Synthesis and biological activity of chloroethyl pyrimidine nucleosides.

PubMed

Colombeau, Ludovic; Teste, Karine; Hadj-Bouazza, Amel; Chaleix, Vincent; Zerrouki, Rachida; Kraemer, Michel; Catherine, Odile Sainte

2008-02-01

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

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2012-09-01

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surrogate endpoints for overall survival in advanced non-small-cell lung cancer patients with mutations of the epidermal growth factor receptor gene.

PubMed

Yoshino, Reiko; Imai, Hisao; Mori, Keita; Takei, Kousuke; Tomizawa, Mai; Kaira, Kyoichi; Yoshii, Akihiro; Tomizawa, Yoshio; Saito, Ryusei; Yamada, Masanobu

2014-09-01

Subsequent therapies confound the ability to discern the effect of first-line chemotherapy on overall survival (OS). We investigated whether progression-free survival (PFS), post-progression survival (PPS) and tumor response were valid surrogate endpoints for OS following first-line chemotherapy in individual patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epidermal growth factor receptor gene mutations. We retrospectively analyzed 35 patients with advanced NSCLC treated with first-line gefitinib. The associations of PFS, PPS and tumor response with OS were analyzed. PPS was found to be strongly correlated with OS, unlike PFS and tumor shrinkage. The factors significantly associated with PPS were performance status (PS) after first-line treatment, best response to second-line treatment and number of regimens used after disease progression. PPS may be a surrogate for OS in this patient population and further therapy after disease progression following first-line chemotherapy may significantly affect OS. However, a larger study is required to validate these results.

Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, R.; Sanford, K.K.; Parshad, R.

1987-03-01

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containingmore » the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.« less

Adhesion molecules affected by treatment of lung cancer cells with epidermal growth factor.

PubMed

Fonseca, Fernando L A; Azzalis, Ligia A; Feder, David; Nogoceke, Everson; Junqueira, Virginia B C; Valenti, Vitor E; de Abreu, Luiz Carlos

2011-10-01

Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, β-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

Duox, Flotillin-2, and Src42A Are Required to Activate or Delimit the Spread of the Transcriptional Response to Epidermal Wounds in Drosophila

PubMed Central

Juarez, Michelle T.; Patterson, Rachel A.; Sandoval-Guillen, Efren; McGinnis, William

2011-01-01

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. We have developed a variety of fluorescent reporters that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. We also find that four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-ß-cyclodextrin, and hydrogen peroxide) are sufficient to globally activate epidermal wound response genes in Drosophila embryos. We explore the epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals. Our results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration. PMID:22242003

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

PubMed

Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

2016-02-01

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor 2-positive breast cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis and biological activity of mustard derivatives of thymine.

PubMed

Hadj-Bouazza, Amel; Teste, Karine; Colombeau, Ludovic; Chaleix, Vincent; Zerrouki, Rachida; Kraemer, Michel; Sainte Catherine, Odile

2008-05-01

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

PW1 gene/paternally expressed gene 3 (PW1/Peg3) identifies multiple adult stem and progenitor cell populations

PubMed Central

Besson, Vanessa; Smeriglio, Piera; Wegener, Amélie; Relaix, Frédéric; Nait Oumesmar, Brahim; Sassoon, David A.; Marazzi, Giovanna

2011-01-01

A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization. PMID:21709251

The xipotl Mutant of Arabidopsis Reveals a Critical Role for Phospholipid Metabolism in Root System Development and Epidermal Cell Integrity

PubMed Central

Cruz-Ramírez, Alfredo; López-Bucio, José; Ramírez-Pimentel, Gabriel; Zurita-Silva, Andrés; Sánchez-Calderon, Lenin; Ramírez-Chávez, Enrique; González-Ortega, Emmanuel; Herrera-Estrella, Luis

2004-01-01

Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity. PMID:15295103

A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

PubMed Central

Hammiller, Brianna O.; El-Abaseri, Taghrid Bahig; Dlugosz, Andrzej A.; Hansen, Laura A.

2015-01-01

Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation. PMID:26284198

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin.

PubMed

Hatt, P J; Moriniere, M; Oberlander, H; Porcheron, P

1994-10-01

During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.

Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

PubMed Central

Bermudez, Yira; Benavente, Claudia A.; Meyer, Ralph G.; Coyle, W. Russell; Jacobson, Myron K.; Jacobson, Elaine L.

2011-01-01

Background Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through Gi-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Results Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional Gi-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. Conclusions The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis. PMID:21655214

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

PubMed Central

Tomshine, Jin C.; Severson, Sandra R.; Wigle, Dennis A.; Sun, Zhifu; Beleford, Daniah A. T.; Shridhar, Vijayalakshmi; Horazdovsky, Bruce F.

2009-01-01

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature. PMID:19570984

The availability of filament ends modulates actin stochastic dynamics in live plant cells

PubMed Central

Li, Jiejie; Staiger, Benjamin H.; Henty-Ridilla, Jessica L.; Abu-Abied, Mohamad; Sadot, Einat; Blanchoin, Laurent; Staiger, Christopher J.

2014-01-01

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament–filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls. PMID:24523291

Pigment cell mechanism of postembryonic stripe pattern formation in the Japanese four-lined snake.

PubMed

Murakami, Arata; Hasegawa, Masami; Kuriyama, Takeo

2016-02-01

Postembryonic changes in the dermal and epidermal pigment cell architecture of the striped and nonstriped morph of the Japanese four-lined snake Elaphe quadrivirgata were examined to reveal stripe pattern formation after hatching. The striped and nonstriped morphs were distinguishable at the hatching, suggesting that the basis of stripe pattern was formed during embryonic development. In the striped morph, the color of stripes changed from red-brown in juveniles to vivid dark-brown in adults, and density of dermal melanophore increased much more in the stripe than background dorsal scales with growth. This increase in density of dermal melanophore was accompanied not only by the increased epidermal melanophore density but also by the change in vertical structures of dermal melanophore. By contrast, the density of epidermal and dermal melanophore evenly increased over the dorsal scales in the nonstriped morph. Thus, the increased vividness of the stripe pattern after hatching is achieved through localized increase of melanophore density particularly in the stripe region but not over the whole dorsal scales. © 2015 Wiley Periodicals, Inc.

