Human Epidermoid Carcinoma Cell Line Research Articles

Isolation and cytotoxic activity of some secoiridoids from Gentiana olivieri

The genus Gentiana (Gentianaceae) is represented by 12 species in Turkish Flora with important biological effects such as antipyretic, stomachic, antidepressant, anticonvulsant, hepatoprotective and antidiabetic activities. Iridoids, secoiridoids, C-glycosylflavones and xanthones are considered to be the most promising groups of compounds responsible for the pharmacological activities of the Gentiana species. In this study swertiamarin, sweroside and gentiopicroside were isolated from Gentiana olivieri and their cytotoxic activities were evaluated against HEP-2 (human larynx epidermoid carcinoma) cell lines based on MTT assay for growth inhibition.

Mitochondrial Pathway of α-Tocopheryl Succinate-Induced Apoptosis in Human Epidermoid Carcinoma A431 Cells

Vitamin E derivatives are known to act as agents exhibiting cytotoxity against tumor cells. The effect of vitamin E succinate on human epidermoid carcinoma cell line A431 was investigated in this study using live imaging, immunocytochemistry, and transmission electron microscopy. α-Tocopheryl succinate-induced apoptotic cell death in A431 cells was shown to be both dose- and time-dependent. The hyperproduction of reactive oxygen species, changes in size, shape and ultrastructural characteristics of mitochondria followed by the release of cytochromecfrom mitochondria to cytosol were observed. These results suggest that α-tocopheryl succinate induces apoptosis that occurs via the mitochondrial pathway. Mitochondria are shown to be crucial targets in α-tocopheryl succinate-induced caspase-dependent cell death in human carcinoma A431 cells.

Indications for an alternative effective treatment of head and neck squamous cell carcinoma with temsirolimus plus bevacizumab

Head and neck squamous cell carcinoma (HNSCC) is a group of tumors known to be sensitive to chemotherapy and radiotherapy in patients who are treatment naive. However, when recurrences do occur, these tumors generally become resistant and objective responses to therapy at that point tend to be less effective. There has been an increasing interest in developing novel molecular-targeted agents that specifically modulate growth factor and signaling pathways that are unregulated in HNSCC tumor cells. Combinations of vascular endothelial growth factor and mammalian target of rapamycin inhibitors have been used in some types of neoplasms, but no such efforts have been made in HNSCC. In this study, we investigated the in vitro, in vivo, and clinical effects of the temsirolimus (mammalian target of rapamycin inhibitor, Tem) and bevacizumab (antivascular endothelial growth factor antibody, Bev) combination. In-vitro studies were carried out on the A431 human squamous epidermoid carcinoma cell line and in-vivo studies were carried out on A431 tumor cells implanted on female Nu/Nu*nuBR (athymic nude) mice. Also, the effectiveness of the Tem and Bev combination was tested clinically in two separate clinical cases of chemoresistant HNSCC. The in-vitro, in-vivo, and clinical results showed that this combination can be significantly effective. In conclusion, we discuss the theoretical basis of the molecular pharmacological interactions between Bev and Tem that could explain these good results. If the therapeutic index is ultimately well determined, the antitumor effect of Bev and Tem is very likely to yield fruitful results.

Efficient DNA cleavage mediated by mononuclear mixed ligand copper(II) phenolate complexes: The role of co-ligand planarity on DNA binding and cleavage and anticancer activity

The new mononuclear copper(II) complexes [Cu(L)(H2O)2]+1 and [Cu(L)(diimine)]+2–6, where LH=2-[(2-dimethylaminoethylimino)methyl]phenol and diimine=2,2′-bipyridine (bpy) (2), or 1,10-phenanthroline (phen) (3), or dipyrido[3,2-f:2′,3′-h]quinoxaline (dpq) (4) or dipyrido[3,2-a:2′,3′-c]phenazine (dppz) (5) or 11,12-dimethyldipyrido[3,2-a:2′,3′-c]phenazine (dmdppz) (6), have been isolated and characterized. The X-ray crystal structures of 2 contains the monomeric complex molecule with a trigonal bipyramidal distorted square pyramidal (TBPDSP) coordination geometry, while 4 and 6 with square pyramidal distorted trigonal bipyramidal (SPDTBP) coordination geometry. The amine nitrogen of −NMe2 group of the tridentate primary ligand is located at one of the corners of the square plane in 2 and 6 but in the axial position in 4. The interaction of the complexes with calf thymus DNA has been investigated using UV-visible and fluorescence spectroscopy, and viscosity measurements to understand the effect of diimine co-ligands on the mode and extent of DNA binding. The complexes 4 and 5 interact with calf thymus DNA more strongly than the other complexes through partial intercalation of the extended planar ring of the dpq (4) and dppz (5) co-ligands with the DNA base stack. All the complexes, except 1, effect the double strand DNA cleavage of plasmid DNA and 5 cleaves plasmid DNA in the absence of a reductant at a concentration (40μM) lower than 4. It is remarkable that all the complexes display cytotoxicity against human breast cancer cell lines (MCF-7) and human cervical epidermoid carcinoma cell lines (ME 180) with potency higher than the currently used chemotherapeutic agent cisplatin and that 5 exhibits cytotoxicity higher than the other complexes.

