Molecular events associated with epithelial to mesenchymal transition of nasopharyngeal carcinoma cells in the absence of Epstein-Barr virus genome

Epithelial-mesenchymal transition (EMT) is an important process in tumor metastasis. The EMT-related events associated with metastasis of NPC in the absence of EBV have not been elucidated. We established an EBV-negative NPC cell line from a bone marrow biopsy of an NPC patient. Using a Matrigel system we isolated an invasive and non-invasive sublines, designated NPC-BM29 and NPC-BM00. NPC-BM29 acquired an invasive-like phenotype characterized by EMT, marked by down-regulation of E-cadherin and β-catenin with concomitant increased expression of Ets1. NPC-BM29 cells expressed ≥ 10-fold higher of MMP-9 than NPC-BM00 cells. NPC-BM29 cells grew better in 2% serum than NPC-BM00 cells, with a population doubling-time of 26.8 h and 30.7 h, respectively. A marked reduction in colony-formation ability of NPC-BM00 cells compared to NPC-BM29 was observed. Wound-healing assay revealed that NPC-BM29 cells displayed higher motility than NPC-BM00 and the motility was further enhanced by cell treatment with TPA, a PKC activator. Cell surface markers and tumor-associated molecules, AE3, MAK6 and sialyl-Tn, were up-regulated in NPC-BM29 cells, whereas the expression of HLA-DR and CD54 was significantly increased in NPC-BM00 cells. NPC-BM29 consistently released higher levels of IL-8 and IL-10 than NPC-BM00, with low levels of IL-1α expression in both cell lines. Higher level of VEGF production was detected in NPC-BM00 than NPC-BM29 cells. These data show that EBV is not required for exhibiting multiple metastatic phenotypes associated with EMT. More studies that target right molecules/signalings associated with the EMT may offer new therapeutic intervention options for NPC invasion and metastasis

Transcription within the replication origin of polyomavirus DNA

Polyomavirus large T antigen binds to four adjacent sites on viral DNA, denoted 1/2, A, B and C. Binding to site 1/2 leads to the initiation of viral DNA replication. RNA polymerases transcribing the late strand of the circular, 5.3 kb viral DNA are blocked just upstream of the replication origin and stalled RNA polymerases accumulate in that region (Skarnes et al., J. Mol. Biol. 203: 153-171, 1988). This accumulation of stalled RNA polymerases depends on binding of large T antigen to site A but not B or C (Bertin et al., J. Virol. 67:5766-5775, 1993). To investigate whether this accumulation depends on sequences other than site A, we cloned an insert containing sites A and 1/2, and the adjacent enhancer region, in the small T antigen intron or between two polyadenylation signals of a polyomavirus mutant.Most of the insertion mutants located in the small T antigen intron were nonviable. However, those located between the two polyadenylation signals were viable. Although stalling did not occur within the inserts, a major transcript within the replication origin was found while examining runon RNAs with viral transcription complexes isolated from cells infected with insertion mutants. Labelled RNA was hybridized with a single-stranded DNA, complementary to late-strand RNA, that extends through the replication origin; a major 80-nt RNA species was protected against nuclease treatment. Hybridization with two other DNAs that extend 18 and 41 nt farther in the direction of late transcription protected 98- and 121-nt RNAs, respectively. These results place the 5$sp prime$ end of this transcript near the early side of site 1/2. Control experiments were carried out in which labelled RNAs synthesized in vitro were hybridized with the same DNAs; no RNAs with these 5$sp prime$ ends were detected.S1 mapping of nuclear RNA from virus-infected cells detected no such transcript. The RNA polymerases that synthesize this RNA could terminate in vivo shortly downstream of the initiation site, or the RNA could be unstable. This "ori-RNA" is sensitive to $alpha$-amanitin, resistant to aphidicolin, and independent of functional large T antigen. Ori-RNA synthesis could help stabilize the unwinding of DNA at the replication origin and enhance initiation of replication

