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Porphyrazines (Pzs) are porphyrin derivatives that show potential application as photosensitizers for photodynamic therapy (PDT), but are still far less explored in the literature. In this work, we evaluate how the photophysics and phototoxicity of the octakis(trifluoromethylphenyl)porphyrazine (H2Pz) against tumor cells can be modulated by coordination with Mg(II), Zn(II), Cu(II) and Co(II) ions. Fluorescence and singlet oxygen quantum yields for the Pzs were measured in organic solvents and in soy phosphatidylcholine (PC) liposomes suspended in water. While H2Pz and the respective complexes with Cu(II) and Co(II) showed very low efficiency to fluoresce and to produce 1O2, the Mg(II) and Zn(II) complexes showed significantly higher quantum yields in organic solvents. The fluorescence of these two Pzs in the liposomes was sensitive to the fluidity of the membrane, showing potential use as viscosity markers. The cytotoxicity of the compounds was tested in HaCaT (normal) and A431 (tumor) cells using soy PC liposomes as drug carriers. Despite the low 1O2 quantum yields in water, the Mg(II) and Zn(II) complexes showed IC50 values against A431 cells in the nanomolar range when activated with low doses of red LED light. Their phototoxicity was ca. three times higher for the tumor cells compared to the normal ones, showing promising application as photosensitizers for PDT protocols. Considering that H2Pz and the respective Co(II) and Cu(II) complexes were practically non-phototoxic to the cells, we demonstrate the importance of the central metal ion in the modulation of the photodynamic activity of porphyrazines. [ABSTRACT FROM AUTHOR]

Background: First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, have been shown to target tumors with L858R (exon 21) and exon 19 deletions, resulting in significant clinical benefits. However, acquired resistance often occurs due to EGFR mutations. Therefore, novel therapeutic strategies for treatment of patients with EGFR-positive tumors are needed. Berberine (BBR) is an active alkaloid extracted from pharmaceutical plants such as Coptis chinensis. Berberine has been shown to significantly inhibit EGFR activity and mediate anticancer effects in multiple preclinical studies. We investigated whether combining BBR with erlotinib could augment erlotinib-induced cell growth inhibition of EGFR-positive cells in a mouse xenograft model. Methods: We examined the antitumor activities and potential mechanisms of erlotinib in combination with berberine in vitro and in vivo using the MTT assay, immunoblotting, flow cytometry, and tumor xenograft models. Results: In vitro studies with A431 cells showed that synergistic cell growth inhibition by the combination of BBR and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, and inhibition of cyclin D and Bcl-2 expression compared to that observed in response to BBR or erlotinib alone. The efficacy of the combination treatment was also investigated in nude mice. Consistent with the in vitro results, BBR plus erlotinib significantly reduced tumor growth. Conclusion: Our data supported use of BBR in combination with erlotinib as a novel strategy for treatment of patients with EGFR positive tumors. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression levels of long noncoding RNA UCA1 in A431 cells and its effects on proliferation, invasion and epithelial-mesenchymal transition of A431 cells. Methods: The expression levels of lncRNA UCA1 in skin squamous cell carcinoma, paracancerous tissues, A431 cells and HaCaT cells were assessed by Quantitative Real-time PCR.Following down-regulation of lncRNA UCA1 with siRNA, proliferation, invasion and EMT of A431 cells were measured by methyl thiazolyl tetrazolium, Transwell and Western blot, respectively. The relationship between lncRNA UCA1 and microRNA-143 was analyzed by miRanda and double luciferase reporter gene assay. RT-qPCR was used to determine the expression levels of miR-143 in A431 cells and HaCaT cells. To determine whether the effects of lncRNA UCA1 is via miR-143, proliferation, invasion and EMT of A431 cells were analyzed following simultaneous upregulation of lncRNA UCA1 and miR-143. The relationship between miR-143 and Hexokinase II was analyzed by TargetScan and double luciferase reporter gene assay. RT-qPCR was used to detect HK2 expression in A431 cells and HaCaT cells. Proliferation, invasion and EMT of A431 cells were analyzed following down-regulation of HK2 with siRNA. Results: In A431 cells, the expression levels of both lncRNA UCA1and HK2 were upregulated (P

Abstract This study investigated the anti-cancer potential of a near-infrared fluorescence (NIRF) molecule conjugated with Cetuximab (Cetuximab-NIRF) in six-week-old female BALB/c athymic (nu+/nu+) nude mice. A431 cells were cultured and injected into the animals to induce solid tumors. Paclitaxel (30 mg/kg body weight (BW)), Cetuximab (1 mg/kg BW), and Cetuximab-NIRF (0.25, 0.5 and 1.0 mg/kg BW) were intraperitoneally injected twice a week into the A431 cell xenografts of the nude mice. Changes in BW, tumor volume and weight, fat and lean mass, and diameter of the peri-tumoral blood vessel were determined after two weeks. Tumor volumes and weights were significantly decreased in the Cetuximab-NIRF (1 mg/kg BW) group compared with the control group (P