Abstract 2436: [18F]FDG accumulation in tumor microenvironment -Microautoradiographic study combined with immunohistochemistry in mice-

Abstract [Purpose] Tumor microenvironment is characterized by the conditions including hypoxia, low nutrition, stromal cell infiltration, and acidosis due to incomplete blood vessel network. Tumor microenvironment has been recognized as a major factor influencing not only the response to anti-cancer therapies but also malignant progression and metastasis. Non-invasive imaging of such a microenvironment by positron emission tomography (PET) will provide more therapeutic merits. Accumulation of FDG, 2-deoxy-2-[F-18]fluoro-D-glucose, a most-often used PET probe, in tumor has been reported to correlate with the metastasis and malignancy. However, the FDG uptake profile in the microenvironment has remained unknown. In this study, we simultaneously visualized the intratumoral FDG distribution and the microenvironment by using microautoradiography in microscopic spatial resolution combined with immunohistochemistry. [Methods] Human epidermoid carcinoma cell line A431 was transplanted into the back of female SCID mice, and FDG was injected into the mice 2 weeks after the transplantation. The tumors were removed from mice 45 minutes after the FDG administration and the frozen sections were obtained. Overall image of the intratumoral FDG distribution was obtained by conventional autoradiography. FDG distribution and microenvironment in microscopic levels were visualized by microautoradiography followed by immunohistochemical detection of hypoxic condition and cell-specific markers. [Result & Conclusion] Conventional autoradiography and microautoradiography showed the similar pattern in FDG distribution in the tumor. Furthermore, microautoradiographic study with immunohistochemistry revealed that grains at high-density were observed in pimonidazol (hypoxia marker)-accumulated areas showing infiltration of many S100A4-immunopositive fibroblasts. These results suggested that FDG accumulation was prominent in stromal cells involved in hypoxic condition in tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2436. doi:1538-7445.AM2012-2436

Efficient Nonviral Gene Therapy Using Folate-Targeted Chitosan-DNA Nanoparticles In Vitro

Nonviral cationic polymers like chitosan can be combined with DNA to protect it from degradation. The chitosan is a biocompatible, biodegradable, nontoxic, and cheap polycationic polymer with low immunogenicity. The objective of this study was to synthesize and then assess different chitosan-DNA nanoparticles and to select the best ones for selective in vitro transfection in human epidermoid carcinoma (KB) cell lines. It revealed that different combinations of molecular weight, the presence or absence of folic acid ligand, and different plasmid DNA sizes can lead to nanoparticles with various diameters and diverse transfection efficiencies. The intracellular trafficking, nuclear uptake, and localization are also studied by confocal microscopy, which confirmed that DNA was delivered to cell nuclei to be expressed.

Antiviral Furanosesquiterpenes from Commiphora erythraea

The crude methanolic extract obtained from C. erythraea resin was chromatographed on silica gel with solvent of increasing polarity. The extract and fractions were evaluated for cytotoxicity and antiviral activity [parainfluenza type 3 virus (PIV3)] by plaque forming units (PFU) reduction assay using HEp-2 cells (human larynx epidermoid carcinoma cell line). From the active fraction, five compounds were isolated and tested. Only two of these showed anti-PIV3 activity with a selectivity index (SI) of 66.6 and 17.5, respectively. Both the compounds are furanosesquiterpenoids.

Membrane processes application on the Symphytum officinale and Geranium robertianum extracts concentration to obtain high antioxidative activity compounds

The paper reports the successful application of membranes processes to obtain the good quality extracts with compounds of high antioxidative activity and therapeutic value. This study provided investigations on the phenolic compounds from two plant species used in Romanian ethno-medicine and evaluated the antioxidant and cytotoxic activities. Three extracts prepared from Geranium robertianum and Symphytum officinale were concentrated by microfiltration and ultrafiltration. The levels of phenolic compounds and flavonoids were determined by UV-Vis spectroscopy and HPLC. The freeradical scavenging activity of concentrated extracts was determined by DPPH method. The preliminary tests of cytotoxic activity for the concentrated extracts have been carried out on human epidermoid laryngeal carcinoma cell line (Hep-2p) and normal monkey kidneys cells (RM). The results showed that all concentrated extracts had a very low cytotoxicity against healthy cells, but a significant cytotoxic effect on Hep-2p tumor cells. The concentrated extracts have a high antioxidant activity (% DPPH inhibition > 80%).