Fractalkine receptor is expressed in mature ovarian teratomas and required for epidermal lineage differentiation

PubMed Central

2013-01-01

Background The goal of this study was to determine a predominant cell type expressing fractalkine receptor (CX3CR1) in mature ovarian teratomas and to establish functional significance of its expression in cell differentiation. Methods Specimens of ovarian teratoma and human fetal tissues were analyzed by immunohistochemistry for CX3CR1expression. Ovarian teratocarcinoma cell line PA-1 was used as a model for cell differentiation. Results We found that the majority of the specimens contained CX3CR1-positive cells of epidermal lineage. Skin keratinocytes in fetal tissues were also CX3CR1- positive. PA-1 cells with downregulated CX3CR1 failed to express a skin keratinocyte marker cytokeratin 14 when cultured on Matrigel in the presence of a morphogen, bone morphogenic protein 4 (BMP-4), as compared to those expressing scrambled shRNA. Conclusions Here we demonstrate that CX3CR1 is expressed in both normally (fetal skin) and abnormally (ovarian teratoma) differentiated keratinocytes and is required for cell differentiation into epidermal lineage. PMID:23958497

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer.

PubMed

Shields, Sarah; Conroy, Emer; O'Grady, Tony; McGoldrick, Alo; Connor, Kate; Ward, Mark P; Useckaite, Zivile; Dempsey, Eugene; Reilly, Rebecca; Fan, Yue; Chubb, Anthony; Matallanas, David Gomez; Kay, Elaine W; O'Connor, Darran; McCann, Amanda; Gallagher, William M; Coppinger, Judith A

2018-03-20

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

PubMed Central

Shields, Sarah; Conroy, Emer; O’Grady, Tony; McGoldrick, Alo; Connor, Kate; Ward, Mark P.; Useckaite, Zivile; Dempsey, Eugene; Reilly, Rebecca; Fan, Yue; Chubb, Anthony; Matallanas, David Gomez; Kay, Elaine W.; O’Connor, Darran; McCann, Amanda; Gallagher, William M.; Coppinger, Judith A.

2018-01-01

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC. PMID:29644001

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

PubMed Central

Yashiro, M.; Chung, Y. S.; Nishimura, S.; Inoue, T.; Sowa, M.

1995-01-01

Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression. Images Figure 1 Figure 5 Figure 6 Figure 7 Figure 10 PMID:7577468

Epidermal cyst mimicking incision line metastasis.

PubMed

Gündoğdu, Ramazan; Ayhan, Erhan; Çolak, Tahsin

2017-01-01

Epidermal cysts are cystic tumors lined with keratinized squamous layer and filled with keratin debris. Epidermal cysts may develop by implantation of surface epidermal layer into the dermis or subcutaneous tissue after trauma or surgical procedures. Cervix cancer spreads either directly or via the vascular and lymphatic systems. Distant skin metastasis of endometrium or cervix cancer is very rare. In this case report, a patient who had a history of cervix cancer operation 11 years ago and presented with a mass that mimicked incision line metastasis and was histopathologically diagnosed with epidermal cyst is presented.

Epidermal cyst mimicking incision line metastasis

PubMed Central

Gündoğdu, Ramazan; Ayhan, Erhan; Çolak, Tahsin

2017-01-01

Epidermal cysts are cystic tumors lined with keratinized squamous layer and filled with keratin debris. Epidermal cysts may develop by implantation of surface epidermal layer into the dermis or subcutaneous tissue after trauma or surgical procedures. Cervix cancer spreads either directly or via the vascular and lymphatic systems. Distant skin metastasis of endometrium or cervix cancer is very rare. In this case report, a patient who had a history of cervix cancer operation 11 years ago and presented with a mass that mimicked incision line metastasis and was histopathologically diagnosed with epidermal cyst is presented. PMID:28740968

BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinitt, C.A.M.; Wood, J.; Lee, S.S.

2010-08-01

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF)more » in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.« less

Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

PubMed Central

Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

1987-01-01

The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

Signalling in the epidermis: the E2F cell cycle regulatory pathway in epidermal morphogenesis, regeneration and transformation.

PubMed

Ivanova, Iordanka A; D'Souza, Sudhir J A; Dagnino, Lina

2005-01-01

The epidermis is the outermost layer in the skin, and it is the first line of defence against the environment. The epidermis also provides a barrier against loss of fluids and electrolytes, which is crucial for life. Essential in the maintenance of this tissue is its ability to continually self-renew and regenerate after injury. These two characteristics are critically dependent on the ability of the principal epidermal cell type, the keratinocyte, to proliferate and to respond to differentiation cues. Indeed, the epidermis is a multilayered tissue composed of keratinocyte stem cells and their differentiated progeny. Central for the control of cell proliferation is the E2F transcription factor regulatory network. This signaling network also includes cyclins, cdk, cdk inhibitors and the retinoblastoma (pRb) family of proteins. The biological importance of the E2F/pRb pathway is emphasized by the fact that a majority of human tumours exhibit alterations that disrupt the ability of pRb proteins to inhibit E2F, leading to permanent activation of the latter. Further, E2F is essential for normal epidermal regeneration after injury. Other member of the E2F signaling pathway are also involved in epidermal development and pathophysiology. Thus, whereas the pRb family of proteins is essential for epidermal morphogenesis, abnormal regulation of cyclins and E2F proteins results in tumorgenesis in this tissue. In this review, we discuss the role of each member of this important growth regulatory network in epidermal formation, homeostasis and carcinogenesis.

Signalling In The Epidermis: The E2f Cell Cycle Regulatory Pathway In Epidermal Morphogenesis, Regeneration And Transformation

PubMed Central

2005-01-01

The epidermis is the outermost layer in the skin, and it is the first line of defence against the environment. The epidermis also provides a barrier against loss of fluids and electrolytes, which is crucial for life. Essential in the maintenance of this tissue is its ability to continually self-renew and regenerate after injury. These two characteristics are critically dependent on the ability of the principal epidermal cell type, the keratinocyte, to proliferate and to respond to differentiation cues. Indeed, the epidermis is a multilayered tissue composed of keratinocyte stem cells and their differentiated progeny. Central for the control of cell proliferation is the E2F transcription factor regulatory network. This signaling network also includes cyclins, cdk, cdk inhibitors and the retinoblastoma (pRb) family of proteins. The biological importance of the E2F/pRb pathway is emphasized by the fact that a majority of human tumours exhibit alterations that disrupt the ability of pRb proteins to inhibit E2F, leading to permanent activation of the latter. Further, E2F is essential for normal epidermal regeneration after injury. Other member of the E2F signaling pathway are also involved in epidermal development and pathophysiology. Thus, whereas the pRb family of proteins is essential for epidermal morphogenesis, abnormal regulation of cyclins and E2F proteins results in tumorgenesis in this tissue. In this review, we discuss the role of each member of this important growth regulatory network in epidermal formation, homeostasis and carcinogenesis. PMID:15951853

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

PubMed

Wechsler, Erin Isaacson; Tugizov, Sharof; Herrera, Rossana; Da Costa, Maria; Palefsky, Joel M

2018-05-01

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

Impact of clinical parameters and systemic inflammatory status on epidermal growth factor receptor-mutant non-small cell lung cancer patients readministration with epidermal growth factor receptor tyrosine kinase inhibitors.