Cutaneous squamous cell carcinoma (CSCC) is the second most common malignant skin tumor and significantly affects patients' quality of life and health. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway activation is involved in CSCC development. Radix Tetrastigma hemsleyani flavone (RTHF) is an active Radix Tetrastigma extract (RTE), which was recently reported to have promising inhibitory effects on CSCC. However, the underlying functional mechanisms of this inhibition remain unknown. In the present study, A431 cells or SCL-1 cells were incubated with 1, 5, and 10 mg/mL RTHF for 48 h, respectively. A significantly increased wound closure rate, decreased number of migrated and invaded cells, decreased colony number, and elevated apoptotic rate were observed after treatment with 1, 5, and 10 mg/mL RTHF. Furthermore, after incubation with RTHF, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 levels were drastically reduced. An A431 xenograft model was constructed, followed by oral administration of 15, 30, or 60 mg/kg RTHF for 21 consecutive days. A significantly lower increase in tumor volume and reduced tumor weight were observed in all RTHF-treated groups. In addition, JAK/STAT3 signaling was drastically repressed in tumor tissues. Collectively, RTHF inhibited CSCC progression, which may be associated with JAK/STAT3 pathway inactivation. [ABSTRACT FROM AUTHOR]

New water soluble conjugates of chlorin e 6 derivative–zinc complex with β- d -galactose or β- d -glucose have been synthesized as potential agents for the photodynamic therapy of cancer. They exhibit photoinduced cytotoxicity at low micromolar concentrations with IC 50(dark) /IC 50(light) ratio of ∼50. [ABSTRACT FROM AUTHOR]

Gallic acid (GA) and its derivative methyl gallate (MG) are well studied plant phenolics. They have exhibited anticancer effects in several cancer cell lines. However, the presence of GA/MG in the seed coats of Givotia rottleriformis and their inhibitory effect on human epidermoid carcinoma (A431) skin cancer cells were not reported. In this study we have isolated and chemically characterized the bioactive compounds GA and MG from the bioassay guided methanolic (MeOH) seed coat extracts of G. rottleriformis. The fractions obtained from open silica column chromatography were subjected to in vitro enzymatic assays. Among seven fractions we found that only fractions 5 and 6 showed significant inhibition activity toward COX-1 with an IC50 value of 28 μg/mL and 9.3 μg/mL and COX-2 with an IC50 value of 35 μg/mL and 7.0 μg/mL respectively. However, we could not find 5-LOX enzyme inhibition activity. MG (10 mg/g DW) and GA (6 mg/g DW) were the major compounds of seed coats. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which showed that GA/MG significantly reduced the growth of A431 cells with an IC50 value of 25 μg/mL and 53 μg/mL and 11 μg/mL and 43 μg/mL at 24 h and 48 h, respectively. However the cytotoxic effect of GA/MG on HaCaT normal skin keratinocyte cell line was found to be less. Western blot analysis has shown that GA/MG treatment down regulated Bcl-2 and up regulated cleaved caspase-3 with respect to increasing doses. Our results conclude that GA and MG have potential anticancer effects and can be used as therapeutic agents for skin cancers.

Abstract Introduction Indigofera tinctoria widely used in Indian and Chinese medicinal system to cure various diseases. Based on the ethnomedicinal value of this plant, the present investigation was aimed to isolate the bioactive compound from Indigofera tinctoria and evaluate the antiproliferative activity against human epidermoid carcinoma (A431) cells. Methods Chrysin (CH) was purified from the chloroform extract of I. tinctoria leaves by silica gel column chromatography and its antiproliferative effect against A431 cells was evaluated by MTT assay. Apoptotic effects were analysed by DNA fragmentation, nucleic acid content and acridine orange/ethidium bromide staining. Cell cycle arrest was analysed by flow cytometry. The apoptotic markers such as cytochrome c, caspase 9, caspase 3 and inhibitor of caspase activated DNase (ICAD) expression were analysed by western blot. Results MTT assay results revealed that the CH has inhibited the proliferation of A431 cells with IC 50 value of 23.52 μg/mL and exhibited potent cytotoxicity; where as there is no promising toxic effect against normal HaCaT cells. DNA fragmentation analysis indicated that the CH treated A431 cells displayed the fragmented DNA. Further, CH induced remarkable cell death through apoptosis (87.65 ± 1.80%) and arrested the cell cycle at Sub G 1 phase. Moreover, the CH could enhance the expression of cytochrome c, caspase 9, caspase 3 and inhibit the ICAD in a dose dependent manner. Conclusion The present investigation concluded that the CH purified from I. tinctoria has potentially inhibited the proliferation of human epidermoid carcinoma (A431) cells and induce the apoptosis through caspase dependent manner. [ABSTRACT FROM AUTHOR]

In recent years, nanotechnology has gained serious attention for diagnosis, prevention and treatment roles. In this study we synthesized nanoceria or CeO2NPs (cerium oxide nanoparticles) and compared toxicity of cerium oxide powder in nano and bulk forms in two cancerous and one normal cell lines. The cell lines were cultured in a standard humidified incubator, at 37 °C in a 5% CO2 atmosphere, in RPMI 1640 medium. The cells were incubated with different concentrations of cerium oxide (from 2 μg/mL to 64 μg/mL) in bulk and nano forms. To determine the effect of cerium oxide on cell viability after 24 h, 48 h, and 72 h incubation, a MTT assay was performed using SKBR3 (human breast cancer cell line), A431 (Human epidermoid carcinoma cell line) and C2Cl2 (ATCC mouse skeletal muscle cell line) cells. Analysis of variance followed by Sidak posthoc test, shows the toxicity of nanoceria is significantly deferent from bulk form on three cell lines in this study and is more on cancerous cells in compared to normal cells especially in higher level of concentrations after 24, 48 and 72 hours (All P<0.05). Additionally, the effect of cell lines, cerium oxide forms and concentrations cerium oxide leads in significantly the lowest amount of viability after 72 hours compared with 24 hours and 48 hours. [ABSTRACT FROM AUTHOR]

