Epidermal Carcinoma Cells Research Articles

Detection of refractive index changes in individual living cells by means of surface plasmon resonance imaging

Real time imaging of living cell activation is an increasing demand in disciplines of life science and medicine. We previously reported that surface plasmon resonance (SPR) sensors detect large changes of refractive index with living cells, such as mast cells, keratinocyte, human basophils and B-cells activated by biological stimuli. However, conventional SPR sensors detect only an average change of refractive index with thousands of cells at detectable area on a sensor chip. In this study, we developed an SPR imaging (SPRI) sensor with a CMOS camera and an objective lens in order to analyze refractive index of individual living cells and their changes upon stimuli. The SPRI sensor could detect reactions of individual rat basophilic leukemia (RBL-2H3) cells, mouse keratinocyte (PAM212) cells, and human epidermal carcinoma (A431) cells in response to either specific or non-specific stimuli, such as antigen, phorbol ester or epidermal growth factor, with or without their inhibitors, resembling signals obtained by a conventional SPR sensor. Moreover, we distinguished reactions of different type cells, co-cultured on a sensor chip, and revealed that the increase of refractive index around nuclei is rapid and potent as compared to that in peripheries in the reaction of RBL-2H3 cells against antigen. This system may be a useful tool to investigate the mechanism of refractive index-changes evoked in near-membrane fields of living cells, and to develop a system of high-throughput screening for clinical diagnosis.

Paclitaxel-Loaded, Folic-Acid-Targeted and TAT-Peptide-Conjugated Polymeric Liposomes: In Vitro and In Vivo Evaluation

Folic acid and TAT peptide were conjugated on the octadecyl-quaternized, lysine-modified chitosan-cholesterol polymeric liposomes (FA-TATp-PLs) to investigate their potential feasibility for tumor-targeted drug delivery. FA-TATp-PLs encapsulating paclitaxel or calcein were synthesized and characterized. Cellular uptake of PLs, FA-PLs, TATp-PLs and FA-TATp-PLs was studied by confocal laser scanning microscopy (CLSM) in folate receptor (FR)-positive KB nasopharyngeal epidermal carcinoma cells and FR-deficient A549 lung cancer cells. In vitro and in vivo antitumor activity of paclitaxel-loaded FA-TATp-PLs were also evaluated in KB and A549 cells as well as in a murine KB xenograft model. Our data showed that 80% paclitaxel released from FA-TATp-PLs in 2 weeks. Different from other various PLs, CLSM analyses showed that FA-TATp-PLs had a significantly high efficient intracellular uptake in both KB and A549 cells. These data revealed the targeting effects of folate decoration, the transmembrane ability of TAT peptide as well as a synergistic interaction between them. In addition, paclitaxel-loaded FA-TATp-PLs exhibited a more superior antitumor effect in vitro and in vivo as compared to that with Taxol. FA-TATp-PLs possessing both targeting effect and transmembrane ability may serve as a promising carrier for the intracellular delivery of therapeutic agents.

Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Fatty Acid-binding Protein 5 and PPARβ/δ Are Critical Mediators of Epidermal Growth Factor Receptor-induced Carcinoma Cell Growth

Epidermal growth factors and their receptors (EGFRs) promote breast cancer cell proliferation and can drive tumorigenesis. However, the molecular mechanisms that mediate these effects are incompletely understood. We previously showed that mammary tumor development in the mouse model of breast cancer MMTV-neu, a model characterized by amplification of the EGFR ErbB2 in mammary tissue, correlates with a marked up-regulation of fatty acid-binding protein 5 (FABP5). FABP5 functions to deliver ligands to and enhance the transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta), a receptor whose target genes include genes involved in cell growth and survival. We show here that in MCF-7 mammary carcinoma cells, EGFR signaling directly up-regulates the expression of FABP5. The data demonstrate that treatment of these cells with the EGFR ligand heregulin-beta1 signals through the ERK and the phophatidylinositol-3-kinase cascades, resulting in activation of the transcription factor NF-kappaB. In turn, NF-kappaB induces the expression of FABP5 through two cognate response elements in the promoter of this gene. The observations further demonstrate that FABP5 and PPARbeta/delta are critical mediators of the ability of EGFR to enhance cell proliferation, indicating that this transcriptional pathway plays a key role in EGFR-induced tumorigenesis. Additional observations indicate that the expression of FABP5 is down-regulated by the Krüppel-like factor KLF2, suggesting a tumor suppressor activity for this factor.

Phospholipase C-γ1 is required for the epidermal growth factor receptor-induced squamous cell carcinoma cell mitogenesis

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-γ1 (PLC-γ1) is a downstream target of EGFR signaling, but the role and necessity of PLC-γ1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-γ1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-γ1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-γ1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-γ1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-γ1 is independent of its lipase activity.

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signaling is the natural role of reactive oxygen species, such as hydrogen peroxide (H2O2), as beneficial messengers in redox signaling pathways. Despite growing evidence, the nature of H2O2 signaling is confounded by difficulties in tracking it in living systems both spatially and temporally at low concentrations. Here we develop an array of fluorescent single-walled carbon nanotubes that can selectively record in real time the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells that are stimulated by epidermal growth factor (EGF). We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that EGF induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signaling of reactive oxygen species at the cellular level.

Study of some cytogenetic effects of Aerolysin Produced by Aeromonas hydrophila on normal and tumor cells in vitro.

This study aimed to find the cytotoxicity of the aerolysin produced by Aeromonas hydrophila on rat embryo fibroblast, mouse mammary adeno carcinoma and human larynx epidermal carcinoma cell lines by estimating the cells inhibitory and proliferation rates . Cells were exposed to ten concentrations of aerolysin toxin for 24, 48 and 72 hours. Then the inhibitory and proliferation rates were calculated. The results showed that the effect of the toxin depends on concentration and exposure time.

Anti-proliferation activity and phytochemical screening of Thai medicinal plant recipes

Thai medicinal plant recipes were surveyed and divided into two groups including the books of Thai traditional medicine and Thai traditional healers. 12 recipes were found from the books of Thai traditional medicine and 28 recipes from 14 questionnaires. 10 recipes were selected from 40 recipes of the books and questionnaires to test the anti-proliferation activity on human mouth epidermal carcinoma (KB) cell lines by Sulforhodamine B (SRB) assay. The anti-proliferation activity and phytochemical screening of 9 herbs in recipe 9 was investigated. The results found that Hydnophyum formicarum exhibited growth inhibit activity against KB cell lines with GI 50 value 5.54µg/ml, which were about 1/277 times of doxorubicin (GI50=0.02µg/ml). Hydnophyum formicarum contain tannin, alkaloid, flavonone and xanthone. The results from this study will be beneficial for further research and development of anti-cancer drug.

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK*Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phosphorylated tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phosphorylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phosphorylation-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phosphorylation-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phosphorylation of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK*Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK*Grb7 complexes and Grb7 phosphorylation by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK*Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK*Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.

Synthesis and Antitumor Evaluation of Novel Bis-Triaziquone Derivatives

Aziridine-containing compounds have been of interest as anticancer agents since late 1970s. The design, synthesis and study of triaziquone (TZQ) analogues with the aim of obtaining compounds with enhanced efficacy and reduced toxicity are an ongoing research effort in our group. A series of bis-type TZQ derivatives has been prepared and their cytotoxic activities were investigated. The cytotoxicity of these bis-type TZQ derivatives were tested on three cancer lines, including breast cancer (BC-M1), oral cancer (OEC-M1), larynx epidermal cancer (Hep2) and one normal skin fibroblast (SF). Most of these synthetic derivatives displayed significant cytotoxic activities against human carcinoma cell lines, but weak activities against SF. Among tested analogues the bis-type TZQ derivative 1a showed lethal effects on larynx epidermal carcinoma cells (Hep2), with an LC50 value of 2.02 mM, and also weak cytotoxic activity against SF cells with an LC50 value over 10 mM for 24 hr treatment. Comparing the viability of normal fibroblast cells treated with compound 1a and TZQ, the LC50 value of the latter was 2.52 mM, indicating more toxicity than compound 1a. This significantly decreased cytotoxicity of compound 1a towards normal SF cells, while still maintaining the anticancer activity towards Hep2 cells is an interesting feature. Among the seven compounds synthesized, compound 1c has similar toxicity effects on the three cancer cell lines and SF normal cells as the TZQ monomer.

Antimicrobial, antioxidant and anticancer activities of Thai local vegetables.

Thai local vegetables (20 species) were screened for their antimicrobial and antioxidant activities. Methanolic extracts of Cassia siamea, Garcinia cowa,Limnophila aromatica and Polygonum odoratum exhibited strong antioxidant and antibacterial activities. The sensitive bacteria to these extracts were Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Among all plant extracts, the extract of P. odoratum had the highest phenolic content and antioxidant activity with EC50 value of 315.35 mg extract/mg DPPH (2,2-diphenyl-1-picrylhydrazyl). These plant extracts were also tested for cytotoxicity against human oral epidermal carcinoma (KB), breast adenocarcinoma (MCF-7) and small cell lung carcinoma (NCI-H187). P. odoratum extract was moderately active against MCF-7 (IC50 value of 6.01 mg/ml). Major types of active compounds in P. odoratum extract were identified by HPLC method. Flavonoids found in this plant were rutin, catechin, quercetin, kaempferol and isorhamnetin. Among these compounds, rutin was found in the highest amount (3.77% w/w dry extract). Key words: Polygonum odoratum, Cassia siamea, Garcinia cowa, Limnophila aromatica, phenolic compounds, flavonoids.

Suppressive effect of tobacco smoke extracts on oral P-glycoprotein function and its impact in smoke-induced insult to oral epidermal cells

P-glycoprotein (Pgp) participates in the export of numerous toxins, drugs, and physiological compounds. To examine the involvement of Pgp in smoke-induced oral cell insult, the effects of extracts of the mainstream tobacco smoke (TS) on Pgp were studied in an oral epidermal carcinoma cell line, OECM-1. TS was first extracted with cyclohexane (CTS) and the residues were further extracted with isopropanol (ITS). For comparison, cells were exposed to CTS and ITS at the concentrations according to their relative extraction yield. ITS but not CTS decreased the efflux of a Pgp substrate, rhodamine (Rh) 123, in a concentration- and time-dependent manner. The efflux was also decreased by co-exposure to CTS and ITS. However, immunoblot analysis revealed that the protein level of Pgp was not affected by ITS. Naphthalene, mainly detected in the ITS, decreased Rh 123 efflux. However, the efflux activity was not affected by benzo(a)pyrene and nicotine, which were present in the CTS and both extracts, respectively. Co-exposure to CTS in combination with ITS, naphthalene, or verapamil enhanced cell insult compared to single exposure. These results demonstrated that smoke and its constituent, naphthalene, diminished Pgp-mediated efflux. The reduction in Pgp function could be a stimulatory factor of TS-induced oral cell insult.

Positive contrast visualization for cellular magnetic resonance imaging using susceptibility-weighted echo-time encoding.

The objective of this study was to investigate a method to generate positive contrast, selective to superparamagnetic iron oxide (SPIO) labeled cells, using the susceptibility-weighted echo-time encoding technique (SWEET). SPIO-labeled human epidermal carcinoma (KB) cells were placed in a gel phantom. Positive contrast from the labeled cells was created by subtraction between conventional spin-echo images and echo-time shifted susceptibility-weighted images. SPIO-labeled cells were injected into the left dorsal flank and hind limb of nude mice, and unlabeled cells were placed on the right side as controls. Tumor growth was monitored using the proposed method, and a histological analysis was used to confirm the presence of the labeled cells. Based on in vitro testing, we could detect 5000 labeled cells at minimum and the number of pixels with positive contrast increased proportionally to the number of labeled cells. Animal experiments also revealed the presence of tumor growth from SPIO-loaded cells. We demonstrated that the proposed method, based on the simple principle of echo-time shift, could be readily implemented in a clinical scanner to visualize the magnetic susceptibility effects of SPIO-loaded cells through a positive-contrast mechanism.

Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of α6β1 integrin

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial–mesenchymal interconversions leading to the disruption of cell–cell contact and modulation of cell–extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in α2, α6 and β1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of α6 and β1 integrin subunits. Neutralizing antibodies against α6 and β1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of α6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475–85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and α6β1 integrin-mediated signalling in ovarian carcinomas.

A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.

Synthesis and in vitro antitumor activities of novel 4-anilinoquinazoline derivatives

A series of 6, 7-dialkoxy-4-anilinoquinazolines were designed, synthesized by substituting different heterocycles on 6-position and a variety of anilines on 4-position of the quinazoline. These novel quinazoline compounds were screened for their cytotoxic effect on epidermal growth factor receptor overexpressing skin epidermoid carcinoma cell line (A431), by using nonoverexpressing tumor cells as negative control (breast adeno carcinoma cell line MCF-7). 2-Butyl-4-chloro-1-{3-[7-methoxy-4-(3-(trifluoromethyl)phenylamino)quinazolin-6-yloxy]-propyl}-1 H-imidazole-5-carboxaldehyde ( 30) and 2-butyl-4-chloro-1-{3-[4-(3-iodophenyl amino)-7-methoxyquinazolin-6-yloxy]propyl}-1 H-imidazole-5-carboxaldehyde ( 33) were found to be more potent against A431 cell line (IC 50 3.5 and 3 μM) and their activities are comparable to gefitinib. Insilico docking experiments with human EGFR Tyrosine kinase domain (PDB id-2gs2) indicated that 33 docks at the same position as that of gefitinib involving Val702, Ala719, Ser696, and Lys721.

Synthesis and Biological Activity of Mustard Derivatives of Thymine

The synthesis and biological activity of a novel DNA cross-linking antitumor agent is presented. The new alkylating agent significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.

Disruption of microfilaments by cytochalasin B decreases accumulation of cisplatin in human epidermal carcinoma and liver carcinoma cell lines

Although cisplatin is a frequently used cancer chemotherapeutic drug, its effectiveness is hindered by the development of resistance in cancer cells. In order to understand the reason(s) for this resistance, the mechanism of uptake of cisplatin into cells must be characterized. While several previous studies showed structural differences between cisplatin-sensitive and resistant cells, the influence of microfilaments, known to affect transport of molecules into cells, and the influence of certain biophysical characteristics of the plasma membrane needed clarification. We show that resistant human epidermal carcinoma (KB-CP20) and liver carcinoma (BEL-7404-CP20) cells become relatively more resistant if their already weak microfilaments are degraded by cytochalasin B treatment (.5-2 microM). The sensitive counterparts of these cells with intact microfilaments are not significantly affected by this treatment. We also show that the "fluidity" of the plasma membrane and the membrane potential of the sensitive and resistant cells studied do not appear to influence the uptake of cisplatin into the cells. Our results suggest that the status of the microfilament system influences the mechanism of uptake of cisplatin into cells.

Synthesis and Biological Activity of Chloroethyl Pyrimidine Nucleosides

The synthesis and biological activity of chloroethyl pyrimidine nucleosides is presented. One of these new nucleosides analogues significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.

CD151 dynamics in carcinoma–stroma interaction: integrin expression, adhesion strength and proteolytic activity

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and α3β1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5CD151−), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5CD151−, striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. α3β1 integrin was internalized in part, and α6β4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5CD151−. Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma–stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.