PubMed

Chen, Yu-Mu; Lai, Chien-Hao; Rau, Kun-Ming; Huang, Cheng-Hua; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Chung, Yu-Hsiu; Wang, Yi-Hsi; Su, Mao-Chang; Huang, Kuo-Tung; Liu, Shih-Feng; Chen, Hung-Chen; Chang, Ya-Chun; Chang, Yu-Ping; Wang, Chin-Chou; Lin, Meng-Chih

2016-11-08

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) readministration to lung cancer patients is common owing to the few options available. Impact of clinical factors on prognosis of EGFR-mutant non-small cell lung cancer (NSCLC) patients receiving EGFR-TKI readministration after first-line EGFR-TKI failure and a period of TKI holiday remains unclear. Through this retrospective study, we aimed to understand the impact of clinical factors in such patients. Of 1386 cases diagnosed between December 2010 and December 2013, 80 EGFR-mutant NSCLC patients who were readministered TKIs after failure of first-line TKIs and intercalated with at least one cycle of cytotoxic agent were included. We evaluated clinical factors that may influence prognosis of TKI readministration as well as systemic inflammatory status in terms of neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR). Baseline NLR and LMR were estimated at the beginning of TKI readministration and trends of NLR and LMR were change amount from patients receiving first-Line TKIs to TKIs readministration. Median survival time since TKI readministration was 7.0 months. In the univariable analysis, progression free survival (PFS) of first-line TKIs, baseline NLR and LMR, and trend of LMR were prognostic factors in patients receiving TKIs readministration. In the multivariate analysis, only PFS of first-line TKIs (p < 0.001), baseline NLR (p = 0.037), and trend of LMR (p = 0.004) were prognostic factors. Longer PFS of first-line TKIs, low baseline NLR, and high trend of LMR were good prognostic factors in EGFR-mutant NSCLC patients receiving TKI readministration.

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

PubMed

Barton, Kiah A; Wozny, Michael R; Mathur, Neeta; Jaipargas, Erica-Ashley; Mathur, Jaideep

2018-01-29

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 ( gl2 ) and immutans ( im ), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment. © 2018. Published by The Company of Biologists Ltd.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

PubMed Central

Wang, Lihong; Cao, Hailong; Lu, Ning; Liu, Liping; Wang, Bangmao; Hu, Tianhui; Israel, Dawn A.; Peek, Richard M.; Polk, D. Brent; Yan, Fang

2013-01-01

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells. PMID:23457600

Genistein enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nuclear factor kappa B in nonsmall cell lung cancer cell lines.

PubMed

Gadgeel, Shirish M; Ali, Shadan; Philip, Philip A; Wozniak, Antoinette; Sarkar, Fazlul H

2009-05-15

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown modest clinical benefit in patients with relapsed nonsmall cell lung cancer (NSCLC). Down-regulation of Akt appears to correlate with the antitumor activity of EGFR-TKIs. Akt activates nuclear factor kappa B (NF-kappaB), which transcribes genes important for cell survival, invasion, and metastasis. The authors hypothesized that genistein, through the inhibition of NF-kappaB, could enhance the activity of EGFR-TKIs in NSCLCs. Three NSCLC cell lines with various EGFR mutation status and sensitivities to EGFR-TKIs were selected: H3255 (L858R), H1650 (del E746-A750), and H1781 (wild-type EGFR). Cells were treated with erlotinib, gefitinib, genistein, or the combination of each of the EGFR-TKIs with genistein. Cell survival and apoptosis were assessed, and expression levels of EGFR, pAkt, cyclooxygenase-2 (COX-2), E-cadherin, prostaglandin E(2) (PGE(2)), and NF-kappaB were measured. Both EGFR-TKIs demonstrated growth inhibition and apoptosis in each of the cell lines, but H1650 and H1781 were much less sensitive. Genistein demonstrated some antitumor activity in all cell lines, but enhanced growth inhibition and apoptosis when combined with the EGFR-TKIs in each of the cell lines. Both combinations down-regulated NF-kappaB significantly more than either agent alone in H3255. In addition, the combinations reduced the expression of EGFR, pAkt, COX-2, and PGE(2,) consistent with inactivation of NF-kappaB. The authors concluded that genistein enhances the antitumor effects of EGFR-TKIs in 3 separate NSCLC cell lines. This enhanced activity is in part because of greater reduction in the DNA-binding activity of NF-kappaB when EGFR-TKIs were combined with genistein.

Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

PubMed

Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

2016-05-31

Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-based pharmacophore design and virtual screening for novel potential inhibitors of epidermal growth factor receptor as an approach to breast cancer chemotherapy.

PubMed

Mahernia, Shabnam; Hassanzadeh, Malihe; Sharifi, Niusha; Mehravi, Bita; Paytam, Fariba; Adib, Mehdi; Amanlou, Massoud

2018-02-01

Cancer cells are described with features of uncontrolled growth, invasion and metastasis. The epidermal growth factor receptor subfamily of tyrosine kinases (EGFR-TK) plays a crucial regulatory role in the control of cellular proliferation and progression of various cancers. Therefore, its inhibition might lead to the discovery of a new generation of anticancer drugs. In the present study, structure-based pharmacophore modeling, molecular docking and molecular dynamics simulations were applied to identify potential hits, which exhibited good inhibition on the proliferation of MCF-7 breast cancer cell line and favorable binding interactions on EGFR-TK. Selected compounds were examined for their anticancer activity against the Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line which overexpresses EGFR using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. Compounds 1 and 2, with an isoindoline-1-one core, induced significant inhibition of breast cancer cells proliferation with IC[Formula: see text] values 327 and 370 nM, respectively.

Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens.

