Human HEp-2 Research Articles

Identification and characterisation of a novel adhesin Ifp in Yersinia pseudotuberculosis

BackgroundIn order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.ResultsWe termed YPTB1572 Ifp (Intimin family protein) and show that it is able to bind directly to human HEp-2 epithelial cells. Cysteine and tryptophan residues in the C-terminal region of intimin that are essential for function in EPEC and EHEC are conserved in Ifp. Protein binding occurred at distinct foci on the HEp-2 cell surface and can be disrupted by mutation of a single cysteine residue at the C-terminus of the protein. Temporal expression analysis using lux reporter constructs revealed that ifp is expressed at late log phase at 37°C in contrast to invasin, suggesting that Ifp is a late stage adhesin. An ifp defined mutant showed a reduction in adhesion to HEp-2 cells and was attenuated in the Galleria mellonella infection model.ConclusionA new Y. pseudotuberculosis adhesin has been identified and characterised. This Ifp is a new member in the family of invasin/intimin outer membrane adhesins.

In VitroSensitivity of Human Metapneumovirus to Type I Interferons

Human metapneumovirus (hMPV) has been recognized as an important respiratory pathogen. Due to its relatively recent discovery, only limited information is available on the relationship between hMPV and type I interferons (IFN). This study was designed to determine whether in vitro hMPV is sensitive to the antiviral activity of IFN-β, leukocyte IFN-α, and several IFN-α subtypes in a human Hep-2 cell line. The results showed that 50% inhibitory concentration values against hMPV for the various type I IFN preparations were significantly higher than those against the IFN-sensitive vesicular stomatitis virus, and some IFN-α subtypes appeared to be more active against hMPV than others, with IFN-α subtypes 5, 6, 8, and 10 being the most potent, and IFN-α2, 17, and 21 the least potent. The results show that hMPV grown in Hep-2 is partially resistant to the antiviral activity of type I IFNs. Additional studies are required to understand whether and to what extent the relatively low sensitivity of hMPV to IFNs influences the clinical outcomes of infected individuals.

Virulence properties and integron-associated antibiotic resistance of Klebsiella mobilis strains isolated from clinical specimens

This study examined Klebsiella mobilis isolates cultured from clinical specimens for virulence-associated properties and antibiotic resistance. The strains produced a number of siderophores, including enterobactin, aerobactin and yersiniabactin. All isolates were able to adhere to and invade epithelial cells. They had cytotoxic activity, which caused destruction of human laryngeal epithelial HEp-2 cells and evoked lysis of murine macrophage J774 cells. Analyses of HEp-2 and J774 cellular morphology and DNA fragmentation in the cells showed features typical of cells undergoing apoptosis. Some K. mobilis strains harboured class 1 integrons carrying the aadA1 gene encoding an aminoglycoside adenyltransferase.

Study on synthesis of thalidomide analogues and their bioactivities; inhibition on iNOS pathway and cytotoxic effects

Synthetic thalidomide analogues (compounds 1–35), including phenylphthalimide, pyridylphthalimide, aminobenzylphthalimide, and diphenylazophthalimide, were tested for their cytotoxic effects on human cancer cell lines Hep2 (Human Larynx Carcinoma Cells), HL-60 (Human Myeloid Leukemia Cells), NUGC (Human Gastric Carcinoma Cells), and HONE-1 (Human Nasopharyngeal Carcinoma Cells) because the incidence rate is more prominent in Asian countries than in Western countries. Compounds 17, 27, 28, and 35 were found to have antitumor activity in Hep2 and HL-60 cell lines. Compounds 2, 4, 15, 17, 19, 20, 23, and 27 can inhibit nitric oxide (NO) synthase activity by more than 90%. These thalidomide analogues were found to be potent inducible nitric oxide synthase (iNOS) inhibitors, and the iNOS inhibiting potential of compounds 17 and 27 might be an advantage for anticancer therapy. In conclusion, inhibition of NO synthesis is a new development in cancer therapy for now and in the future. We modified the structures of the thalidomide analogues to have a stronger anticancer effect and a good therapeutic effect.

MicroRNA‐16 targets zyxin and promotes cell motility in human laryngeal carcinoma cell line HEp‐2

MicroRNAs are small noncoding RNAs that negatively regulate target mRNAs at the posttranscription level. Here, we show that miR-16 is upregulated in human laryngeal carcinoma tissues. Inhibition of miR-16 in HEp-2 laryngeal cancer cell line could suppress cell migration and enhance cell-cell adhesion. Subsequently, zyxin, whose expression is negatively regulated by miR-16, is confirmed to be a direct target gene of miR-16. Furthermore, overexpression of zyxin could also restrain cell movement and enhance cell-cell adhesion. The study of miR-16 and its target zyxin will shed light on diagnosis and therapy of laryngeal cancer.

Anti-tumor and Anti-inflammatory Properties of Ankaflavin and Monaphilone A from Monascus purpureus NTU 568

An azaphilonidal derivative monaphilone A (MA) was recently isolated from the fermented products of Monascus purpureus NTU 568 by our laboratory. We report here the exploration of apoptosis-related and anti-inflammatory properties of MA and ankaflavin (AK) by some experiments about inducing death of human laryngeal carcinoma cell line HEp-2 and reducing inflammatory responses on murine macrophage RAW 264.7 cells. We employed a ssDNA enzyme-linked immunosorbent assay (ELISA) kit to investigate the nuclear changes of early apoptosis induced by AK and MA on HEp-2 cells and used a western blot and an enzyme activity assay to demonstrate the activation of caspase-3, caspase-8, and caspase-9 by MA and AK. Our studies revealed that AK and MA may decrease lipopolysaccharide (LPS)-induced inflammatory responses, including nitrite productions and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. All evidence support that azaphilonidal derivatives from M. purpureus NTU 568, such as AK and MA, are suitable for the development of chemotherapy or chemopreventive agents.

Promoting effect of Chlamydia pneumoniae infection on human laryngeal carcinoma HEp-2 cell adhesion and migration

To explore the effect of Chlamydia pneumoniae (C.pn) infection on human laryngeal carcinoma cell line HEp-2 cell adhesion and migration, to further clarify the role and mechanism of C.pn infection in tumor metastasis. HEp-2 cells were infected with C.pn after the culture and propagation of C.pn. The cytopathic effect was observed by microscopy. Morphological characteristics of C.pn inclusions in HEp-2 cells were examined by fluorescence microscopy and acridine orange staining. The ultrastructural changes of C.pn inclusions in the HEp-2 cells were examined by transmission electron microscopy (TEM). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of HEp-2 cells to collagen I. Wound-healing assay and transwell assay were performed to explore the effect of C.pn infection on HEp-2 cell migration. At 72 h post-infection, C.pn infected-HEp-2 cells were swollen and partially desquamated. Numerous vacuoles (inclusions) were observed and C.pn inclusions occupied almost the whole cytoplasm of the HEp-2 cells. Grape-like C.pn inclusions were observed in the HEp-2 cells stained with acridine orange under a fluorescence microscope at 72 h after infection. Under TEM, there were more mature pear-shaped elementary bodies, but less larger and round reticulate bodies in the HEp-2 cells infected with C.pn for 72 h. In the cell adhesion assay, the A value in C.pn infection group was 0.669 ± 0.011, significantly higher than that in the control group (0.558 ± 0.005) at 2 h after infection (P < 0.001). The cell adhesion ratio in the C.pn infection group was 119.89%. The migration distance of C.pn infected-HEp-2 cells in the wound-healing assay was significantly longer than that of control cells at 24 h after infection (P < 0.05). HEp-2 cells infected with C.pn for 12 h migrated more than the control cells in the transwell assay (23.40 ± 2.41 vs 10.40 ± 1.67) (P < 0.001). C.pn infection can significantly promote HEp-2 cell adhesion to collagen I and migration of HEp-2 cells, indicating that C.pn infection may play an important role in promoting the metastasis of laryngeal cancer.

Rapid functional screening of effective siRNAs against Plk1 and its growth inhibitory effects in laryngeal carcinoma cells

Plk 1 is overexpressed in many human malignancies including laryngeal carcinoma. However, its therapeutic potential has been never examined in laryngeal carcinoma. In the present study, a simple cellular morphology-based strategy was firstly proposed for rapidly screening the effective siRNAs against Plk1. Furthermore, we investigated the effects of Plk1 depletion via a novel identified effective siRNA against Plk1, Plk1 siRNA-607, on human laryngeal carcinoma Hep-2 cells. The results indicated that Plk1 siRNA-607 transfection resulted in a significant inhibition in Plk1 expression in cells, and subsequently caused a dramatic mitotic cell cycle arrest followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth and viability of the laryngeal carcinoma cells. Taken together, our present study not only suggests a simple strategy for rapidly screening effective siRNAs against Plk1 but also implicates that Plk1 may serve as a potential therapeutic target in human laryngeal carcinoma.

Linkage between cellular adherence and biofilm formation in Escherichia coli O157:H7 EDL933

During the establishment of Escherichia coli O157:H7 infection, its capacity to adhere to host intestinal epithelial cells is the critical first step in pathogenesis. It also has the capability to form biofilms, and because both are surface activities, we sought to gain insight into a potential linkage between biofilm formation and adherence to epithelial cells. We conducted an adherence assay with 51 biofilm-negative mutants and two human epithelial cell lines, T84 and HEp2. Our results show that unlike wild-type cells, biofilm-negative mutants adhere poorly to epithelial cells. Some adhesin-negative mutants were fully competent in biofilm formation, however. Thus, biofilm-forming activity in E. coli O157:H7 EDL933 is required for adherence to T84 and HEp2 cells, but it is not sufficient.

Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin

Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli-carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep-2 cells. Moreover, internalization of non-invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

Somatic Hypermutation In Stereotyped Subset 4 BCRs/mAbs of CLL Patients, Expressing IGHV4-34 gene, Edit Anti-DNA Reactivity

AbstractAbstract 2444Extensive analysis of immunoglobulin gene sequences from chronic lymphocytic leukemia (CLL) patients reveals that ~30% carry stereotyped B-cell antigen receptors (BCRs) with highly homologous HCDR3s, over-represented in selected IGHV/IGHD/IGHJ rearrangements involving such IGHVs as 1–69, 4–34, and 3–21. This finding along with other structurally unique features of CLL BCRS suggests that antigens or superantigens or both may play an active role in the disease.The mutational status of IGHV genes is one of the most powerful prognostic molecular markers in CLL. Patients with IGHV genes with less than 2% differences from the germ line (“unmutated”; U-CLL) have a more aggressive clinical course compared to those with more than 2% differences (“mutated”; M-CLL). In subsets of sequences with stereotyped HCDR3s certain amino acid changes appear to be “CLL-biased”. One of the most distinctive SHM patterns has been described in sequences expressing stereotyped IGHV4-34 BCRs belonging to Subset 4, that are also characterized by long HCDR3s enriched in basic residues (especially arginine).The IGHV4-34 gene encodes antibodies which are autoreactive due to their recognition of the N-acetyllactosamine (NAL) antigenic determinant of the I/i blood group antigen, also expressed on a 220-kDa CD45 B cell-specific isoform; this recognition is mediated in a superantigenic fashion via a motif in HFR1 of the IGHV4-34 sequence. In addition, IGHV4-34 antibodies with HCDR3 sequence motifs enriched in basic (electropositive) amino acids have been shown to react against B cells and DNA, although HCDR3 electropositivity per se does not universally endow IGHV4-34 B cells with this property.Along these lines, we wanted to investigate if the modifications of subset 4 IGHV4-34 by SHM in precursors of the CLL clones could have affected the binding properties if the respective IG receptors, especially against B cells and DNA. We therefore recombinantly expressed 4 mAbs with IGHV4-34 rearrangements, from our CLL patients sample library; mAbs CLL183, CLL240, CLL342 belong to stereotypic subset 4 and have mutated heavy chain immunoglobulin genes, whereas CLL141 expresses an unmutated gene without belonging to any described subset.As previously reported, we tested reactivity of these mAbs with apoptotic and viable B and T cells. All 4 mAbs reacted significantly only with viable cells; the 3 mAbs belonging to subset 4 bound only viable B cells, with a preference for naïve B cells. We also tested the reactivity of these mAbs with single stranded DNA (ssDNA) and double stranded (dsDNA). We used common assays for identifying nuclear autoantigens in autoimmune disorders: 1) for binding total anti-nuclear antigens, human epithelial cell line HEp-2, and 2) for binding dsDNA, Crithidia Luciliae (CL). None of these tests had a strong positive result.To identify a role played by SHM in editing the anti-DNA reactivity, we chose to revert to germline 9 amino acid positions mutated in the IGHV and 1 in the IGKV sequences of CLL240. We either made revertant antibodies retromutated at single positions or at all mutated positions (completely unmutated and germline-like). Enzyme-linked assay (ELISA) for the semi-quantitative detection of ssDNA and dsDNA showed that the complete revertant antibody regained the ability to strongly bind ssDNA and partially dsDNA. The light chain did not appear to play a crucial role in the reacquisition of the binding, because the recombinant mAb expressing only the IGKV in the germline sequence did not bind ssDNA or dsDNA, as did the original recombinant mAb. Some of the revertants also showed a stronger binding to normal human B cells, with a few of them acquiring a partial ability to also bind T cells. The role of the single revertants is being further analyzed.Few IGHV4-34 CLL sequences are altered in the specific HFR1 motif responsible for NAL binding. This suggests that these IGHV4-34 expressing CLL cells could still bind and be stimulated for clonal expansion by NAL epitopes present in self and exogenous antigens. However, the findings of the present study also indicate that the distinctive modifications introduced by SHM in the stereotyped IGHV4-34 BCRs of subset 4 CLL likely represent a censoring mechanism for avoiding intense self-reactivity, mediated by the arginine-rich HCDR3s, and subsequent clonal deletion. Disclosures:No relevant conflicts of interest to declare.