Ring-Oxidative Biotransformation and Drug Interactions of Propofol in the Livers of Rats

Propofol, an intravenous anesthetic agent, is widely used for inducing and maintaining anesthesia during surgical procedures and for sedating intensive care unit patients. In the clinic, rapid elimination is one of the major advantages of propofol. Meanwhile, the biotransformation and drug interactions of propofol in rat livers are still little known. In this study, we evaluated the ring-oxidative metabolism of propofol in phenobarbital-treated rat livers and possible drug interactions. Administration of phenobarbital to male Wistar rats significantly increased levels of hepatic cytochrome P450 (CYP) 2B1/2 and microsomal pentoxyresorufin O-dealkylase (PROD) activity. Analyses by high-performance liquid chromatography and liquid chromatography mass spectroscopy revealed that propofol was metabolized by phenobarbital-treated rat liver microsomes into 4-hydroxypropofol. In comparison, PROD activity and 4-hydroxy-propofol production from propofol metabolism were suppressed by orphenodrine, an inhibitor of CYP2B1/2, and a polyclonal antibody against rat CYP2B1/2 protein. Furthermore, exposure of rats to propofol did not affect the basal or phenobarbital-enhanced levels of hepatic CYP2B1/2 protein. Meanwhile, propofol decreased the dealkylation of pentoxyresorufin by phenobarbital-treated rat liver microsomes in a concentration-dependent manner. Taken together, this study shows that rat hepatic CYP2B1/2 plays a critical role in the ring-oxidative metabolism of propofol into 4-hydroxypropofol, and this anesthetic agent can inhibit CYP2B1/2 activity without affecting protein synthesis

Cinnamomum verum component 2-methoxycinnamaldehyde: a novel antiproliferative drug inducing cell death through targeting both topoisomerase I and II in human colorectal adenocarcinoma COLO 205 cells

Background: Cinnamomum verum is used to manufacture the spice cinnamon. In addition, the plant has been used as a Chinese herbal medication. Methods: We investigated the antiproliferative effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the cortex of the plant, and the molecular biomarkers associated with tumorigenesis in human colorectal adenocarcinoma COLO 205 cells. Specifically, cell viability was evaluated by colorimetric assay; apoptosis was determined by flow cytometry and morphological analysis with bright field, acridine orange, and neutral red stainings, as well as comet assay; topoisomerase I activity was determined by assay based upon DNA relaxation and topoisomerase II by DNA relaxation plus decatentation of kinetoplast DNA; lysosomal vacuolation and volume of acidic compartments (VACs) were determined by neutral red staining. Results: The results demonstrate that 2-MCA inhibited proliferation and induced apoptosis as implicated by mitochondrial membrane potential (ΔΨm) loss, activation of both caspase-3 and -9, increase of annexin V+PI+ cells, as well as morphological characteristics of apoptosis. Furthermore, 2-MCA also induced lysosomal vacuolation with elevated VAC, cytotoxicity, and inhibitions of topoisomerase I as well as II activities. Additional study demonstrated the antiproliferative effect of 2-MCA found in a nude mice model. Conclusions: Our data implicate that the antiproliferative activity of 2-MCA in vitro involved downregulation of cell growth markers, both topoisomerase I and II, and upregulation of pro-apoptotic molecules, associated with increased lysosomal vacuolation. In vivo 2-MCA reduced the tumor burden that could have significant clinical impact. Indeed, similar effects were found in other tested cell lines, including human hepatocellular carcinoma SK-Hep-1 and Hep 3B, lung adenocarcinoma A549 and squamous cell carcinoma NCI-H520, and T-lymphoblastic MOLT-3 (results not shown). Our data implicate that 2-MCA could be a potential agent for anticancer therapy

Indomethacin Inhibits Cancer Cell Migration via Attenuation of Cellular Calcium Mobilization

Non-steroidal anti-inflammatory drugs (NSAIDs) were shown to reduce the risk of colorectal cancer recurrence and are widely used to modulate inflammatory responses. Indomethacin is an NSAID. Herein, we reported that indomethacin can suppress cancer cell migration through its influence on the focal complexes formation. Furthermore, endothelial growth factor (EGF)-mediated Ca2+ influx was attenuated by indomethacin in a dose dependent manner. Our results identified a new mechanism of action for indomethacin: inhibition of calcium influx that is a key determinant of cancer cell migration

The Primary Resistance of Helicobacter pylori in Taiwan after the National Policy to Restrict Antibiotic Consumption and Its Relation to Virulence Factors-A Nationwide Study.