PubMed

Sakakibara, Keiko; Nishiyama, Tomoaki; Sumikawa, Naomi; Kofuji, Rumiko; Murata, Takashi; Hasebe, Mitsuyasu

2003-10-01

Differentiation of epidermal cells is important for plants because they are in direct contact with the environment. Rhizoids are multicellular filaments that develop from the epidermis in a wide range of plants, including pteridophytes, bryophytes, and green algae; they have similar functions to root hairs in vascular plants in that they support the plant body and are involved in water and nutrient absorption. In this study, we examined mechanisms underlying rhizoid development in the moss, Physcomitrella patens, which is the only land plant in which high-frequency gene targeting is possible. We found that rhizoid development can be split into two processes: determination and differentiation. Two types of rhizoids with distinct developmental patterns (basal and mid-stem rhizoids) were recognized. The development of basal rhizoids from epidermal cells was induced by exogenous auxin, while that of mid-stem rhizoids required an unknown factor in addition to exogenous auxin. Once an epidermal cell had acquired a rhizoid initial cell fate, expression of the homeodomain-leucine zipper I gene Pphb7 was induced. Analysis of Pphb7 disruptant lines showed that Pphb7 affects the induction of pigmentation and the increase in the number and size of chloroplasts, but not the position or number of rhizoids. This is the first report on the involvement of a homeodomain-leucine zipper I gene in epidermal cell differentiation.

CPK3-phosphorylated RhoGDI1 is essential in the development of Arabidopsis seedlings and leaf epidermal cells

PubMed Central

Wu, Yan

2013-01-01

The regulation of Rho of plants (ROP) in morphogenesis of leaf epidermal cells has been well studied, but the roles concerning regulators of ROPs such as RhoGDIs are poorly understood. This study reports that AtRhoGDI1 (GDI1) acts as a versatile regulator to modulate development of seedlings and leaf pavement cells. In mutant gdi1, leaf pavement cells showed shorter lobes in comparison with those in wild type. In GDI1-14 seedlings (GDI1-overexpression line) the growth of lobes in pavement cells was severely suppressed and the development of seedlings was altered. These results indicate that GDI1 plays an essential role in morphogenesis of epidermal pavement cells through modulating the ROP signalling pathways. The interaction between GDI1 and ROP2 or ROP6 was detected in the leaf pavement cells using FRET analysis. Dominant negative, not constitutively active, DN-rop6 could weaken the effect caused by overexpression of GDI1; because the pleiotropic phenotype of GDI1-14 plants was eliminated in the hybrid line GDI1-14 DN-rop6. GDI1 could be phosphorylated by CPK3. Three conserved Ser/Thr residues in GDI1 were determined as targeted amino acids for CPK3. Overexpression of GDI1(3D), not GDI1(3A), could rescue the abnormal growth phenotypes of gdi1-1 seedlings, demonstrating the impact of GDI1 phosphorylation in the development of Arabidopsis. In summary, these results suggest that GDI1 regulation in morphogenesis of seedlings and leaf pavement cells could be undergone through modulating the ROP signalling pathways and the phosphorylation of GDI1 by CPK3 was required for the developmental modulation in Arabidopsis. PMID:23846874

Near Infrared Optical Visualization of Epidermal Growth Factor Receptors Levels in COLO205 Colorectal Cell Line, Orthotopic Tumor in Mice and Human Biopsies

PubMed Central

Cohen, Gadi; Lecht, Shimon; Oron-Herman, Mor; Momic, Tatjana; Nissan, Aviram; Lazarovici, Philip

2013-01-01

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner. PMID:23857061

Near infrared optical visualization of epidermal growth factor receptors levels in COLO205 colorectal cell line, orthotopic tumor in mice and human biopsies.

PubMed

Cohen, Gadi; Lecht, Shimon; Oron-Herman, Mor; Momic, Tatjana; Nissan, Aviram; Lazarovici, Philip

2013-07-12

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6-9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

Effectiveness and cost-effectiveness of erlotinib versus gefitinib in first-line treatment of epidermal growth factor receptor-activating mutation-positive non-small-cell lung cancer patients in Hong Kong.

PubMed

Lee, Vivian W Y; Schwander, Bjoern; Lee, Victor H F

2014-06-01

To compare the effectiveness and cost-effectiveness of erlotinib versus gefitinib as first-line treatment of epidermal growth factor receptor-activating mutation-positive non-small-cell lung cancer patients. DESIGN. Indirect treatment comparison and a cost-effectiveness assessment. Hong Kong. Those having epidermal growth factor receptor-activating mutation-positive non-small-cell lung cancer. Erlotinib versus gefitinib use was compared on the basis of four relevant Asian phase-III randomised controlled trials: one for erlotinib (OPTIMAL) and three for gefitinib (IPASS; NEJGSG; WJTOG). The cost-effectiveness assessment model simulates the transition between the health states: progression-free survival, progression, and death over a lifetime horizon. The World Health Organization criterion (incremental cost-effectiveness ratio <3 times of gross domestic product/capita:

Diminished survival of human cytotrophoblast cells exposed to hypoxia/reoxygenation injury and associated reduction of heparin-binding epidermal growth factor-like growth factor.

PubMed

Leach, Richard E; Kilburn, Brian A; Petkova, Anelia; Romero, Roberto; Armant, D Randall

2008-04-01

The antiapoptotic action of heparin-binding epidermal growth factor (HBEGF)-like growth factor and its regulation by O(2) constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/R in cytotrophoblast cells. A transformed human first-trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O(2) followed by 20% O(2)) and assessed for HBEGF expression and cell death. Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O(2). H/R significantly reduced proliferation and increased cell death when compared with trophoblast cells cultured continuously at 2% or 20% O(2). Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either human epidermal receptor (HER)-1 or HER4, its cognate receptors. These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, whereas restoration of HBEGF signaling improves trophoblast survival.

Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer

PubMed Central

Herrmann, Amanda C.; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J.; Hong, Waun Ki; Perez-Soler, Roman

2016-01-01

Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

PubMed

Chandler, L A; Sosnowski, B A; McDonald, J R; Price, J E; Aukerman, S L; Baird, A; Pierce, G F; Houston, L L

1998-09-25

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Dacomitinib (PF-00299804), a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor, demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro.

PubMed

Zhu, Liancheng; Lopez, Salvatore; Bellone, Stefania; Black, Jonathan; Cocco, Emiliano; Zigras, Tiffany; Predolini, Federica; Bonazzoli, Elena; Bussi, Beatrice; Stuhmer, Zachary; Schwab, Carlton L; English, Diana P; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Santin, Alessandro D

2015-07-01

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that carries an extremely poor prognosis. Up to 35 % of USC may overexpress the epidermal growth factor receptor-2 (HER2/neu) at strong (i.e., 3+) level by immunohistochemistry (IHC) or harbor HER2/neu gene amplification by fluorescence in situ hybridization (FISH). In this study, we assessed the sensitivity of a panel of USC cell lines with and without HER2/neu gene amplification to dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor tyrosine kinase inhibitor. Eight primary cell lines (i.e., four harboring HER2/neu gene amplification by FISH and four FISH- cell lines), all demonstrating similar in vitro growth rates, were evaluated in viability/proliferation assays. The effect of dacomitinib on cell growth, cell cycle distribution, and signaling was determined using flow cytometry-based assays. Dacomitinib caused a significantly stronger growth inhibition in HER2/neu FISH+ USC cell lines when compared to FISH- USC (dacomitinib half maximal inhibitory concentration (IC50) mean ± SEM = 0.02803 ± 0.003355 μM in FISH+ versus 1.498 ± 0.2209 μM in FISH- tumors, P < 0.0001). Dacomitinib growth inhibition was associated with a significant and dose-dependent decline in phosphorylated HER2/neu and S6 transcription factor and a dose-dependent and time-dependent cell cycle arrest in G0/G1 in FISH+ USC. Dacomitinib is remarkably effective against chemotherapy-resistant HER2/neu gene-amplified USC. Clinical studies with dacomitinib in HER2/neu FISH+ USC patients resistant to standard salvage chemotherapy are warranted.

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines. Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment. Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck .

Developmental patterning of sub-epidermal cells in the outer integument of Arabidopsis seeds

PubMed Central

Fiume, Elisa; Coen, Olivier; Xu, Wenjia; Lepiniec, Loïc

2017-01-01

The seed, the reproductive unit of angiosperms, is generally protected by the seed coat. The seed coat is made of one or two integuments, each comprising two epidermal cells layers and, in some cases, extra sub-epidermal cell layers. The thickness of the seed-coat affects several aspects of seed biology such as dormancy, germination and mortality. In Arabidopsis, the inner integument displays one or two sub-epidermal cell layers that originate from periclinal cell divisions of the innermost epidermal cell layer. By contrast, the outer integument was considered to be two-cell layered. Here, we show that sub-epidermal chalazal cells grow in between the epidermal outer integument cell layers to create an incomplete three-cell layered outer integument. We found that the MADS box transcription factor TRANSPARENT TESTA 16 represses growth of the chalaza and formation of sub-epidermal outer integument cells. Finally, we demonstrate that sub-epidermal cells of the outer and inner integument respond differently to the repressive mechanism mediated by FERTILIZATION INDEPENDENT SEED Polycomb group proteins and to fertilization signals. Our data suggest that integument cell origin rather than sub-epidermal cell position underlies different responses to fertilization. PMID:29141031

Galvanic zinc-copper microparticles inhibit melanogenesis via multiple pigmentary pathways.

PubMed

Won, Yen-Kim; Lin, Connie B; Seiberg, Miri; Chen, Nannan; Hu, Yaping; Rossetti, Dianne; Saliou, Claude; Loy, Chong-Jin

2014-01-01

The endogenous electrical field of human skin plays an important role in many skin functions. However, the biological effects and mechanism of action of externally applied electrical stimulation on skin remain unclear. Recent study showed that galvanic zinc-copper microparticles produce electrical stimulation and reduce inflammatory and immune responses in intact skin, suggesting the important role of electrical stimulation in non-wounded skin. The objective of this study is to investigate the biological effect of galvanic zinc-copper microparticles on skin pigmentation. Our findings showed that galvanic zinc-copper microparticles inhibited melanogenesis in a human melanoma cell line (MNT-1), human keratinocytes and melanoma cells co-cultures, and in pigmented epidermal equivalents. Treatment of galvanic zinc-copper microparticles inhibited melanogenesis by reducing the promoter transactivation of tyrosinase and tyrosinase-related protein-1 in human melanoma cells. In a co-culture Transwell system of keratinocytes and melanoma cells, galvanic zinc-copper microparticles reduced melanin production via downregulation of endothelin-1 secretion from keratinocytes and reduced tyrosinase gene expression in melanoma cells. In addition, exposure of pigmented epidermal equivalents to galvanic zinc-copper microparticles resulted in reduced melanin deposition. In conclusion, our data demonstrated for the first time that galvanic zinc-copper microparticles reduced melanogenesis in melanoma cells and melanin deposition in pigmented epidermal equivalents by affecting multiple pigmentary pathways.

Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification.

PubMed

Coumar, Mohane Selvaraj; Chu, Chang-Ying; Lin, Cheng-Wei; Shiao, Hui-Yi; Ho, Yun-Lung; Reddy, Randheer; Lin, Wen-Hsing; Chen, Chun-Hwa; Peng, Yi-Hui; Leou, Jiun-Shyang; Lien, Tzu-Wen; Huang, Chin-Ting; Fang, Ming-Yu; Wu, Szu-Huei; Wu, Jian-Sung; Chittimalla, Santhosh Kumar; Song, Jen-Shin; Hsu, John T-A; Wu, Su-Ying; Liao, Chun-Chen; Chao, Yu-Sheng; Hsieh, Hsing-Pang

2010-07-08

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

Neratinib reverses ATP-binding cassette B1-mediated chemotherapeutic drug resistance in vitro, in vivo, and ex vivo.

PubMed

Zhao, Xiao-qin; Xie, Jing-dun; Chen, Xing-gui; Sim, Hong May; Zhang, Xu; Liang, Yong-ju; Singh, Satyakam; Talele, Tanaji T; Sun, Yueli; Ambudkar, Suresh V; Chen, Zhe-Sheng; Fu, Li-wu

2012-07-01

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Neratinib Reverses ATP-Binding Cassette B1-Mediated Chemotherapeutic Drug Resistance In Vitro, In Vivo, and Ex Vivo

PubMed Central

Zhao, Xiao-qin; Xie, Jing-dun; Chen, Xing-gui; Sim, Hong May; Zhang, Xu; Liang, Yong-ju; Singh, Satyakam; Talele, Tanaji T.; Sun, Yueli; Ambudkar, Suresh V.; Chen, Zhe-Sheng

2012-01-01

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [125I]iodoarylazidoprazosin in a concentration-dependent manner (IC50 = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function. PMID:22491935

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection

PubMed Central

Bourke, Claire D.; Prendergast, Catriona T.; Sanin, David E.; Oulton, Tate E.; Hall, Rebecca J.; Mountford, Adrian P.

2015-01-01

Keratinocytes constitute the majority of cells in the skin’s epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45−, CD326−, CD34+) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin−) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67+ nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1β production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those observed in epidermal wound healing. PMID:25575749

Epidermal stem cells: location, potential and contribution to cancer.

PubMed

Ambler, C A; Määttä, A

2009-01-01

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells.

Thalidomide inhibits tumor necrosis factor-alpha production and antigen presentation by Langerhans cells.

PubMed

Deng, Liang; Ding, Wanhong; Granstein, Richard D

2003-11-01

Thalidomide is an effective treatment for several inflammatory and autoimmune disorders including erythema nodosum leprosum, Behcet's syndrome, discoid lupus erythematosus, and Crohn's disease. Thalidomide is believed to exert its anti-inflammatory effects, at least in part, by inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We studied the effects of thalidomide on epidermal Langerhans cells (LC). LCs are epidermal antigen-presenting dendritic cells that play important roles in skin immune responses. Using the murine epidermis-derived dendritic cell lines, XS106A from A/J mice and XS52 from BALB/c mice as surrogates for LC, we found that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysis revealed that thalidomide significantly decreased the peak-induced mRNA level of TNF-alpha in XS106A cells and XS52 cells. We then examined the effect of thalidomide on fresh LC enriched to approximately 98% using positive selection of Ia+ cells with antibodies conjugated to magnetic microspheres. TNF-alpha production was reduced by 67.7% at a thalidomide concentration of 200 microg per mL. Thalidomide also had a profound inhibitory effect on the ability of LC to present antigen to a responsive TH1 clone. Thalidomide inhibits TNF-alpha production and the antigen-presenting ability of epidermal LCs. These mechanisms may contribute to the therapeutic effects observed with this agent.

H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalizationmore » analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.« less

[Progress in epidermal stem cells].

PubMed

Wang, Li-Juan; Wang, You-Liang; Yang, Xiao

2010-03-01

Mammalian skin epidermis contains different epidermal stem cell pools which contribute to the homeostasis and repair of skin epithelium. Epidermal stem cells possess two essential features common to all stem cells: self-renewal and differentiation. Disturbing the balance between self-renewal and differentiation of epidermal stem cell often causes tumors or other skin diseases. Epidermal stem cell niches provide a special microenvironment that maintains a balance of stem cell quiescence and activity. This review primarily concentrates on the following points of the epidermal stem cells: the existing evidences, the self-renewal and differentiation, the division pattern, the signal pathways regulating self-renewal and differentiation, and the microenvironment (niche) and macroenvironment maintaining the homeostasis of stem cells.

Differentiated thyroid cancer cell invasion is regulated through epidermal growth factor receptor-dependent activation of matrix metalloproteinase (MMP)-2/gelatinase A

PubMed Central

Yeh, Michael W; Rougier, Jean-Philippe; Park, Jin-Woo; Duh, Quan-Yang; Wong, Mariwil; Werb, Zena; Clark, Orlo H

2008-01-01

Mechanisms of invasion in thyroid cancer remain poorly understood. We hypothesized that signaling via the epidermal growth factor receptor (EGFR) stimulates thyroid cancer cell invasion by altering the expression and cleavage of matrix metalloproteinases (MMPs). Papillary and follicular carcinoma cell lines were treated with EGF, the EGFR tyrosine kinase inhibitor AG1478, and the MMP inhibitors GM-6001 and Col-3. Flow cytometry was used to detect EGFR. In vitro invasion assays, gelatin zymography, and quantitative reverse transcription-PCR were used to assess the changes in invasive behavior and MMP expression and activation. All cell lines were found to overexpress functional EGFR. EGF stimulated invasion by thyroid cancer cells up to sevenfold (P<0.0001), a process that was antagonized completely by AG1478 and Col-3, partially by GM-6001, but not by the serine protease inhibitor aprotinin. EGF upregulated expression of MMP-9 (2.64– to 8.89-fold, P<0.0001) and membrane type-1 MMP (MT1-MMP, 1.97- to 2.67-fold, P<0.0001). This effect was blocked completely by AG1478 and partially by Col-3. The activation of MMP-2 paralleled MT1-MMP expression. We demonstrate that MMPs are critical effectors of invasion in the papillary and follicular thyroid cancer cell lines studied. Invasion is regulated by signaling through EGFR, an effect mediated by augmentation of gelatinase expression and activation. MMP inhibitors and growth factor antagonists may be effective tumoristatic agents for the treatment of aggressive thyroid carcinomas. PMID:17158762

Pemetrexed Singlet Versus Nonpemetrexed-Based Platinum Doublet as Second-Line Chemotherapy after First-Line Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor Failure in Non-small Cell Lung Cancer Patients with EGFR Mutations.

PubMed

Park, Sehhoon; Keam, Bhumsuk; Kim, Se Hyun; Kim, Ki Hwan; Kim, Yu Jung; Kim, Jin-Soo; Kim, Tae Min; Lee, Se-Hoon; Kim, Dong-Wan; Lee, Jong Seok; Heo, Dae Seog

2015-10-01

Platinum-based doublet chemotherapy is the treatment of choice for patients with non-small cell lung cancer (NSCLC); however, the role of a platinum-based doublet as second-line therapy after failure of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for NSCLC patients has not yet been elucidated. The purpose of this study was to compare the clinical efficacy of pemetrexed versus a platinum-based doublet as second-line therapy after failure of EGFR TKI used as first-line therapy for NSCLC patients with EGFR mutations. We designed a multicenter retrospective cohort study of 314 NSCLC patients with EGFR mutations who received an EGFR TKI as first-line palliative chemotherapy. Our analysis included 83 patients who failed EGFR TKI therapy and received second-line cytotoxic chemotherapy. Forty-six patients were treated using a platinum-based doublet and 37 patients were treated using singlet pemetrexed. The overall response rates of patients receiving a platinum-based doublet and patients receiving pemetrexed were17.4% and 32.4%, respectively (p=0.111). The median progression-free survival (PFS) of patients receiving pemetrexed was significantly longer than that of patients receiving a platinum-based doublet (4.2 months vs. 2.7 months, respectively; p=0.008). The hazard ratio was 0.54 (95% confidence interval, 0.34 to 0.86; p=0.009). Our retrospective analysis found that second-line pemetrexed singlet therapy provided significantly prolonged PFS compared to second-line platinum-based doublet chemotherapy for NSCLC patients with EGFR mutations who failed first-line EGFR TKI. Conduct of prospective studies for confirmation of our results is warranted.

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

PubMed

Lamorte, Louie; Rodrigues, Sonia; Naujokas, Monica; Park, Morag

2002-10-04

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression.

PubMed

Beckham, Carla J; Olsen, Jayme; Yin, Peng-Nien; Wu, Chia-Hao; Ting, Huei-Ju; Hagen, Fred K; Scosyrev, Emelian; Messing, Edward M; Lee, Yi-Fen

2014-08-01

High grade bladder cancer is an extremely aggressive malignancy associated with high rates of morbidity and mortality. Understanding how exosomes may affect bladder cancer progression could reveal novel therapeutic targets. Exosomes derived from human bladder cancer cell lines and the urine of patients with high grade bladder cancer were assessed for the ability to promote cancer progression in standard assays. Exosomes purified from the high grade bladder cancer cell line TCC-SUP and the nonmalignant urothelial cell line SV-HUC were submitted for mass spectrometry analysis. EDIL-3 was identified and selected for further analysis. Western blot was done to determine EDIL-3 levels in urinary exosomes from patients with high grade bladder cancer. shRNA gene knockdown and recombinant EDIL-3 were applied to study EDIL-3 function. Exosomes isolated from high grade bladder cancer cells and the urine of patients with high grade bladder cancer promoted angiogenesis and migration of bladder cancer cells and endothelial cells. We silenced EDIL-3 expression and found that shEDIL-3 exosomes did not facilitate angiogenesis, and urothelial and endothelial cell migration. Moreover, exosomes purified from the urine of patients with high grade bladder cancer contained significantly higher EDIL-3 levels than exosomes from the urine of healthy controls. EDIL-3 activated epidermal growth factor receptor signaling while blockade of epidermal growth factor receptor signaling abrogated this EDIL-3 induced bladder cell migration. Exosomes derived from the urine of patients with bladder cancer contains bioactive molecules such as EDIL-3. Identifying these components and their associated oncogenic pathways could lead to novel therapeutic targets and treatment strategies. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1.

PubMed

Ambler, Carrie A; Watt, Fiona M

2010-11-01

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

Protective effects of amifostine and cyclooxygenase-1 inhibitor against normal human epidermal keratinocyte toxicity induced by methotrexate and 5-fluorouracil.

PubMed

Maiguma, Takayoshi; Kaji, Hiroaki; Makino, Kazutaka; Teshima, Daisuke

2009-07-01

Our study aimed to find more effective protective agents against mucosa toxicity induced by methotrexate and 5-fluorouracil. We focused on the relationship between oral mucositis and keratinocyte injury and examined methotrexate and 5-fluorouracil-induced cytotoxicity in normal human epidermal keratinocyte cell lines. Cell viability and superoxide radical activity were measured based on converting WST-1 (4-[3-(4-indophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzen disulfonate) to a water-soluble formazan dye. DNA synthesis by 5-bromo-2'-deoxyuridine incorporation was measured as an indirect parameter of cell proliferation. Allopurinol and amifostine were used as the radical scavengers. l-glutamine was used as a mucosa-protective agent. A cyclooxygenase inhibitor interrupting the production of hydroxyl radicals in the arachidonic acid cascade was also examined. 5-fluorouracil and methotrexate caused cytotoxicity due to the activation of intracellular superoxide radicals specifically on normal human epidermal keratinocytes. From the electron spin resonance study, it was found that allopurinol was a superoxide radical scavenger, while amifostine was hydroxyl radical scavenger. Allopurinol showed no effect on the cytotoxicity due to 5-fluorouracil and methotrexate. The cell injury induced by methotrexate was restored by amifostine. However, the cell injury induced by 5-fluorouracil was markedly recovered by a selective cyclooxygenase-1 inhibitor compared to amifostine. It was suggested that amifostine and cyclooxygenase-1 inhibitor could be useful protective agents against methotrexate and 5-fluorouracil chemotherapeutic toxicity. Additionally, this in vitro cell injury model using normal human epidermal keratinocytes may be useful for understanding the pathophysiology of oral mucositis induced by chemotherapeutic agents.

Protease-Activated Receptor-2 Is Associated with Terminal Differentiation of Epidermis and Eccrine Sweat Glands

PubMed Central

Shin, Yong-Sup; Kim, Hyung Won; Kim, Chang Deok; Kim, Hyun-Woo; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Ko, Young-Kwon

2015-01-01

Background Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin. Objective To evaluate the basic physiological role of PAR-2 in skin. Methods We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes. Results Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker. Conclusion Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands. PMID:26273149

Protease-Activated Receptor-2 Is Associated with Terminal Differentiation of Epidermis and Eccrine Sweat Glands.

PubMed

Shin, Yong-Sup; Kim, Hyung Won; Kim, Chang Deok; Kim, Hyun-Woo; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Ko, Young-Kwon; Lee, Young Ho

2015-08-01

Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin. To evaluate the basic physiological role of PAR-2 in skin. We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes. Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker. Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands.

Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

NASA Astrophysics Data System (ADS)

Lee, L. S.

1981-02-01

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1

PubMed Central

Ambler, Carrie A.; Watt, Fiona M.

2010-01-01

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin. PMID:20940224

[Enhanced lymphocyte proliferation in the presence of epidermal cells of HIV-infected patients in vitro].

PubMed

Kappus, R P; Berger, S; Thomas, C A; Ottmann, O G; Ganser, A; Stille, W; Shah, P M

1992-07-01

Clinical observations show that the HIV infection is often associated with affections of the skin. In order to examine the involvement of the epidermal immune system in the HIV infection, we determined accessory cell function of epidermal cells from HIV-1-infected patients. For this we measured the proliferative response of enriched CD(4+)-T-lymphocytes from HIV-infected patients and noninfected controls to stimulation with anti-CD3 and IL-2 in the presence of epidermal cells; the enhancement of the response is dependent on the presence of functionally intact accessory cells. The capacity of epidermal cells to increase the anti-CD3-stimulated T-cell proliferative response was significantly enhanced in HIV patients (CDC III/IVA) as compared with noninfected donors. It is discussed, whether the increased activity of epidermal cells from HIV-infected patients may be responsible for several of the dermal lesions in the course of an HIV infection as due to an enhanced production and release of epidermal cell-derived cytokines.

Morphological and functional studies on the epidermal cells of amphioxus ( Branchiostoma belcheri tsingtauense) at different developmental stages

NASA Astrophysics Data System (ADS)

Mao, Bing-Yu; Sun, Xiao-Yang; Zhang, Hong-Wei; Zhang, Shi-Cui; Wu, Xian-Han

1997-09-01

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24 96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means.