Evaluation of antioxidative, protective effect against H2O2 induced cytotoxicity, and cytotoxic activities of three different Quercus species

Quercus species are used as antidiarrheic, for the treatment of hemorrhoid, oral and anal mucosa inflammation. These tree species have been of interest to researchers because of their usage in folk medicine, consumption as food, beverage and especially usage of oak woods for construction in wine barrels. The DPPH, SO and NO radical scavenging activities, protective effect against H2O2 induced cytotoxicity as well as their cytotoxic activity against Hep-2 human larynx epidermoid carcinoma cell line of the MeOH and water extracts of the barks of Quercus cerris var. cerris, Quercusmacranthera subsp. syspirensis and Quercus aucheri were investigated for the first time. Total phenolic content of the extracts was also evaluated by Folin–Ciocalteu method. Results demonstrated that the extracts showed strong radical scavenging activity comparable to those of standard compounds. Extracts also showed good protective effect against H2O2 induced cytotoxicity on human erythrocytes comparing to ascorbic acid. On the other hand, while each extract showed dose dependent cytotoxic activity, MeOH extract of Q.macranthera subsp. syspirensis showed the strongest cytotoxicity against the tested cell line. Taken together, the results showed that Quercus species may be a promising alternative to synthetic substances as natural compound with high antioxidant and antiproliferative activities.

Antibacterial activity, cytotoxicity and chemical constituents of Hydnora johannis roots

In Sudan, the roots of Hydnora johannis (Hydnoraceae) are traditionally used for the treatment of dysentery, diarrhea, cholera and swelling tonsillitis. The ethnomedicinal value of H. johannis was investigated through phytochemical study, in vitro antibacterial activity and preliminary cytotoxic tests. Determination of total phenols, flavonoids and proanthocyanidins was carried out using spectrometric methods. The antibacterial activity of the water and ethanolic extracts was determined using the microdilution method. Pure compounds were isolated from the ethyl acetate extract by chromatographic methods and their structures were established by spectroscopic methods. Cytotoxicity assay was performed against selected human mouth epidermoid carcinoma cell line (KB), and non-cancer human fetal lung cell line (MRC-5). Both water and ethanol (70%) extracts were found to contain the same amount of total phenols and proanthocyanidins, whereas the level of flavonoids was higher in the ethanol extract. The water extract was found to possess antibacterial activity against Enterococcus faecalis (MIC value of 16 μg/mL), Bacillus subtilis, B. cereus and Staphylococcus aureus (MIC values of 64 μg/mL) but not against bacteria mainly responsible for diarrhea. This leads to the suggestion that, the mode of action of water extract which is rich in tannins was not connected to their inhibition to the diarrhea bacteria but to their action on the digestive tract. Reduction in potency of the water and ethanol (70%) extracts when fractionated was observed. The ethyl acetate fraction obtained from fractionation of ethanol extract possessed only activity against the two strains of S. aureus with MIC values of 128 μg/mL. In addition, six compounds were isolated from the ethyl acetate extract as cirsiliol (3′,4′,5-trihydroxy-6-7-dimethoxy flavone) ( 1), trans 3′5-dihydroxy-4′7-dimethoxydihydroflavonol ( 2), oleic acid ( 3), vanillin (4-hydroxy-3-methoxybenzaldehyde) ( 4), protocatechuic acid (3,4 dihydroxy benzoic acid) ( 5) and dl catechin (trans (+) 2-(3,4-dihydroxyphenyl-3,4-dihydro-2H-1-benzopyran-3,5,7-triol)) ( 6). Four compounds from the ethyl acetate extract were also identified by GCMS as stigmasterol ( 7), oleic acid ( 3), myristic acid ( 8), and palmitic acid ( 9). Little cytotoxicity is reported against the cell lines used. Thus, the safety of this plant in the traditional medicine should be verified by much further testing, including in vivo experiments and clinical studies.

Nuclear localization of maspin is essential for its inhibition of tumor growth and metastasis

Maspin (mammary serine protease inhibitor or SerpinB5) acts as a tumor suppressor when overexpressed in aggressive cancer cell lines. However, its role in human cancer is controversial. Maspin expression has been associated with a poor prognosis in some studies, whereas in others, with favorable outcome. The clinical data suggest, however, that nuclear-localized maspin is associated with improved survival. We hypothesized that the tumor suppressor activity of maspin may require nuclear localization, and that the discordance between clinical and experimental reports is a consequence of the variable subcellular distribution of maspin. Furthermore, we surmized that nuclear maspin could function as a tumor suppressor through the regulation of genes involved in tumor growth and invasion. Maspin or maspin fused to a nuclear export signal were expressed in metastatic human breast and epidermoid carcinoma cell lines. We found that pan-cellular localized maspin inhibited in vivo tumor growth and metastasis when assessed in xenograft chicken embryo and murine mammary fat pad injection models. However, when maspin was excluded from the nucleus via a nuclear exclusion signal, it no longer functioned as a metastasis suppressor. Using chromatin immunoprecipitation, we show that nuclear maspin was enriched at the promoter of colony-stimulating factor-1 (CSF-1) and associated with diminished levels of CSF-1 mRNA. Our findings demonstrate that the nuclear localization of maspin is required for its tumor and metastasis suppressor functions in vivo, and suggest that its mechanism of action involves, in part, direct association of maspin with target genes.

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Ursolic acid derivatives from Bangladeshi medicinal plant, Saurauja roxburghii: Isolation and cytotoxic activity against A431 and C6 glioma cell lines

Ursene-type pentacyclic triterpenes, including the ursolic acid, corosolic acid, and a new ursene-type pentacyclic triterpene, 7,24-dihydroxy ursolic acid, were isolated from the methanolic extract of the leaves of the Bangladeshi medicinal plant, Saurauja roxburghii, a higher plant indigenous to South East Asia and some part of North America. They were tested for the cytotoxicity against C6 rat glioma and A431 human epidermoid carcinoma cell lines. Although they have the same ursene-type pentacyclic triterpene core structure, the position and numbers of hydroxyl groups on the terpene structure significantly affected the activity and the selectivity to the cell lines.

Abstract 2367: RhoA GTPase but not cortactin is required for tumor cell migration of human epidermoid carcinoma cells in vivo

Abstract Metastasis contributes to over 90% of cancer mortalities. Metastatic disease is a dynamic, multi-step process that requires invasion and migration of cancer cells from the primary tumour site and leads to colonization at a distant location. Migration events mediated by the family of Rho GTPases are facilitated by acto-myosin contraction at the cell rear. In vitro studies have demonstrated that knockdown of RhoA causes insufficient actomyosin contraction and decreased cell detachment. Migration of cancer cells in vivo does not only require detachment from the primary tumor, but also relies on invasion of the cell into the surrounding stromal tissue. Formation of functional invadopodia drives invasion and a key component of invadopodia is cortactin. The objective of this study is to combine inhibitory short hairpin RNA (shRNA) technology with an in vivo metastasis model to identify mechanisms of cancer cell migration that have not previously been studied in an animal model. I hypothesize that inhibition of mediators required for cellular rear detachment and invadopodia formation, such as RhoA and cortactin respectively, will prevent migration of human fibrosarcoma (HT1080) and human epidermoid carcinoma (Hep3) cell lines in vivo. Inhibition of RhoA and cortactin was performed by lentiviral infection of Hep3 and HT1080 cell lines with individual pLOK.1-puro vectors containing a 21-mir sequence to target and decrease mRNA expression. The migratory ability of both cell lines following RNA inhibition was analyzed in vitro using a scratch wound assay and in vivo using a migration and metastasis model in the shell-less chicken embryo, whereby transduced HT1080 and Hep3 cells are injected intravenously and subsequently arrest in the chorioallantoic membrane (CAM). We used a microscope mounted incubator and spinning disc confocal microscopy to visualize cells in real time and quantify their migration parameters. Results obtained from in vitro migration assays indicate that inhibition of cortactin does not affect the migration of Hep3 and HT1080 cell lines across a rigid substratum. Diminished levels of cortactin also had no significant effect on the migration of these cells in vivo. Importantly, inhibition of RhoA caused a significant decrease in cell migration in vitro for both cell lines and in vivo for Hep3 cells (p<0.05). This data suggests that RhoA, a key mediator of cellular rear detachment, is required for in vivo migration of human epidermoid carcinoma cells while cortactin, implicated in invadopodia structures, is not required. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2011-2367

Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells

Divalent lead cations (Pb 2+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb 2+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A 2 as well as the hormone-like lipid compound prostaglandin E 2. To further clarify the mechanism by which Pb 2+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 μM) of Pb 2+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb 2+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb2 + ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway.