The Taiwan Government issued a policy to restrict antimicrobial usage since 2001. We aimed to assess the changes in the antibiotic consumption and the primary resistance of H. pylori after this policy and the impact of virulence factors on resistance.The defined daily dose (DDD) of antibiotics was analyzed using the Taiwan National Health Insurance (NHI) research database. H. pylori strains isolated from treatment naïve (N=1395) and failure from prior eradication therapies (N=360) from 9 hospitals between 2000 and 2012 were used for analysis. The minimum inhibitory concentration was determined by agar dilution test. Genotyping for CagA and VacA was determined by PCR method.The DDD per 1000 persons per day of macrolides reduced from 1.12 in 1997 to 0.19 in 2008, whereas that of fluoroquinolones increased from 0.12 in 1997 to 0.35 in 2008. The primary resistance of amoxicillin, clarithromycin, metronidazole, and tetracycline remained as low as 2.2%, 7.9%, 23.7%, and 1.9% respectively. However, the primary levofloxacin resistance rose from 4.9% in 2000-2007 to 8.3% in 2008-2010 and 13.4% in 2011-2012 (p=0.001). The primary resistance of metronidazole was higher in females than males (33.1% vs. 18.8%, p<0.001), which was probably attributed to the higher consumption of nitroimidazole. Neither CagA nor VacA was associated with antibiotic resistance.The low primary clarithromycin and metronidazole resistance of H. pylori in Taiwan might be attributed to the reduced consumption of macrolides and nitroimidazole after the national policy to restrict antimicrobial usage. Yet, further strategies are needed to restrict the consumption of fluoroquinolones in the face of rising levofloxacin resistance

Development of a Gd(III)-Based Receptor-Induced Magnetization Enhancement (RIME) Contrast Agent for β‑Glucuronidase Activity Profiling

β-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ([Gd­(DOTA-FPβGu)]) for molecular imaging of β-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a β-glucuronidase substrate (β-d-glucopyranuronic acid). The binding association constant (<i>K</i>_A) of [Gd­(DOTA-FPβGu)] is 7.42 × 10², which is significantly lower than that of a commercially available MS-325 (<i>K</i>_A = 3.0 × 10⁴) RIME contrast agent. The low <i>K</i>_A value of [Gd­(DOTA-FPβGu)] is due to the pendant β-d-glucopyranuronic acid moiety. Therefore, [Gd­(DOTA-FPβGu)] can be used for detection of β-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd­(DOTA-FPβGu)] was elucidated by LC-MS. The kinetics of β-glucuronidase catalyzed hydrolysis of [Eu­(DOTA-FPβGu)] at pH 7.4 best fit the Miechalis–Menten kinetic mode with <i>K</i>_m = 1.38 mM, <i>k</i>_cat = 3.76 × 10³, and <i>k</i>_cat/<i>K</i>_m = 2.72 × 10³ M^–1 s^–1. The low <i>K</i>_m value indicates high affinity of β-glucuronidase for [Gd­(DOTA-FPβGu)] at physiological pH. Relaxometric studies revealed that <i>T</i>₁ relaxivity of [Gd­(DOTA-FPβGu)] changes in response to the concentration of β-glucuronidase. Consistent with the relaxometric studies, [Gd­(DOTA-FPβGu)] showed significant change in MR image signal in the presence of β-glucuronidase and HSA. <i>In vitro</i> and <i>in vivo</i> MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase