Human Epidermoid Carcinoma Cell Line Research Articles

Cellular internalisation, bioimaging and dark and photodynamic cytotoxicity of silica nanoparticles doped by {Mo6I8}4+ metal clusters.

Silica nanoparticles (SNPs) doped by hexanuclear molybdenum cluster complexes [{Mo6X8}L6]n (X = Cl, Br, or I; L = various inorganic or organic ligands) have been recently suggested as materials with high potential for biomedical applications due to both their outstanding photoluminescence properties and their ability to efficiently generate singlet oxygen upon photoirradiation. However, no studies were undertaken so far to prove this concept. Therefore, here we examined the potential of photoluminescent SNPs doped by {Mo6I8}4+ for applications such as bioimaging and photodynamic therapy using the human epidermoid larynx carcinoma (Hep-2) cell line as a model. Our results demonstrated both: (i) significant luminescence from cells with internalised molybdenum cluster-doped SNPs combined with the low cytotoxicity of particles in the darkness and (ii) significant cytotoxicity of the particles upon photoirradiation. Thus, this research provides strong experimental evidence for high potential of molybdenum-cluster-doped materials in biomedical applications such as optical bioimaging, biolabeling and photodynamic therapy.

Isolation, cytotoxic evaluation, and simultaneous quantification of eight bioactive secondary metabolites fromCicer microphyllumby high-performance thin-layer chromatography

Chemical investigation of Cicer microphyllum resulted in the isolation and characterization of eight natural products viz. Stigmasterol, Oleanolic acid-3-acetate, Oleanolic acid, Biochanin A, Genistein, Pratensein, Chrysoeriol, and Luteolin. Herein, we report a novel, accurate, and cost-effective high-performance thin-layer chromatography method for the simultaneous quantification of the isolated natural products on silica-gel 60F254 plates using the solvent system n-hexane/ethyl acetate/formic acid (9.0:6.5:0.8, v/v/v). Natural products were quantified after postchromatographic derivatization with ceric ammonium sulfate. The method was validated as per the International Conference on Harmonization guidelines. All calibration curves showed a good linear relationship (r > 0.9943) within the test range. Precision was assessed by intra- and interday tests with relative standard deviations <1.82%, accuracy validation recovery 98.38-99.57% with relative standard deviations <1.00%. On quantification, Pratensein was a major constituent (0.921%). The screening for cytotoxic activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay resulted into identification of Luteolin as potent molecule with IC50 3.5 and 25.6 μg/mL against murine melanoma and human epidermoid carcinoma cell lines, respectively.

Abstract LB-098: Antitumor studies: Design, synthesis, antitumor activity and molecular docking study of novel 2-deoxo-2-substituted-5-deazaalloxazines

Abstract Cancer is the major threat to the public health worldwide, thereby we have a considerable interest to get potential antitumor agents via computational design, synthesis, functional elucidation, and biological evaluation of different deazaalloxazine analogs. Many of these compounds revealed higher selectivities against different tumor cell lines. In the study the structure activity relationships (SAR) of the proposed derivatives was investigated, by applying structure based drug design (SBDD) using most advanced molecular modeling tool programs, namely: AutoDock 4.2 and Accelrys Discovery studio 2.0. These computational approaches aim to increase the speed and efficiency in the drug discovery process. The reasonable drug candidates were subjected to the chemical synthesis and biological in vitro test against different tumor cell lines. The docking study of the synthesized and the rationally designed derivatives was carried out using PTKs as target enzymes which was early reported as a proposed pathway for inhibition of cancer. The main outcome of this study is the synthesis of novel 2-methylthio, 2-(substituted alkyl amino), 2-(heterocyclic substituted), 2-amino, 2,4-dioxo and 2-deoxo-5-deazaalloxazine derivatives. Their antitumor activities against human T-cell acute lymphoblastoid leukemia cell line (CCRF-HSB-2), human oral epidermoid carcinoma cell line (KB), human breast cancer cell line (MCF-7) and human cervical cancer cell line (Hela) have been investigated in vitro. Many compounds showed promising antitumor activities. Furthermore, AutoDock study has been done by binding of the 5-deazaalloxazine analogs into c-kit PTK (PDB code: 1t46), where a good correlation between their IC50 and AutoDock binding free energy was exhibited. Citation Format: Sawsan A. Mahmoud, Mosaad S. Mohamed, Nageh A. Abou Taleb, Tomohisa Nagamatsu, Hamed I. Ali. Antitumor studies: Design, synthesis, antitumor activity and molecular docking study of novel 2-deoxo-2-substituted-5-deazaalloxazines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2015-LB-098

Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail-induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the down-regulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954cm−1 which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. From the Clinical EditorThis method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering.

Detection and imaging of aggressive cancer cells using an epidermal growth factor receptor (EGFR)-targeted filamentous plant virus-based nanoparticle.

Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of diseases and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Virus-based nanoparticles are a promising platform because their scaffold can be manipulated both genetically and chemically to simultaneously display targeting ligands while carrying payloads for diagnosis or therapeutic intervention. Here, we displayed a 12-amino-acid peptide ligand, GE11 (YHWYGYTPQNVI), on nanoscale filaments formed by the plant virus potato virus X (PVX). Bioconjugation was used to produce fluorescently labeled PVX-GE11 filaments targeted toward the epidermal growth factor receptor (EGFR). Cell detection and imaging was demonstrated using human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231), all of which upregulate EGFR to various degrees. Nonspecific uptake in ductal breast carcinoma (BT-474) cells was not observed. Furthermore, co-culture experiments with EGFR(+) cancer cells and macrophages indicate successful targeting and partitioning toward the cancer cells. This study lays a foundation for the development of EGFR-targeted filaments delivering contrast agents for imaging and diagnosis, and/or toxic payloads for targeted drug delivery.

Aggregation of nanoparticles in endosomes and lysosomes produces surface-enhanced Raman spectroscopy

The purpose of this study was to explore the use of surface-enhanced Raman spectroscopy (SERS) to image the distribution of epidermal growth factor receptor (EGFR) in cells. To accomplish this task, 30-nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per mL) were incubated with cells (106 per mL) of the A431 human epidermoid carcinoma and normal human bronchial epithelial cell lines. Using the 632.8-nm excitation line of a He-Ne laser, Raman spectroscopy measurements were performed using a point mapping scheme. Normal cells show little to no enhancement. SERS signals were observed inside the cytoplasm of A431 cells with an overall enhancement of 4 to 7 orders of magnitude. Raman intensity maps of the 1450 and 1583 cm−1 peaks correlate well with the expected distribution of EGFR and AuNPs, aggregated following uptake by endosomes and lysosomes. Spectral features from tyrosine and tryptophan residues dominate the SERS signals.

Synthesis of chromonylthiazolidines and their cytotoxicity to human cancer cell lines.

Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compounds 3a and 3b showed the most selective cytotoxic effects against human epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL) and breast cancer (IC50 32.8 ± 1.4 μg/mL) cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost, and selective anticancer agents.

Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line.

Background:Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2).Materials and Method:The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability.Results:The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line.Conclusion:Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line).

Synthesis of 6-Azapurines by Transformation of Toxoflavins and Reumycins (7-Azapteridines) and their Cytotoxicities

This paper describes a reliable and facile synthesis of 6-azapurines, 1,5-dimethyl-1H-imidazo[4,5-e][1,2,4]triazin-6(5H)-ones and 5-methyl-5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-ones, by treatment of toxoflavins and reumycins with 10 % aqueous or ethanolic sodium hydroxide at 5–70°C or reflux, followed by decarboxylation and oxidation by air along with a benzilic acid type rearrangement. Furthermore, heating the produced 6-azapurines in 10 % ethanolic sodium hydroxide afforded the corresponding 1-methyl-5,6-dioxo-1,4,5,6-tetrahydro-1,2,4-triazines with 1-methylurea. The antitumour activities of the 6-azapurines against CCRF-HSB-2 (human T-cell acute lymphoblastoid leukemia) and KB (human oral epidermoid carcinoma) cell lines were also investigated in vitro and some of the compounds showed prospective antitumour activities.

Green synthesis of silver nanoparticles using the mushroom fungus Schizophyllum commune and its biomedical applications