Purpose:To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters. Materials and methods:An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method. Results:Significant decreasing cellular proliferation rate (p < 0.05) as well as reduced survival fraction (p < 0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G2/M phase (32.3 ± 1.5%) in the irradiated group as compared with untreated cells (8.22 ± 1.2%) was detected. Also, a significant (p < 0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found. Conclusion:HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G2/M cell cycle arrest. [ABSTRACT FROM AUTHOR]

Stylosanthes guianensis is a fodder legume native from South America and widely grown worldwide. Dried plant material was purchased on the web and taxonomically identified by light and SEM microscopy, and morphological analysis of plants germinated from seeds. The plant was extracted with dichloromethane:2-propanol (9:1). Bioguided fractionation using calcein-AM cytotoxicity assay on HeLa and A431 tumor cells allowed to isolate a lipophilic fraction, endowed with strong cytotoxicity. By means of 1- and 2-D NMR, HPLC–MS, and HR-ESIMS it could be seen that the fraction was an inseparable mixture of complex lipids, mainly consisting of esterified 3-hydroxy fatty acids. Acidic methanolysis of the mixture yielded 3-OH C10 and C12 carboxylic acids, together with palmitic, stearic, and arachidonic acids. Mass values indicate the presence of dimeric and trimeric combinations of 3-hydroxy, C10/C12 acids, and C16/C18/C20 acids, linked via ester bond. Monomeric hydroxyl-fatty acids were also observed, in particular derivatives of mono-hydroxy and di-hydroxy linolenic, linoleic, and oleic acids. 3-O-acylated, esterified fatty acids are unusual in higher plants, and recall motifs of Gram-negative endotoxin lipid A. These oxylipins are likely to be responsible for the antiproliferative activity of S. guianensis , suggesting possible use of the plant in the development of antitumor drugs. [ABSTRACT FROM AUTHOR]

The Epidermal Growth Factor Receptor (EGFR) is a cell surface receptor with primary implications in cell growth in both normal and malignant tissue. Paradoxically, cell lines that hyperexpress the EGFR have been documented to undergo receptor-mediated apoptosis. The underlying mechanism by which EGF-induced apoptosis occurs however remains inexplicit. In an attempt to identify this mechanism, we assessed downstream effectors of EGFR in MDA-MB-468 cells during conditions of EGF-induced apoptosis. The effector assessment revealed STAT3 as a potential mediator of EGF-induced apoptosis. Alternative strategies for activating STAT3, independent of EGFR stimulation, resulted in the induction of the apoptotic pathways. A reduction in STAT3 expression via RNAi resulted in a significant attenuation of EGF-induced PARP cleavage. Our findings support STAT3 as a positive mediator of EGF-induced apoptosis in MDA-MB-468 cells. [ABSTRACT FROM AUTHOR]

This work reports the preparation of water-soluble leucine capped gold nanoparticles by two single-step synthesis methods. The first procedure involves a citrate reduction approach where the citrate is used as reducing agent and leucine as capping/stabilizing agent. Different sizes of gold nanoparticles, citrate reduced and stabilized by leucine, Leu-AuNPs-C, with the mean diameters in the range of 21-56 nm, were obtained by varying the macroscopic parameters such as: concentration of the gold precursor solution, Au (III):citrate molar ratio and leucine pH. In the second procedure, leucine acts both as reducing and stabilizing agent, allowing us to obtain spherical gold nanoparticles, Leu-AuNPs, with a majority of 80 % (with the mean diameter of 63 nm). This proves that leucine is an appropriate reductant for the formation of water-soluble and stable gold nanoparticles colloids. The characterization of the leucine coated gold nanoparticles was carried out by TEM, UV-Vis and FT-IR analysis. The cytotoxic effect of Leu-AuNPs-C and Leu-AuNPs was also evaluated. [ABSTRACT FROM AUTHOR]

NF-κB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-κB activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-κB proteins RelA/p65 and p50 in relation to the inhibitor IκB-α, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-κB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NF-κB was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IκB-α. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IκB-α in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IκB-α in the membrane. Furthermore, EGF stimulation for 30 min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-κB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IκB-α. Moreover, we discovered the existence of a dynamic, IκB-α-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-κB stimulation. [ABSTRACT FROM AUTHOR]

In medicine, the use of silica nanoparticles (SiO2 NPs) offers new perspectives in biosensor, drug delivery and cancer therapy. However, questions about potential toxic and deleterious effects of SiO2 NPs have also been raised. The aim of this study was to investigate the induction of cytotoxicity, oxidative stress and apoptosis by SiO2 NPs (size 15 nm) in human skin epithelial (A431) and human lung epithelial (A549) cells. SiO2 NPs (concentration range 25–200 µg/ml) induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) and lactate dehydrogenase leakage assays. SiO2 NPs were also found to induce oxidative stress in a dose-dependent manner, indicated by depletion of glutathione and induction of reactive oxygen species (ROS) generation and lipid peroxidation. Quantitative real-time polymerase chain reaction analysis showed that following the exposure of cells to SiO2 NPs, the messenger RNA level of apoptotic genes (caspase-3 and caspase-9) were upregulated in a dose-dependent manner. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in both kinds of cells exposed to SiO2 NPs. This study suggested that SiO2 NPs induce cytotoxicity and apoptosis in A431 and A549 cells, which is likely to be mediated through ROS generation and oxidative stress. [ABSTRACT FROM PUBLISHER]