Induction of Apoptosis in Lung Cancer Cells by TRAIL and L-leucyl-L-leucine Methyl Ester

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that induces apoptosis in many tumor cells. Previous studies suggested that TRAIL treatment might also cause release of lysosomal cathepsin proteases to the cytosol, thus further promoting apoptosis. L-leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosome-destabilizing agent that may cause release of cathepsins into the cytosol and ensuing apoptosis. We hypothesized that a combination of TRAIL and LeuLeuOMe may synergistically promote apoptosis in lung cancer cells. The human epidermoid lung carcinoma cell line Calu-1 (TRAIL-resistant) and human large cell lung carcinoma cell line NCI-H460 (TRAIL-sensitive) were assayed for sensitivity to TRAIL and LeuLeuOMe, given alone or in different combination doses. Each agent alone induced a dose-dependent cytotoxicity, with substantially different efficacies of the two agents for the two cell types. When both agents were combined, synergistic cytotoxicity was achieved even in the TRAIL-resistant cells. TRAIL-induced cytotoxicity was completely inhibited by pan-caspase inhibitor z-VAD-fmk, but not by cysteine protease inhibitor E-64d. Conversely, E-64d totally blocked LeuLeuOMe-induced cytotoxicity. TRAIL caused mitochondrial damage, while enlarged lysosomes and lysosomal rupture were observed in LeuLeuOMe-treated cells. Our data suggest that, while TRAIL and LeuLeuOMe cause apoptosis through pathways that differ in their involvement of lysosomal cysteine proteases, mitochondrial and lysosomal desta-bilization have converging pro-apoptotic effects. Thus, the synergy of TRAIL and LeuLeuOMe may be used therapeutically to promote apoptosis in lung cancers, even those with intrinsic or acquired resistance to TRAIL.

Fischerisin A and B, Cytotoxic Sesquiterpenoid-Geranylhydroquinones from Ligularia fischeri

During the course of screening natural sesquiterpenoids for new antitumor agents, two novel compounds, fischerisin A (1) and fischerisin B (2), were isolated from the roots of Ligularia fischeri. Their structures were elucidated by interpretation of their IR, high resolution-mass spectrometry (HR-MS), 1D- and 2D-NMR data. Fischerisin A and B are the first representatives of a novel sesquiterpenoid-geranylhydroquinone hybrid, and both compounds exhibited in vitro cytotoxicity against cultured human oral epidermoid carcinoma (KB) and human breast cancer (MCF-7) cell lines with IC(50) values of 9.7 and 10.2 µM, and 9.8 and 17.8 µM, respectively.

Synthesis and preliminary evaluation of curcumin analogues as cytotoxic agents

A series of curcumin analogues with different substituents at the 4-position of the phenyl group were synthesized and screened for in vitro cytotoxicity against a panel of human cancer cell lines. Several novel curcumin analogues, especially 32 and 34, exhibited selective and potent cytotoxic activity against human epidermoid carcinoma cell line A-431 and human glioblastoma cell line U-251, implying their specific potential in the chemoprevention and chemotherapy of skin cancer and glioma. The preliminary SAR information extracted from the results suggested that introduction of appropriate substituents to the 4'-positions could be a promising approach for the development of new cytotoxic curcumin analogues with special selectivity for A-431 and U-251 cell lines.

Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis

Essential oils are complex mixtures of odorous and volatile compounds derived from secondary plant metabolism. They can be isolated from many plants by mechanical pressing or hydro- and steam-distillation and are known to induce a wide range of biological effects through their antibacterial, antifungal, cytotoxic, antioxidant and antimutagenic activities. In order to explore their beneficial properties on human skin cells, we investigated the effects of an essential oil from rosewood Aniba rosaeodora (REO) on the human epidermoid carcinoma cell line A431, on immortal HaCaT cells thought to represent an early stage of skin carcinogenesis, on transformed normal HEK001 keratinocytes and on primary normal NHEK keratinocytes. In a defined range of concentrations, REO selectively killed A431 and HaCaT cells. The same treatments had only a minor cytotoxic effect on HEK001 and NHEK cells. Preferentially in A431 and HaCaT cells, REO triggered the production of reactive oxygen species, induced depolarization of the mitochondrial membrane and caused caspase-dependent cell death characterized by phosphatidylserine externalization, an early marker of apoptosis. Both intrinsic and extrinsic apoptotic pathways were implicated in REO-induced cell death. The identification of selective induction of apoptosis in precancerous and cancerous skin cells by REO highlights the potential anticancer activity of this essential oil.

Multiplexed Evaluation of a Cell-Based Assay for the Detection of Antidrug Neutralizing Antibodies to Panitumumab in Human Serum Using Automated Fluorescent Microscopy

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay's sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 microg/mL for the positive control antibody.