Silver nanoparticles, which are small metallic colloidal particles, ranging 1 ∼ 100 nm in size, have several nano biotechnological applications in medicine, manufacturing and engineering industries. Fungus-mediated synthesis of silver nanoparticles is an ecofriendly, green process. Further, extracellular enzymes and proteins elaborated by fungi are involved in the synthesis of the silver nanoparticle, which makes the downstream processing relatively simpler. In the present investigation, Schizophyllum commune, a mushroom fungus, was tested for its ability to synthesize extracellular as well as intracellular silver nanoparticles. When the fungus was challenged with 1 mM silver nitrate, a change in colour of the broth and the mycelium was observed, indicative of extracellular and intracellular synthesis of silver nanoparticles. The presence of silver nanoparticles was confirmed by studying its Surface Plasmon Resonance absorption band in the visible wavelength. FTIR spectrum analysis of the silver nanoparticles indicated the presence of biomolecules in association with the reduction of silver ions. Scanning Electron Microscopic analysis of the silver nanoparticles revealed the nanorange dimensions of both the extracellular and the intracellular silver nanoparticles. Analysis of biological activities of the silver nanoparticles disclosed their significant antibacterial activity against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas fluorescens. Additionally, the silver nanoparticles inhibited the growth of the dermatophytic fungal pathogens viz. Trichophyton simii, Trichophyton mentagrophytes and Trichophyton rubrum significantly. Anticancer activity of silver nanoparticles, assayed through MTT cytotoxicity assay, uncovered that 27.2 and 64% mortality could be obtained in Human Epidermoid Larynx Carcinoma (HEP -2) cell lines at concentrations between 10 and 100 µg/mL, respectively. The results obtained indicate that Schizophyllum commune is capable of synthesizing silver nanoparticles in shaken broth cultures (120 rpm) at 25 ± 2℃ and pH 7.

Abstract 3139: Nurr1, a novel target of 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane for inhibition of the initiation and progression of skin cancer tumorigenesis

Abstract The objective of this study was to demonstrate the chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) in skin cancer using an in vitro and in vivo models. In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line (A431) and normal human epidermal keratinocytes (NHEK) respectively. Apoptosis induction and accumulation of ROS following UVB exposure in DIM-D pretreated NHEK cells (2 hr prior) was also evaluated. Immunocytochemistry and western blot analysis were performed to determine proapoptotic and proinflammatory markers expressions in DIM-D treated A431 cells and in UVB irradiated NHEK cells. For in vivo experiments, DIM-D was encapsulated within nanocarriers by hot melt homogenization using Nano DeBEE. Nanocarriers were surface modified with oleic acid and incorporated into a gel using established procedures (G-NOD) and characterized for particle size, zeta potential, entrapment efficiency, rheology, skin permeation and drug release. The chemo-preventive efficiency of G-NOD was evaluated using UVB-induced skin cancer model in SKH hairless mice and skin samples were collected and investigated for lipid peroxidation, protein carbonyls, and molecular markers by western blot. The IC50 values of DIM-D were 24.5±2.6, 17.2±3.6and 4.1±1.1 whilst for EGCG were 192.1±3.8, 85.3±2.4 and 26.0 ±1.4 for 24, 48 and 72 hr treatments respectively. DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). Nanocarriers containing DIM-D were 188.00±8.00 nm in size with polydispersity of 0.59±0.01. The zeta potential and entrapment efficiency were 37.17±0.90mV and 89±0.50%, respectively. There was significant (p&amp;lt;0.05) reduction in tumor size/number for G-NOD pretreated group (2 tumors/mouse, average diameter, 2±0.50 mm) compared to DIM-PG (4 tumors/mouse, average diameter, 4±0.45mm) and EGCG PEG-gel (EGCG-PG; 6 tumors/mouse, average diameter, 5±0.25mm). IHC and western blot results indicated significant increase (p&amp;lt;0.05) in expression of Nurr1 in G-NOD pretreated group compared to EGCG-PG. There was however pronounced reduction in expression of STAT3, NFkB, cleaved Caspase-3, BCl2 and 8-OHdG for G-NOD pretreated group compared to DIM-PG and EGCG-PG. Lipid peroxidation assay revealed decrease in amount of Malondialehyde (MDA) produced in G-NOD pretreated group prior to UV exposure by 2 fold compared to DIM-PG and 1.3 fold compared to EGCG-PG treatments. Therefore, findings in both in vitro and in vivo studies suggest that the enhanced percutaneous delivery of DIM-D reduces UVB-induced damage to skin lipids and protein and inhibits initiation and progression of skin photocarcinogenesis in SKH mice via the transactivation of Nurr1. Citation Format: Ravi Doddapaneni, Cedar Boakye, Punit Shah, Apurva R. Patel, Chandraiah Godugu, Stephen Safe, Santosh Katiyar, Mandip Sachdeva. Nurr1, a novel target of 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane for inhibition of the initiation and progression of skin cancer tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2014-3139

A new bisanthraquinone and cytotoxic xanthones from Cratoxylum cochinchinense

A new bisanthraquinone has been isolated from the stems of Cratoxylum cochinchinense together with vismiaquinone C and 16 known xanthones. Their structures were characterised by using spectroscopic methods. Two of the isolated compounds are modified xanthones (6 and 15) and exhibited strong cytotoxicity against human epidermoid carcinoma (A341: IC50 2.01, 1.78 μg/mL) and human breast cancer cell lines (SKBR3: IC50 1.54, 0.69 μg/mL).

Detection of Enteroviruses in Water Samples from Yopougon, Côte d’Ivoire by Cell Culture and Polymerase Chain Reaction

The objective of this study was to compare sensitivities of enterovirus isolation from wastewater in different cell lines as well as to compare the sensitivity and specificity of isolation in cell culture with direct detection by reverse transcription polymerase chain reaction (RT-PCR). Sixty-eight samples of wastewaters were collected between September 2008 and January 2009 in Yopougon, Abidjan. Enteroviruses were concentrated according to World Health Organization recommendations. Viruses were inoculated into various cell lines while direct RT-PCR was performed on water concentrates. The buffalo green monkey kidney cell line was the most sensitive with 58.8% of viral isolation. This was followed by the rhabdomyosarcoma cell line with sensitivity of 51.6%, with human epidermoid carcinoma cell line showing sensitivity of 50% and fibroblastic cells derived from transgenic mice LTK-1 (L20B) cell showing 23.50% sensitivity. However, a lower specificity of 2.9% was observed with the L20B cell line. 44.1% of the samples were positive by direct RT-PCR detection while 51.47% samples were positive by using RT-PCR on infected cell cultures. No difference in percentage positivity was observed using RT-PCR on infected tissue culture isolates or using RT-PCR directly on wastewater samples.

Synthesis and cytotoxicity of (3β)-3-acetyloxy-5(6)-androsten-7-one oxime and 3,5(6)-androstadien-7-one oxime

(3β)-3-Acetyloxy-5(6)-androsten-7-one oxime and 3,5(6)-androstadien-7-one oxime were synthesized and their in vitro cytotoxic activities against human epidermoid carcinoma cell line KB, human cervical carcinoma cell line HeLa, human gastric carcinoma cell line MKN-28, and human breast carcinoma cell line MCF-7 were evaluated. The compound (3β)-3-acetyloxy-5(6)-androsten-7-one oxime exhibited significant growth inhibitory properties against cell lines tested and afforded IC50 value 26.0 ± 1.6, 12.8 ± 1.0, 18.1 ± 0.2, 10.2 ± 1.2 μM for KB, HeLa, MKN-28, and MCF-7, respectively.

Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor, NR4A2 (Nurr1)

BackgroundThe objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.MethodsIn vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.ResultsThe IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.ConclusionOur results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.

Abstract 5170: Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 .

Abstract The p38 enzyme, also known as mitogen activated protein kinase or MAPK (pT180/pY182), is responsive to cellular stress such as osmotic shock, heat shock, and irradiation, and when activated, modulates the cell cycle, activation of DNA repair, regulation of protein translation and initiation of immune response. The p38 pathway consists of a cascade of protein kinases which ultimately leads to its phosphorylation on specific tyrosine and threonine residues. Growth factors play an important role in the activation of p38 MAPK. The binding of EGF or other specific growth factors to external receptors activate its kinase activity. The activation of p38 MAP kinase can directly influence gene transcription. A growing number of transcription factors are known to be direct targets of p38, thus its implication in many forms of cancer. In the A431 human epidermoid carcinoma cell line, proliferation and migration responses to extracellular stimuli require activation of distinct pathways. In this study we describe the use of the BioScale ViBE workstation and AMMP (acoustic membrane microparticle) assay technology to study the expression of p-p38 in lysates of A431 epidermoid carcinoma cells with or without stimulation of surface receptors for epidermal growth factor, compared to xenograft tumors of the same cell line. The AMMP p-p38 assay is robust and reproducible, with CVs less than 6% for within day and between day measurements. The assay demonstrates little interference from cell or tumor lysate matrices. Results from the p-p38 AMMP assays demonstrate that, in a group of ten A431 tumors, all were positive for the activated analyte. There was heterogeneity in the expression of the p-p38 in the tumors but all gave a positive assay response. Recombinant p-p38 was used as a positive control in each experiment. The results of the phospho-p38 AMMP assay were compared to western blots for the detection of p- p38 in EGF stimulated A431 cell lysates and untreated tumor lysates. The western blots were confirmation of presence or absence of the AMMP measured analyte in the samples. p-p38 was detected by western blot with similar intensity for equal protein loading of each sample type. The elucidation of p-p38 in A431 tumors in untreated nu/nu mice was unexpected as the EGF receptor was not activated based on AMMP results for P-EGFR derived from the same tumor sample set. This would indicate that the P38 was activated through a pathway other than the EGFR pathway. The observation of similar signals between the p-p38 levels detected between EGF stimulated A431 cells grown in vitro and in vivo tumors was also unexpected, again pointing to different pathways of activation. These results demonstrate that the BioScale AMMP assay for detection of p-p38 can be a powerful tool for the study of cancer, apoptosis or cell signalling, giving sensitive and reproducible results without interference from matrix components. Citation Format: Lee Anne Beausang, Kristen Leong, W. Matthew Dickerson, Ashley Saab, Edward M. Alderman. Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2013-5170

Abstract 1295: Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ).

Abstract The peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) regulates a variety of biological processes but the function of PPARβ/δ in carcinogenesis remains controversial. Stable human cancer cell lines (A431, MDA-MB-231 and MCF7) that constitutively over-express PPARβ/δ were produced to effectively examine the role of this receptor in human cancer models. Ligand activation of PPARβ/δ both in MDA-MB-231 (ER-negative) and MCF7 (ER-positive) cells over-expressing PPARβ/δ cells caused significantly decreased clonogenicity compared to controls. Over-expression of PPARβ/δ reduced tumor volumes and tumor weights of ectopic xenografts from both MDA-MB-231 and MCF7cells compared to controls. Ligand activation of PPARβ/δ caused an even greater reduction of tumor volumes and tumor weights compared to controls. Interestingly, similar results were observed in the human epidermoid carcinoma cell line A431 and other human cancer cell lines. Ligand activation of PPARβ/δ strongly inhibited clonogenicity in A431 cells over-expressing PPARβ/δ. Xenografts from A431 control cells rapidly developed tumors while tumors were barely detectable in A431 cells over-expressing PPARβ/δ. These observations demonstrate the inhibitory effect of activating or over-expressing PPARβ/δ on tumorigenesis in human cancer cell lines. This suggests that PPARβ/δ suppression of tumorigenicity may be mediated through common mechanisms. Citation Format: Pei-Li Yao, Michael G. Borland, Prasad Krishnan, Bokai Zhu, Frank J. Gonzalez, Jeffrey M. Peters. Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1295. doi:10.1158/1538-7445.AM2013-1295

Anti-Viral Activity of Water Extract of Paeonia lactiflora Pallas Against Human Respiratory Syncytial Virus in Human Respiratory Tract Cell Lines

Paeonia lactiflora Pallas (P. lactiflora, Ranunculaceae) is a common ingredient of Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) and Ge-Gen-Tang (GGT; kakkon-to). SMGGT and GGT are different prescriptions of traditional Chinese medicine with different ingredients designed for airway symptoms. Both SMGGT and GGT have anti-viral activity against human respiratory syncytial virus (HRSV). Therefore, P. lactiflora was hypothesized to be the effective ingredient of both SMGGT and GGT against HRSV. However, P. lactiflora does not have any proven antiviral activity. This study used both human upper (Human larynx epidermoid carcinoma cell line, HEp-2) and lower (human lung carcinoma cell line, A549) respiratory tract cells to test the hypothesis that a hot water extract of P. lactiflora could effectively inhibit plaque formation induced by HRSV infection. The ability of P. lactiflora to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that P. lactiflora was time-dependently and dose-dependently effective against HRSV in HEp-2 and A549 cells, particularly supplemented before viral inoculation (p < 0.0001). 10 μg/ml P. lactiflora had a comparable anti-HRSV activity with 10 μg/ml ribavirin, a broad-spectrum antiviral agent. P. lactiflora was dose-dependently effective against viral attachment (p < 0.0001), with a better effect on A549 cells (p < 0.0001). P. lactiflora was time-dependently (p < 0.0001) and dose-dependently (p < 0.0001) effective against viral penetration. Moreover, P. lactiflora stimulated IFN-β secretion without any effect on TNF-α secretion. Therefore, P. lactiflora could be beneficial at preventing HRSV infection by inhibiting viral attachment, internalization, and stimulating IFN secretion.