Abstract: While epidermal growth factor (EGF) is a well known mitogen, high doses of EGF result in a paradoxical apoptotic response in the cells that overexpress EGF receptor such as A431 epidermoid carcinoma cells. EGF-induced apoptosis in A431 cells is dependent upon activation of transcription factor STAT1. In this study, we demonstrate that p38 MAP kinase is another important mediator of EGF-dependent pro-apoptotic response in A431 cells. By utilizing p38 MAP kinase inhibitors, SB203580 and BIRB0796, we significantly reduced the integral growth-inhibiting as well as pro-apoptotic effects of EGF. Moreover, we observed that inhibition of p38 MAP kinase markedly decreased phosphorylation of tyrosine 701 in STAT1, while neither EGF-induced accumulation nor serine phosphorylation of STAT1 was decreased. We propose that p38 MAP kinase mediates STAT1 tyrosine phosphorylation, thereby enforcing EGF-induced apoptosis. [Copyright &y& Elsevier]

The historical tag of the epidermal growth factor (EGF) as carcinogenesis promoter has not been uniformly reproduced, since, in some experiments, malignant cells are bound to growth inhibition and apoptosis. The present study intends to obtain additional data on the interaction of EGF with cancer cells by analyzing its effect in vitro on the growth of cultured A431 cells, and in vivo on nude mice xenotransplanted with this cell line; identifying, in addition, the gene expression patterns for a selected group of genes related to EGF and cancer pathways in the solid tumor. EGF-treated animals showed significantly smaller tumor volumes than controls, suggesting cellular growth inhibition mediated by this factor. Similar results were obtained regarding the proliferation of cultured A431 when treated with 2.2, 33 and 165 nM of EGF. These results may imply a common mechanism for the EGF-mediated growth inhibition of A431 cells in both biological conditions (in vivo and in vitro). The action of EGF at nanomolar concentrations may trigger a transient recovery of the tumor suppressor ability of tumor cells through the reduction of the biological action of mutated TP53, cell cycle arrest due to a decrease in Cdk4 expression levels and the initiation of caspase through the activation of CASP9. [ABSTRACT FROM AUTHOR]

Enhanced fucosyltransferase IV ( FUT4) expression correlates with increased tumor malignancy in many carcinomas. However, little is known about the regulation of FUT4 expression, and whether FUT4 expression is influenced by the methylation status of the FUT4 promoter is unclear. In this study, we demonstrated that FUT4 expression is negatively correlated with the methylation degree of a CpG island in the FUT4 promoter, suggesting that the methylation status of FUT4 promoter regulates the expression of FUT4. The results indicate that manipulating the methylation status of the FUT4 promoter to regulate FUT4 expression may be a novel approach in the treatment of malignant tumors. [ABSTRACT FROM AUTHOR]

Ultraviolet C (UVC) is a DNA damage inducer, and 20 J/m² of UVC irradiation caused cell growth inhibition and induced cell death after exposure for 24-36 h. The growth of NIH 3T3 cells was significantly suppressed at 24 h after UVC irradiation whereas the proliferation of A431 cells was inhibited until 36 h after UVC irradiation. UVC irradiation increased COX-2 expression and such up-regulation reached a maximum during 3-6 h in NIH 3T3 cells. In contrast, UVC-induced COX-2 reached a maximum after 24-36 h in A431 cells. Measuring prostaglandin E2 (PGE2) level showed a biphasic profile that PGE2 release was rapidly elevated in 1-12 h after UVC irradiation and increased again at 24 h in both cell lines. Treatment with the selective COX-2 inhibitor, SC-791, during maximum expression of COX-2 induction, attenuated the UVC induced-growth inhibition in NIH 3T3 cells. In contrast, SC-791 treatment after UVC irradiation enhanced death of A431 cells. These data showed that the patterns of UVC-induced PGE2 secretion from NIH 3T3 cells and A431 cells were similar despite the differential profile in UVC-induced COX-2 up-regulation. Besides, COX-2 might play different roles in cellular response to UVC irradiation in various cell lines. [ABSTRACT FROM AUTHOR]

Changes in the sensitivity of several transformed and embryonic cells to the lytic activity of natural killers (NK) due to N-acetylcysteine (NAC) has been studied. We found that epidermoid carcinoma A431 cells and murine hepatoma MH22a cells, as well as the transformed mouse fibroblasts 3T3-SV40 treated with 10 mM NAC that we investigated previously normalized their phenotype to the various extent. Like normal cells these cells exposed to NAC are not recognized and destroyed by NK. Murine embryonic fibroblasts (MEFs) similar to transformed cells were destroyed by NK. MEFs treated with 10 mM NAC lost their susceptibility to NK, as did transformed cells. The loss of cell sensitivity to NK cytolytic activity was accompanied by the reorganization of the actin cytoskeleton and generation of well-pronounced stress-fibers. [ABSTRACT FROM AUTHOR]