Anti-Adhesive Activities of Flavan-3-ols and Proanthocyanidins in the Interaction of Group A-Streptococci and Human Epithelial Cells

Bacterial adhesion to epithelial cells is a key step in infections, allowing subsequent colonization, invasion and internalization of pathogens into tissues. Anti-adhesive agents are therefore potential prophylactic tools against bacterial infections. The range of anti-adhesive compounds is largely confined to carbohydrate analogues. Tannins are generously recognized as potent antimicrobials, but little data exist on their anti-adherence potency. Using a model for mucosal pathogenesis with labeled group A-streptococci (GAS) and human laryngeal HEp-2 cells, a series of flavan-3-ols (epicatechin, epigallocatechin, epigallocatechin-3-O-gallate) and highly purified and chemically characterized proanthocyanidin samples including procyanidins based on epicatechin, catechin or ‘mixed’ constituent flavanyl units, prodelphinidins made up of (epi)gallocatechin monomeric unts as well as oligomers possessing A-type units in their molecules was evaluated for anti-adhesive effects. Reduced microbial adherence was observed exclusively for prodelphinidins, suggesting that pyrogallol-type elements, i.e., (epi)gallocatechin units are important structural features. This is the first report on structure-activity relationships regarding the anti-adhesive potency of proanthocyanidins. In addition, the structures of the first chemically defined proanthocyanidins from Pelargonium sidoides are disclosed.

Effects of Epigallocatechin gallate glucoside on antitumor activities in human laryngeal epidermoid carcinoma Hep2

(−)-Epigallocatechin-3-gallate (EGCG) has been reported to inhibit cellular proliferation and induce apoptosis in a range of cancer cells. This study examined the cytotoxicity of EGCG glucoside on human laryngeal epidermoid carcinoma Hep2 cells. The EGCG glucoside treatment decreased the cell viability in Hep2 cells. Furthermore, EGCG glucoside caused apoptotic morphological changes with chromatin condensation and nuclear fragmentation, as observed by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and activated caspase-3 immunohistochemisty. However, the EGCG glucoside did not induce the production of reactive oxygen species (ROS), suggesting that oxidative stress is not involved in the apoptotic response. EGCG glucoside induces apoptosis in Hep2 cells through not the generation of ROS, but the activation of caspase-3.

Familial Alzheimer’s disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer's disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial Hep2 cells. It is assumed that mutations in familial Alzheimer's disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.

