptional activation. Promoter deletion and mutation analysis indicated that HER-2/neu stimulated Jab1 via the T cell factor (TCF) binding site located at the -380/-368 region of the human Jab1 promoter. DNA affinity precipitation assay and chromatin immunoprecipitation assay verified that binding of beta-catenin and TCF-4 to this consensus site was increased by HER-2/neu. In addition, dominant-negative mutant of TCF significantly attenuated the stimulatory effect of HER-2/neu. We also demonstrated that HER-2/neu increased beta-catenin/TCF-mediated Jab1 expression via the AKT signaling pathway because chemical inhibitor or dominant-negative mutant of AKT effectively attenuated the stimulatory action of HER-2/neu. IGF-I, which is a well-known AKT activator, also up-regulated the expression of Jab1 in NIH/3T3 and MCF-7 cells. Knockdown of Jab1 by small interfering RNA (siRNA) preferentially inhibited proliferation of HER-2/neu-overexpressing breast cancer cells. Taken together, our results suggest that HER-2/neu transcriptionally activates Jab1 expression to promote proliferation of breast cancer cells.

Simultaneous separation of anionic and cationic proteins by capillary electrophoresis using high concentration of poly(diallyldimethylammonium chloride) as an additive.

The simultaneous separation of anionic and cationic proteins has been achieved by addition of high concentration of poly(diallyldimethylammonium chloride) (PDDAC) in capillary electrophoresis. A capillary was filled with PDDAC so that it would act as ion-pair reagents in the separation of anionic proteins. On the other hand, the PDDAC can also be used as coating additives for the analysis of cationic proteins. Increasing the concentration of PDDAC in the separation buffer had the ability to improve the separation efficiency, change the electrophoretic mobility, and alter the separation selectivity; however, this was not true in the case of analyzing proteins by using the PDDAC larger than 1.6%. By both using a buffer containing 1.6% PDDAC and applying pH-stepwise techniques, 13 proteins with a wide range of pI (4.7-11.1) and molecular masses (6.5-198.0 kDa) could be separated within 30 min in a single run. In addition to this separation, we observed not only more peaks from alpha-chymotrypsinogen A and aprotinin but also the bovine serum albumin (BSA) dimer and trimer. With the 50 nL protein injection sample, the limits of detections at signal-to-noise of 3 for proteins are in the range of 0.07-0.79 microM. Except for BSA, the relative standard derivation values of migration time and peak height for all proteins were <1.3 and <6.9%, respectively. We suggested that this proposed method is a promising approach for clinical diagnosis and proteomics applications.

Laying traits and underlying transcripts, expressed in the hypothalamus and pituitary gland, that were associated with egg production variability in chickens.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red- (n=12) and Black-feather (n=14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n=17) and L2 (high-egg production; n=14); and a commercial single comb White Leghorn (WL; n=17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P<0.05) and WL strains (P<0.05). However, their rate of yolk accumulation (P<0.05) and follicle rapid growth period (P<0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P<0.05) as well as the rate of yolk accumulation (P<0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P<0.05) or laying rate after first egg (P<0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P<0.05) and/or increasing average clutch length (P<0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P<0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion.

Histone deacetylase (HDAC) inhibitors are known to exert antimetastatic and antiangiogenic activity in vitro and in vivo. RECK is a membrane-anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. In this study, we test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation and cancer cell invasion. Our results showed that trichostatin A (TSA) up-regulated RECK via transcriptional activation in CL-1 human lung cancer cells. Flow cytometric analysis demonstrated that RECK protein on cell surface was increased after treatment of TSA. Moreover, up-regulation of RECK expression by TSA attenuated MMP-2 activity. To explore whether HDAC inhibitor-induced inhibition of MMP-2 activation is indeed mediated via RECK, we used small interference RNA (siRNA) to block RECK expression and found that inhibition of RECK by siRNA abolished the inhibitory effect of TSA on MMP-2 activation. In addition, TSA suppressed the invasive ability of CL-1 cells. Taken together, this study reveals a novel mechanism by which HDAC inhibitors suppress tumor invasion and provides a new strategy for cancer therapy.

Induction of RECK by nonsteroidal anti-inflammatory drugs in lung cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to exert anti-angiogenic and anti-metastatic activity both in vitro and in vivo. Block of angiogenesis and metastasis by NSAIDs has been found to be mediated partly via suppression of matrix metalloproteinase (MMP) activity. However, the molecular mechanism of this inhibitory action has not been well defined. Recent works demonstrated that a membrane-anchored MMP inhibitor RECK may potently suppress MMP-2 and -9 activity to inhibit angiogenesis and metastasis in vitro and in vivo. In this study, we test the possibility that NSAIDs may up-regulate RECK to inhibit MMP activity. RT-PCR analyses showed that NS398 and aspirin up-regulated RECK mRNA level in CL-1 human lung cancer cells. Additionally, NSAIDs increased RECK protein level as detected by immunoblotting. Since RECK is a membrane-anchored glycoprotein, we also performed immunofluorescent staining to assess the expression of RECK on cell surface. Our results showed that fluorescent intensity of RECK was obviously increased after NSAID treatment. Moreover, induction of RECK by NSAIDs was associated with reduction of MMP-2 activity. We also found that NSAID-activated RECK expression might not be mediated via inhibition of cyclo-oxygenases (COXs) because addition of prostaglandin E(2) (PGE(2)) could not counteract the effect of NSAIDs and overexpression of COX-2 could not down-regulate RECK. Taken together, our results suggest that induction of RECK expression may be one of the mechanisms by which NSAIDs suppress MMP activity to block angiogenesis and metastasis.

RECK is a target of Epstein-Barr virus latent membrane protein 1.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) has been suggested to be involved in tumor metastasis. However, the molecular mechanism of LMP1-induced metastasis is largely unknown. In this study, we investigated the effect of LMP1 on the expression of RECK, a metastasis suppressor gene, in an EBV-negative nasopharyngeal carcinoma (NPC) cell line. Our data demonstrated that LMP1 induced downregulation of RECK via transcription repression in TW04 cells. In addition, we found that LMP1 acted via an Sp1 site to inhibit RECK promoter activity. We next studied the signaling pathway that mediated the effect of LMP1 on RECK expression. Our results showed that LMP1 potently stimulated the activity of extracellular signal-regulated kinases (ERKs) and inhibition of ERK activity by PD98059 antagonized LMP1-induced downregulation of RECK. Conversely, the c-Jun N-terminal kinase inhibitor SP600125 and p38(HOG) kinase inhibitor SB203580 had little effect. We also found that the expression of LMP1 increased the invasive ability of TW04 cells. The importance of RECK in LMP1-induced invasiveness was supported by three observations. First, restoration of RECK expression by PD98059 reduced LMP1-induced release of active MMP-9. Second, suppression of PD98059-induced RECK expression by small interference RNA abolished the inhibitory action of PD98059 on LMP1-induced invasiveness. Third, coexpression of RECK with LMP1 in TW04 cells effectively suppressed cell invasiveness induced by LMP1. Taken together, these results suggest that LMP1 inhibits RECK expression via the ERK/Sp1 signaling pathway and this inhibition is a critical step for LMP1-induced tumor metastasis.

Involvement of histone deacetylation in ras-induced down-regulation of the metastasis suppressor RECK.

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Previous study demonstrated that oncogenic ras inhibited RECK expression via an Sp1 binding site in the RECK promoter. In this study, we investigated the molecular mechanism by which ras inhibited RECK expression. Co-transfection assay showed that Sp1 and Sp3 are transactivators, rather than repressors, for RECK gene. So, we tested whether ras activation induced the binding of histone deacetylases (HDACs) to Sp1 to repress RECK expression. Our data showed Sp1-associated HDAC1 in cells was increased after ras induction. By using DNA affinity precipitation assay, we found that induction of oncogenic ras enhanced the binding of HDAC1 to the DNA probe corresponding to the Sp1 site in the RECK promoter. Additionally, a HDAC inhibitor trichostatin A (TSA) potently antagonized the inhibitory action of ras on RECK. The signaling pathway by which ras suppresses RECK was also addressed. Induction of oncogenic ras activated extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38(HOG) kinase in 2-12 cells. Addition of PD98059 or overexpression of dominant-negative mutant of ERK2 indeed reversed ras-mediated inhibition of RECK promoter activity. Taken together, our results suggest that oncogenic ras represses RECK expression via a histone deacetylation mechanism.

Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis.

Cyclooxygenase-2 (COX-2) has been shown to be involved in multiple steps of carcinogenesis. In this study, we examined COX-2 expression in nasopharyngeal carcinoma (NPC). COX-2 mRNA analyzed by reverse-transcription polymerase chain reaction (RT-PCR) was detected in 66% (16 of 24) of tumor tissues. Western blot analysis demonstrated that COX-2 protein level was increased in tumor tissues and was correlated with the expression level of mRNA. Immunohistochemical study showed that COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. Ten histologically normal nasopharyngeal tissues obtained from nasopharyngeal hyperplasia were also investigated. We found that COX-2 mRNA was detectable in three tissues and the COX-2 protein level was very low. The frequency of COX-2 overexpression was significantly higher in patients of the N1-N3 group than in patients of the N0 group (P=0.006). Taken together, these data suggest that COX-2 is overexpressed and is associated with increased lymphatic invasion in NPC.

Enhancement of chemotherapeutic drug-induced apoptosis by a cyclooxygenase-2 inhibitor in hypopharyngeal carcinoma cells.

Our previous study demonstrated that cyclooxygenase-2 (COX-2) was overexpressed in human hypopharyngeal carcinoma. In this study, we tested the effect of a specific COX-2 inhibitor, NS398, on proliferation of hypopharyngeal cancer cells. Our results indicated that NS398 inhibited growth of hypopharyngeal cancer cells and this inhibition is associated with induction of G1 growth arrest. Western blot analysis showed that expression of G1 cyclins or cyclin-dependent kinases (CDKs) was not changed by NS398. On the contrary, NS398 significantly increased expression of CDK inhibitors p21(Waf1) and p27(Kip1). We also found that treatment of NS398 alone could not induce significant apoptosis in hypopharyngeal cancer cells. However, NS398 potently augmented chemotherapeutic drug-induced apoptosis. Caspase activation and DNA fragmentation were clearly detected in cancer cells pretreated with NS398 followed by chemotherapeutic drugs. Collectively, our results suggest that COX-2 inhibitors may suppress proliferation of hypopharyngeal cancer cells via induction of G1 growth arrest and may be useful in combination with chemotherapeutic drugs for the treatment of hypopharyngeal carcinoma.

Involvement of PKA and Sp1 in the induction of p27(Kip1) by tamoxifen.

We have previously shown that tamoxifen (Tam) inhibits proliferation of estrogen receptor-negative human non-small cell lung cancer cells and this inhibition is associated with induction of p27(Kip1). In this study, we investigated the mechanism by which Tam increases p27(Kip1) expression. Because intracellular p27(Kip1) protein level is mainly controlled via posttranslational regulation, we first tested whether Tam might affect protein stability of p27(Kip1). Metabolic labeling and pulse chase assays showed that Tam did not affect the half-life of this protein. We next examined whether Tam enhanced p27(Kip1) expression through transcriptional activation. Our results demonstrated that Tam directly stimulated the p27(Kip1) promoter in lung cancer cells. Deletion and mutation analysis revealed that two Sp1 consensus sites located between -545 and -532bp from the transcription start site were crucial for the induction of p27(Kip1) by Tam. Conversely, mutation in a CTF site (-525/-520) nearby these two Sp1 sites had little effect. Electromobility shift assays showed that Sp1 transcription factor bound to these consensus sites and the DNA binding activity of Sp1 was enhanced by Tam. Our data also demonstrated that induction of p27(Kip1) by Tam was inhibited by protein kinase A inhibitor H89, but not by protein kinase C inhibitor calphostin C and mitogen-activated kinase kinase inhibitor PD98059. Taken together, our results suggest that Tam transcriptionally activates p27(Kip1) expression via the Sp1 consensus sites in the p27(Kip1) promoter and PKA is involved in this process.