Abstract: Polo-like kinase 1 (Plk1), a critical regulator of mitotic entry, progression and exit, has been shown to be involved in a variety of cancers and thus is becoming an attractive target for cancer management. In case of DNA damage, Plk1 not only inhibits p53 independent apoptosis by dysfunctioning p73α but also allows cells to recover from growth arrest. Here, we showed the effects of knocking down plk1 gene through small interference RNA (siRNA) on cell cycle progression, proliferation and chemosensitivity of p53 mutant A431 cells to cisplatin (CDDP). The expression of Plk1 was measured by RT-PCR and Western blotting. Anti-proliferative response accompanied with cell cycle arrest in G(2)/M phase and induction of cell death was recorded following Plk1 knockdown. Furthermore, cells following knockdown of Plk1, which induced increase of Cyclin B1, p-Cdc2 and p73α with a decrease in p-Cdc25C, were more sensitive to CDDP. CDDP treatment induced nuclear translocation and co-localization of Plk1 with p73α whereas combination of CDDP and Plk1siRNA upregulated the expression of p73α protein in a synergistic manner thereby leading to an increase up to ∼5 folds in CDDP-induced cell death. The increase in caspase-3 activity indicated apoptosis as a contributor in the total cell death. Conclusively, plk1 gene silencing can enhance the sensitivity of A431 cells to low doses of CDDP by upregulating p73α expression and thus can be a revolutionary approach in cancer chemotherapy. [Copyright &y& Elsevier]

Abstract: The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A431 cells) are an important target for drug discovery. We have developed a new A431/cell membrane chromatography (A431/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A431 cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A431/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A431 cell growth in vitro. This new A431/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource. [Copyright &y& Elsevier]

Abstract: Aims: Amlodipine, a dihydropyridine Ca2+ channel blocker, inhibits the proliferation of human epidermoid carcinoma A431 cells in vitro and in vivo. This study examined the underlying mechanism of this antiproliferative effect in relation to epidermal growth factor receptor (EGFR) signaling. Main methods: The tyrosine phosphorylated active state of EGFR in A431 cells incubated with the test agents was evaluated by western blot with anti-phosphotyrosine antibody. EGFR phosphorylation levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates. Key findings: In vitro treatment of exponentially growing A431 cells with amlodipine decreased the tyrosine phosphorylation states of EGFR. Amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR and a membrane scaffolding protein, caveolin-1, in serum-starved A431 cells. Amlodipine attenuated the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 without affecting the EGFR/caveolin-1 interaction. Crosslinking experiments showed that amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR predimers. Addition of cholesterol abolished these inhibitory effects of amlodipine plus its inhibition of cell growth. Furthermore, treatment of mice with amlodipine (10mg/kg/day×7days, i.p.) decreased the levels of phosphorylated EGFR in A431 xenograft tumors. Significance: The results indicated that amlodipine inhibits tyrosine phosphorylation of EGFR in vitro and in vivo, possibly via modulating cholesterol-rich, caveolin-1-containing membrane microdomains. [Copyright &y& Elsevier]

Abstract: Since skin cancer incidence and prevalence is constantly rising up the charts despite all efforts, search for newer, better agents for protection and treatment is required. Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as a herbal medicine, given the presence of homologous phenolic ketones, of which [6]-gingerol is the major one. The quantity of [6]-gingerol in the fresh ginger rhizome was found to be 104–965μg/g in common varieties of ginger available in Indian market. Herein, [6]-gingerol was assessed for its anti-apoptotic effects in human epidermoid carcinoma A431 cells. [6]-Gingerol treatment exhibited considerable cytotoxicity as indicated by growth inhibition of A431 cells mediated via generation of reactive oxygen species (ROS). Increase in ROS led to decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results revealed that perturbations in mitochondrial membrane are associated with deregulation of Bax/Bcl-2 ratio at gene transcriptional level as well as protein level, where treatment with [6]-gingerol leads to up-regulation of Cytochrome-c and Apaf-1 subsequently culminating in triggering of Caspase cascade. These firmly suggest that [6]-gingerol can be effectively used for the treatment of skin cancer. [Copyright &y& Elsevier]

Abstract: Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30mJ/cm2) and resveratrol (60μM) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition of A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-κB) pathway by blocking phosphorylation of serine 536 and inactivating NF-κB and subsequent degradation of IκBα, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis. [Copyright &y& Elsevier]

Abstract: A cyclic decapeptide was chemically synthesized that mimics the loop structure of a β-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10μM in the intact cells. [Copyright &y& Elsevier]

Abstract: Preclinical studies have shown the potential antitumour efficacy of monoclonal antibodies (MAbs) directed to the epidermal growth factor receptor (EGFR). In this report, we investigated the cytotoxic effects of the MAb matuzumab (EMD 72000) towards A431 cells and compared it to cetuximab. While cetuximab induced cell cycle arrest and inhibited A431 cell proliferation, matuzumab did not. Both MAbs inhibited growth factor induced EGFR, HER2 and AKT phosphorylation; however, only cetuximab inhibited ERK 1/2 phosphorylation. Taken together, the data indicate that each antibody may elicit different responses on EGFR downstream signalling pathways with a distinct impact on A431 cell line survival. When combined, MAbs synergistically inhibited cell proliferation and induced EGFR down-regulation with a strong inhibition of ERK1/2 and AKT phosphorylation. In addition, both MAbs efficiently inhibited VEGF expression and induced ADCC, highlighting their therapeutic potential in vivo when used either as a single agent or in combination. [Copyright &y& Elsevier]

For production of monoclonal antibody (mAb) against human epidermal growth factor receptor (EGFR), first, five female Balb/c mice 6 to 8 weeks old were immunized against A431 tumoral cells that express more EGFR on its membrane in four periods and the most immune mouse was selected for fusion. The fusion of mouse's spleen immune cells with SP2/0 cells (myeloma cells) were fulfilled in presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Large scale of monoclonal antibodies was produced in vitro and ascetic fluid. In this investigation, 280 clones were obtained, 27 of which displayed absorbance more than 1. Of these, 3 clones represented absorbance about 1.7 and selected for limiting dilution. The yield of limiting dilution was 8 monoclones with absorbance about 2. These results indicate that such monoclonal antibodies against EGFR can be used in diagnosis and treatment of tumors with membranous EGFR. [ABSTRACT FROM AUTHOR]

Abstract: An infamous poison, arsenic also has been used as a drug for nearly 2400 years; in recently years, arsenic has been effective in the treatment of acute promyelocytic leukemia. Increasing evidence suggests that opposite effects of arsenic trioxide (ATO) on tumors depend on its concentrations. For this reason, the mechanisms of action of the drug should be elucidated, and it should be used therapeutically only with extreme caution. Previously, we demonstrated the opposing effects of ERK1/2 and JNK on p21 WAF1/CIP1 (p21) expression in response to ATO in A431 cells. In addition, JNK phosphorylates c-Jun (Ser63/73) to recruit TGIF/HDAC1 to suppress p21 gene expression. Presently, we demonstrated that a high concentration of ATO sustains ERK1/2 phosphorylation, and increases c-Fos biosynthesis and stability, which enhances p21 gene expression. Using site-directed mutagenesis, a DNA affinity precipitation assay, and functional assays, we demonstrated that phosphorylation of the C-terminus of c-Fos (Thr232, Thr325, Thr331, and Ser374) plays an important role in its binding to the p21 promoter, and in conjunction with N-terminus phosphorylation of c-Fos (Ser70) to transactivate p21 promoter expression. In conclusion, a high concentration of ATO can sustain ERK1/2 activation to enhance c-Fos expression, then dimerize with dephosphorylated c-Jun (Ser63/73) and recruit p300/CBP to the Sp1 sites (−84/−64) to activate p21 gene expression in A431 cells. [Copyright &y& Elsevier]

The effect of photodynamic treatment on the activity of regulatory glycolytic enzymes hexokinase (EC 2.7.1.1) and pyruvate kinase (EC 2.7.1.40) has been investigated. The experiments were carried out on human epidermoid carcinoma A431 cells photosensitised with either safranin O (Safr) or mTHPC. An increased concentration of ATP in the treated cells has been observed. The Safr-mediated treatment at CD80 and mTHPC-mediated treatment at CD50 induced an immediate sustained significant reduction of hexokinase and pyruvate kinase activities. No changes were found in the activities of both enzymes when cells were treated with Safr at CD50. The activities of HK and PK were reduced by PDT under conditions involving damage to mitochondrial outer membrane. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the differentiation-promoting effects of terbinafine (Lamisil®, TB) were investigated in human epithelioid squamous carcinoma (A431) cells. The polyhydroxyethylmethacrylate (poly-HEMA)- and type-I collagen-coated culture plate models were adapted to harvest the TB-induced differentiated cells by agitation of the suspension medium. We demonstrated that p27/Kip1, p21/Cip1 and the keratinocyte differentiation marker, human involucrin (hINV), were induced (>25μM) in TB-induced differentiated A431 cells. Animal studies demonstrated that administration of TB (10mg/kg body weight) inhibited A431-xenografted tumor growth through differentiation processes as evidenced by expression of pancytokeratin in tumor tissues. Immunocytochemical staining analysis showed that p27/Kip1, but not p21/Cip1, positive-stained cells were detected in the early-differentiated cells of TB-treated tumor tissues. SP1, which regulates p27/Kip1 expression, was induced by TB (>10μM) in A431 cells. The TB-induced promoter activity and protein expression levels of p27/Kip1 were significantly attenuated by pretreatment with mithramycin A, a SP1 specific inhibitor. We also demonstrated that TB-induced differentiated A431 cells sorted from the poly-HEMA-coated culture plates were arrested in the G1 phase. TB-induced G1 arrest in the suspension-cultured cells was attenuated by mithramycin A pretreatment. Such results suggest that SP1 plays a critical role in the p27/Kip1 gene transcriptional activation that may be subsequently involved in the TB-induced A431 cancer cell differentiation process. [Copyright &y& Elsevier]

Abstract: EGF in high concentrations has a growth-inhibitory effect on human epidermoid carcinoma cells A431. The transcription factor STAT1 is the most probable candidate for mediating this effect. In the present study, we demonstrated a strong reduction of the expression level of STAT1 in EGF-resistant sub-clones of A431 cells. EGF resistance was reversed by introducing wild-type STAT1, but not its Y701F mutant. Moreover, blocking the activity of Src family kinases reduced tyrosine phosphorylation of STAT1 and STAT3 and protected A431 cells from the EGF-induced growth inhibition. To further elucidate roles of STATs in A431 cell growth and survival, clones of A431 cells expressing short hairpin RNA (shRNA) against STAT1 or STAT3 were generated. Neither STAT1 nor STAT3 knockdown exerted any effect on growth rate or apoptotic death of A431 cells in the absence of EGF. However, upon EGF treatment A431 cells with knocked down STAT1 continued to grow and demonstrated a significantly lower level of apoptosis as compared to A431 cells. The knockdown of STAT3 did not alter cell growth or apoptosis. Taken together, our experiments prove the essential role of tyrosine phosphorylated STAT1, but not of STAT3, in EGF-induced apoptosis in A431 cells. [Copyright &y& Elsevier]