Evaluation of an aqueous-ethanolic extract from Pelargonium sidoides (EPs® 7630) for its activity against group A-streptococci adhesion to human HEp-2 epithelial cells

The root extract of Pelargonium sidoides DC (Geraniaceae), EPs® 7630, is currently used to treat respiratory tract infections. The therapeutic benefits are largely related to the modulation of the non-specific immune system. The present study was designed to investigate the anti-adhesive activity of this herbal medicine with Streptococcus pyogenes as model microorganism and to identify the underlying biologically active principle. Adherence of fluorescent-labelled group A-streptococci (GAS) to human epithelial (HEp-2) cells was assessed by flow cytometry. Anti-adhesive properties of the parent extract as well as a methanol-soluble (MSF) and a methanol-insoluble fraction (MIF) derived thereof were examined. Treatment with skin powder produced polyphenol-free samples which were included for comparison. Anti-adherence studies were extended to a series of highly purified proanthocyanidins including homogenous epicatechin- and catechin-based polyflavans, a 'mixed' procyanidin sample, an A-type proanthocyanidin mixture as well as a prodelphinidin test substance. After pre-treatment of GAS with EPs® 7630 or its subfractions MIF and MSF at concentrations of 30 μg/ml, adhesion of the pathogen to HEp-2 cells was inhibited by ca. 45%, ca. 35% and ca. 30%, respectively. However, following preincubation of cells with the extract and the fractions no effect was observed. This finding indicates that the anti-adhesive effects are due to interactions with binding factors on the bacterial surface. Since polyphenol-free samples proved to be inactive, proanthocyanidins appear to represent the anti-adhesive principle. Comparative studies with chemically defined proanthocyanidins revealed that the prodelphinidin nature, i.e. the pyrogallol B-ring elements of constituent flavanyl units, represented an important structural feature of the anti-adhesive potential of this herbal medicine. The current data provide strong evidence for a potent anti-adhesion principle of the Pelargonium sidoides root extract related to specific proanthocyanidins. This finding suggests an interaction with bacterial binding sites in a specific rather than non-specific manner. However, the blocked adhesion molecules remain to be identified. The anti-adhesive mechanism may well contribute to the anti-infective activity of EPs® 7630 at an early time point of a bacterial infection.

Vincristine-resistant human laryngeal carcinoma cells demonstrate increased Rous sarcoma virus promoter activity

Aims Gene therapy is a candidate approach for treating cancer patients whose tumors have developed resistance to some drugs. Our study aims to examine possible alteration in Ad5RSVβgal-mediated transgene expression in a vincristine-resistant cells (VK2) derived from the human laryngeal carcinoma cell line HEp2, and the underlying mechanism(s) thereof. Main methods Adenovirus-mediated transgene expression in HEp2 and VK2 cells was measured by β-gal staining. Semiquantitative PCR was used to evaluate attachment of adenovirus to the cell surface and adenovirus internalization into cells. After transfection of cells with plasmid DNA, promoter activity was measured by semiquantitative RT-PCR. Key findings We show here that VK2 cells exhibited increased Ad5RSVβgal-mediated transgene expression, despite moderately decreased Ad5RSVβgal attachment and internalization, as compared with HEp2 cells. The increased transgene expression was also observed with a virus (Ad5FbΔ639RSVβgal) that does not use the coxsackie-adenovirus receptor (CAR), suggesting that increased transgene expression is independent of CAR. Upon transfection of VK2 cells with a plasmid expressing a reporter gene under the control of the RSV promoter or a plasmid containing the complete Ad5RSVβgal genome, RSV promoter activity was 33- and 4.7-fold higher, respectively, than in HEp2 cells. Significance The increased Ad5RSVβgal-mediated transgene expression in the VK2 cells is due to the increased RSV promoter activity in VK2 cells. Our results point out that (i) drug-resistance may be accompanied with an alteration in promoter activity; (ii) the proper choice of promoter could contribute to a decrease in the vector dose required to achieve a therapeutic effect during gene therapy.

Characterization of the anti-adhesive principle of a Pelargonium sidoides root extract (EPs® 7630) in the interaction of group A-streptococci and human laryngeal epithelia cells

Recently, intriguing experimental research has shown an anti-adhesive capability of the aqueous-ethanolic root extract EPs® 7630 that prevents docking of group A-streptococci on human laryngeal epithelial HEp-2 cells [1]. The present work aimed to gain further insight into the underlying biologically active principle and to identify the components that account for the anti-adhesive activity of this herbal medicine. In a validated flow cytometry-based assay [2], fluorescent-labelled group A-streptococci were incubated with human epithelial (HEp-2) cells. Only after pre-treatment of S. pyogenes, EPs®7630, a methanol-soluble (MSF) and a methanol-insoluble fraction (MIF) inhibited the adhesion of the pathogen to the host cells by ca. 45%, ca. 30% and ca. 34%, respectively. This finding indicated that the anti-adhesive effects were due to interactions with binding factors on the bacterial surface. Treatment of the extracts with skin powder produced polyphenol-free samples. That these samples were devoid of any anti-adhesive activities clearly indicated that the present proanthocyanidins played a decisive role as anti-adhesive components. Comparative studies with chemically defined proanthocyanidins including A- and B-type oligomers identified the presence of pyrogallol B-ring elements of constituent flavanyl units (prodelphinidin nature) as important structural feature of the anti-adhesive principle of this herbal medicine. However, the targets on the bacterial cell surface remain unknown.

Extraction optimisation of daphnoretin from root bark of Wikstroemia indica (L.) C.A. and its anti-tumour activity tests

The bark of Wikstroemia indica is rich in daphnoretin, which has strong antiviral and anti-tumour activities. In order to optimise the extraction conditions of daphnoretin from the bark of W. indica, various extraction parameters were chosen to identify their effects on daphnoretin extraction. Response surface methodology (RSM) was applied to obtain the optimal combination of these parameters. Results showed that the optimisation conditions for daphnoretin extraction were: ethanol concentration 67.44%, extraction time 49.44 min, temperature 60.19 °C and liquid–solid ratio 23.49:1. The maximum extraction of daphnoretin obtained experimentally was 2.18‰. The mathematical model developed was found to fit well with the experimental data. Further, four kinds of human cancer cell lines, HeLa, A549, CNE and HEp-2, were tested in vitro to explore the anti-tumour spectrum of daphnoretin. It was found for the first time that daphnoretin showed significant inhibition on the proliferation of CNE and HeLa cells.

Anti-inflammatory and anticancer compounds isolated from Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.

The aim was to search for anti-inflammatory and anticancer compounds from three medicinal plants, viz. Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn. The NO* scavenging potential of selected plant extracts was determined on LPS/IFN-gamma activated murine peritoneal macrophage cultures, and iNOS and COX-2 expression was evaluated by Western blot analysis. Bio-assay guided fractionation yielded four compounds: physcion and emodin from V. madraspatana, 1-hydroxytectoquinone from R. cordifolia, and oleanonic acid from L. camara. The anti-inflammatory activity of these compounds was tested through the carrageenan-induced rat-paw oedema model. They were then tested against a murine tumour (Ehrlich ascites carcinoma), and three human cancer cell lines, namely A375 (malignant skin melanoma), Hep2 (epidermoid laryngeal carcinoma) and U937 (lymphoma). All four compounds dose dependently inhibited NO* through suppression of iNOS protein without affecting macrophage viability. Physcion and emodin caused 65-68% reduction of oedema volume at 40 mg/kg, which validated their in-vivo anti-inflammatory effect. 1-Hydroxytectoquinone and oleanonic acid exhibited promising cytotoxicity against A375 cells. Ethnomedical reports on these traditional medicinal plants have been rationalised through an insight into the anti-inflammatory as well as anticancer potential of four constituents, characterised to be prospective candidates for designing novel therapeutic agents.

CDNA Expression Analysis of a Human Radiosensitive-Radioresistant Cell Line Model Identifies Telomere Function as a Hallmark of Radioresistance

To achieve a more complete understanding of the molecular mechanisms underlying tumor radioresistance, we established a radioresistant cell line from the human larynx squamous cell carcinoma cell line Hep-2 after long-term radiation induction. The biological features of the resulting cell lines were characterized. cDNA microarray technology was used to measure the alterations of gene expression in the radioresistant cells. We found that certain genes associated with DNA repair, cell cycle, apoptosis, etc. were significantly changed. In particular, genes related to telomeres, such as POT1, were significantly altered. Radioresistant cells had higher telomerase activity and longer telomeres than their parental cells. Our research suggests that telomere function is a novel hallmark of cellular radiosensitivity, and the mechanistic link between telomere maintenance and radiosensitivity may involve the genes and pathways we implicated in this study.