Abstract: The general protein kinase inhibitor staurosporine (STS) has dual effects on human epidermoid cancer cells (A431) and normal rat kidney fibroblasts (NRK). It almost immediately stimulated increased lamellipodial activity of both cell lines and after 2h induced typical signs of apoptosis, including cytoplasmic condensation, nuclear fragmentation, caspase-3 activation and DNA degradation. In the early phase we observed disruption of actin-containing stress fibres and accumulation of monomeric actin in the perinuclear region and cell nucleus. Increased lamellipodial-like extensions were observed particularly in A431 cells as demonstrated by co-localisation of actin and Arp2/3 complex, whereas NRK cells shrunk and exhibited numerous thin long extensions. These extensions exhibited uncoordinated centrifugal motile activity that appeared to tear the cells apart. Both cofilin and ADF were translocated from perinuclear regions to the cell cortex and, as expected in the presence of a kinase inhibitor, all the cofilin was dephosphorylated. Myosin II was absent from the extensions, and a reduction of phosphorylated myosin light chains was observed within the cytoplasm indicating myosin inactivation. Microtubules and intermediate filaments retained their characteristic filamentous organisation after STS exposure even when the cells became rounded and disorganised. Simultaneous treatment of NRK cells with STS and the caspase inhibitor zVAD did not inhibit the morphological and cytoskeletal changes. However, the cells underwent cell death as verified by positive annexin-V-staining. Thus it seems likely that cell death induced by STS may not only be a consequence of the activation of caspase, instead the disruption of the many motile processes involving the actin cytoskeleton may by itself suffice to induce caspase-independent cell death. [Copyright &y& Elsevier]

Abstract: In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules. [Copyright &y& Elsevier]

Abstract: Etoposide inhibits topoisomerase II and induces apoptosis in human epidermoid cancer cells (A431) and normal rat fibroblasts (NRK) as verified by apoptotic morphology and chromatin degradation. Here we examine changes in the localisation of actin, cofilin and the Arp2/3 complex during the apoptotic process in response to etoposide. Twenty-four hours after etoposide addition, a large number of cells of both lines exhibited nuclear and cytoplasmic fragmentation with the formation of numerous blebs typical of apoptosis. Etoposide exposure induces dissolution of stress fibres and an increase in actin and cofilin in membrane patches and apoptotic blebs. The actin is more peripherally located than the cofilin, similar to that reported for lamellipodia of highly motile keratocytes. By contrast, in control cells, cofilin is evenly distributed throughout the cytoplasm, though often enriched around the nucleus. The active form is inferred to be more peripherally localised and to be present in apoptotic blebs, since an antibody specific for phosphorylated cofilin did not stain the cell periphery nor apoptotic blebs. Although immunoblots of 2D gels demonstrate that the ratio of de-phosphorylated to phosphorylated cofilin does not change after etoposide treatment, this does not mean that there are no changes in the turnover of the active and inactive forms. Transfection of both cell lines with EGFP-containing constructs of wild-type cofilin and mutants resembling its activated (S3A) and inactivated (S3D) forms shows that the active form has a more peripheral localisation and is also present in the membrane blebs with a strong colocalisation with actin. We further show that Arp 2/3 also localises in apoptotic blebs and discuss the role of these proteins in apoptosis by analogy with actin-based protrusive motility in lamellipodia. [Copyright &y& Elsevier]

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor (EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2. [Copyright &y& Elsevier]

Purpose. To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. Methods. Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. Results. G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and ≈30 µg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4°C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. Conclusions. G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture. [ABSTRACT FROM AUTHOR]

Eicosanoids, which include prostaglandins, thromboxanes, and leukotrienes, are produced from arachidonic acid by three main pathways in cells, including cyclooxygenases and lipoxygenases, and cytochrome P450 enzymes. Accumulated evidence indicates that a certain peroxide tone is required for the initiation of reaction by lipoxygenases and cyclooxygenases. An endogenous inhibitor of arachidonate oxygenation was suspected in the cytosolic fraction of human epidermoid carcinoma A431 cells. After a series of studies, the existence of this inhibitor was confirmed, while it was purified and characterized. By amino acid sequence analysis, the inhibitor in A431 cells was subsequently identified as a phospholipid hydroperoxide glutathione peroxidase (PHGPx). Depletion of cellular glutathione in cells by diethyl maleate or by dibuthionine-sulfoximine results in an increase in enzyme activities of 12(S)-lipoxygenase and cyclooxygenase, suggesting that glutathione-depleting agents abolish the enzyme activity of PHGPx in cells. Stable transfectants of A431 cells with overexpression and depletion of PHGPx have been constructed, respectively. Reduction of arachidonate metabolism through 12(S)-lipoxygenase and cyclooxygenase 1 and that of the arsenite-induced generation of reactive oxygen species are observed in cells overexpressing PHGPx. On the other hand, enhancement of arachidonate metabolism and the arsenite-induced generation of reactive oxygen species is detected in PHGPx-depleted cells. In conclusion, the endogenous inhibitor of arachidonate metabolism present in A431 cells is a PHGPx, which plays a functional role in the down-regulation of arachidonate oxygenation catalyzed by 12(S)-lipoxygenase and cyclooxygenase 1 through the reduction of the level of intracellular lipid hydroperoxides. The latter acts as the peroxide tone for arachidonate metabolism in A431 cells.Copyright © 2003 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120 nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3—an antisense ODN complementary to an inaccessible site in EGFR mRNA—were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology. [Copyright &y& Elsevier]

Polymeric nanoparticles composed of polystyrene (PS) as core and poly(methacrylic acid) (PMA) as corona were prepared by the dispersion copolymerization. The potential of the nanoparticles as carriers for recombinant human epidermal growth factor (EGF) was investigated. The nanoparticles showed monodispersity and good water-dispersibility. The loading content of EGF to the nanoparticles was very high due to electrostatic interaction between EGF and nanoparticles. EGF was released as a pseudo-zero order pattern after initial burst effect. The nanoparticles were sufficient for A431 cells proliferation. [ABSTRACT FROM AUTHOR]

We have developed a time-resolved fluorescent assay using Wallac’s DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein–tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells. [Copyright &y& Elsevier]

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E[sub 2] significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5′o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels ( Icrac). These conductive properties include extremely low conductance (≈1 pS), very high selectivity for Ca2+ over K+ ( PCa/ PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane. [ABSTRACT FROM AUTHOR]

This paper presents a new method to evaluate the biological significance of glycosphingolipids (GSLs) using a GSL-spedfic enzyme, endoglycoceramidase (EGCase), by which GSL-sugar chains are removed from the cell surface of living cells. In this report, the effects of EGCase on epidermal growth factor (EGF)-dependent tyrosine-specific EGF receptor (EGFR) phosphorylation of A431 cells are described. After treatment of A431 cells with EGCase II (20 mU/ml) in the presence of the activator for 12 h, all acidic GSLs tested were reduced to ∼70% of control, but no hydrolysis occurred on Galα1,4Galcβ1,4Glcβ1,1Cer (Gb3Cer) and GalNAcpβ1 Galα1,4Galβ1,4Glcβ1,1Cer (Gb4Cer). In plasma membrane fractions of A431 cells, the reduction of gangliosides by EGCase II was found to be much faster than that of intact cells and reached 54.8% reduction of total gangliosides after 2 h incubation with the enzyme. EGF-dependent phosphorylation of EGFR of A431 cells was inhibited by the reduction of cell surface GSLs by EGCase when either intact A431 cells or their plasma membrane fractions were used, while EGF binding to their receptors was not changed. Neither hydrolysis of cell surface GSLs nor reduction of EGFR phosphorylation occurred when A431 cells were incubated with the activator or EGCase alone. The exogenous addition of ceramides or sphingosines, and treatment of cell membranes with sphingomyelinase, had no effect on the EGFR phosphorylation of purified membrane fractions, while the inhibitory effect of EGCase II on EGFR phosphorylation was restored by the addition of GM3-sugar chains, but not by lactose or sialic acid. The data presented in this paper indicate that the removal of sugar chains of GSLs, but not the accumulation of ceramides, reduces EGFdependent EGFR phosphorylation of A431 cells, suggesting that endogenous GSLs, possibly GM3, may be one of the integral constituents for supporting (or stimulating) the EGFR phosphorylation of A431 cells in [ABSTRACT FROM PUBLISHER]

The A431 cell line is composed of malignant keratinocytes derived from a vulval epidermoïd carcinoma. These cells have the peculiarity to stop their proliferation when they are treated with physiological concentrations of EGF, which is a mitogen for normal keratinocytes. We reported earlier that EGF induces involucrin accumulation in A431 cells and proposed that the arrest of proliferation triggers differentiation as shown by the induction of this cornified envelope precursor protein. To test this hypothesis, we compared the A431 subclone 15, which is not growth arrested by EGF-treatment, to the parental A431 cells. We found indeed that EGF reduces the involucrin content of clone 15 cells in a dose dependent manner. These opposite effects of EGF on the expression of terminal differentiation marker involucrin in A431 and A431 clone 15 keratinocytes were observed in defined medium as well as in presence of fetal calf serum. Nevertheless, when growth of parental A431 cells was inhibited by treatment with TGF-β or simply when cultures reached confluency, no involucrin accumulation was observed. Therefore growth arrest per se is not directly correlated with the induction of differentiation. [ABSTRACT FROM AUTHOR]

A431 cells grew at similar rates in protein-free Coon's modified Ham's F12 medium (PF-C-F12) with and without added bovine calf serum. The cells secreted a heparin-binding growth factor and a type-β transforming growth factor, but their growth in PF-C-F12 was not affected by these factors, or by DNA synthesis factor from Rhodamine fibrosarcoma, basic fibroblast growth factor, insulin, human transferrin, bovine serum albumin, and their combinations. Growth of A431 cells in PF-C-F12 was not density dependent and was not affected by either addition of conditioned medium or replacement of conditioned medium by fresh medium. These results indicate that A431 cells have an intracellular mechanism for autonomous growth, and that their growth is not affected by factors that they secrete or by exogenous growth factors. [ABSTRACT FROM AUTHOR]

To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. A radionuclide receptor displacement assay demonstrated the bioactivity of these derivatives. Aggregation of the epidermal growth factor receptor was measured by determining the increase in fluorescence energy transfer between receptorbound fluorescein and tetramethylrhodamine-labeled epidermal growth factor. Energy transfer between receptor-bound fluorescent derivatives was reversibly greater at 37 than 4°C, indicating temperature-dependent aggregation of the receptor. The extracellular protrusion of the epidermal growth factor receptor was calculated from the magnitude of energy transfer between receptorbound fluorescein labeled epidermal growth factor and 5-(N-dodecanoylamino)-eosin partitioned into the lipid membrane at 4 and 37°C. No significant change in the distance of closest approach between the N-terminus of epidermal growth factor and the plasma membrane was observed at 4°C (69±2 Å) and 37°C (67±2 Å). Thus, the extracellular protrusion of the occupied epidermal growth factor receptor did not change detectably upon receptor aggregation. [ABSTRACT FROM AUTHOR]