The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis

PubMed Central

Fujiwara, Makoto T.; Kojo, Kei H.; Kazama, Yusuke; Sasaki, Shun; Abe, Tomoko; Itoh, Ryuuichi D.

2015-01-01

Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids. PMID:26500667

Carbon-concentrating mechanisms in seagrasses.

PubMed

Larkum, Anthony William D; Davey, Peter A; Kuo, John; Ralph, Peter J; Raven, John A

2017-06-01

Seagrasses are unique angiosperms that carry out growth and reproduction submerged in seawater. They occur in at least three families of the Alismatales. All have chloroplasts mainly in the cells of the epidermis. Living in seawater, the supply of inorganic carbon (Ci) to the chloroplasts is diffusion limited, especially under unstirred conditions. Therefore, the supply of CO2 and bicarbonate across the diffusive boundary layer on the outer side of the epidermis is often a limiting factor. Here we discuss the evidence for mechanisms that enhance the uptake of Ci into the epidermal cells. Since bicarbonate is plentiful in seawater, a bicarbonate pump might be expected; however, the evidence for such a pump is not strongly supported. There is evidence for a carbonic anhydrase outside the outer plasmalemma. This, together with evidence for an outward proton pump, suggests the possibility that local acidification leads to enhanced concentrations of CO2 adjacent to the outer tangential epidermal walls, which enhances the uptake of CO2, and this could be followed by a carbon-concentrating mechanism (CCM) in the cytoplasm and/or chloroplasts. The lines of evidence for such an epidermal CCM are discussed, including evidence for special 'transfer cells' in some but not all seagrass leaves in the tangential inner walls of the epidermal cells. It is concluded that seagrasses have a CCM but that the case for concentration of CO2 at the site of Rubisco carboxylation is not proven. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The receptor tyrosine kinase ERBB4 is expressed in skin keratinocytes and influences epidermal proliferation.

PubMed

Hoesl, Christine; Röhrl, Jennifer M; Schneider, Marlon R; Dahlhoff, Maik

2018-04-01

The epidermal growth factor receptor (EGFR) and associated receptors ERBB2 and ERBB3 are important for skin development and homeostasis. To date, ERBB4 could not be unambiguously identified in the epidermis. The aim of this study was to analyze the ERBB-receptor family with a special focus on ERBB4 in vitro in human keratinocytes and in vivo in human and murine epidermis. We compared the transcript levels of all ERBB-receptors and the seven EGFR-ligands in HaCaT and A431 cells. ERBB-receptor activity was analyzed after epidermal growth factor (EGF) stimulation by Western blot analysis. The location of the receptors was investigated by immunofluorescence in human keratinocytes and skin. Finally, we investigated the function of ERBB4 in the epidermis of skin-specific ERBB4-knockout mice. After EGF stimulation, all ligands were upregulated except for epigen. Expression levels of EGFR were unchanged, but all other ERBB-receptors were down-regulated after EGF stimulation, although all ERBB-receptors were phosphorylated. We detected ERBB4 at mRNA and protein levels in both human epidermal cell lines and in the basal layer of human and murine epidermis. Skin-specific ERBB4-knockout mice revealed a significantly reduced epidermal thickness with a decreased proliferation rate. ERBB4 is expressed in the basal layer of human epidermis and cultured keratinocytes as well as in murine epidermis. Moreover, ERBB4 is phosphorylated in HaCaT cells due to EGF stimulation, and its deletion in murine epidermis affects skin thickness by decreasing proliferation. ERBB4 is expressed in human keratinocytes and plays a role in murine skin homeostasis. Copyright © 2018 Elsevier B.V. All rights reserved.

[Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines].

PubMed

Ma, Li; Han, Xiao-hong; Wang, Shuai; Wang, Jian-fei; Shi, Yuan-kai

2012-09-25

To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

Differentiated epidermal cells regain the ability to regenerate a skin equivalent by increasing the level of β-catenin in the cells.

PubMed

Zhao, Zhili; Zhang, Cuiping; Fu, Xiaobing; Yang, Rongya; Peng, Chen; Gu, Tingmin; Sui, Zhifu; Wang, Congmin; Liu, Chang

2012-01-01

Epidermal stem cells are of major importance for skin regeneration and tissue engineering, but differentiated epidermal cells lost their proliferative capacity and are no longer able to regenerate a skin equivalent. Here, we investigated the role of β-catenin in regulating regenerative functions of differentiated epidermal cells. Lithium chloride and a highly specific glycogen synthase kinase (GSK)-3β inhibitor were applied to induce the expression of β-catenin in differentiated epidermal cells. After a 6-day induction, the large flat-shaped cells with a small nuclear-cytoplasmic ratio had changed into small round-shaped cells with a large nuclear-cytoplasmic ratio. Phenotypic assays showed a remarkably higher expression of CK19, β(1)-integrin, Oct4 and Nanog in induced cells than in the control group (p < 0.01). In addition, the results of growth and functional investigations demonstrated that the induced epidermal cells exhibited a high colony-forming ability, a long-term proliferative potential and the ability to regenerate a skin equivalent, which were regarded as the most important features of epidermal stem cells. These results suggest that the activation of β-catenin favors the reversion or dedifferentiation of differentiated epidermal cells to an immature or a less differentiated state. This study may also offer a new approach to yield enough epidermal stem cells for skin regeneration and tissue engineering. Copyright © 2012 S. Karger AG, Basel.

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.

Dependence of EGF-Induced Increases in Corneal Epithelial Proliferation and Migration on GSK-3 Inactivation

DTIC Science & Technology

2009-10-01

during epidermal growth factor-stimulated actin nucleation in breast cancer cells. J Biol Chem. 2000;275:3741–3744. 27. Jope RS, Johnson GV. The...injury-induced corneal epi- thelial wound closure.1,2 This cytokine induces increases in cell proliferation and migration through activation of its cog ...occurs between the PI3-K and the ERK pathways in colon cancer cell lines.12 Without a cytokine, GSK-3 is dephosphorylated and constitutively active

Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17

PubMed Central

Guinea-Viniegra, Juan; Zenz, Rainer; Scheuch, Harald; Jiménez, María; Bakiri, Latifa; Petzelbauer, Peter; Wagner, Erwin F.

2012-01-01

Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α–converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs. PMID:22772468

Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells

PubMed Central

Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

2016-01-01

ABSTRACT Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention. PMID:27097375

Stromal cell-derived factor 1 (SDF-1) accelerated skin wound healing by promoting the migration and proliferation of epidermal stem cells.

PubMed

Guo, Rui; Chai, Linlin; Chen, Liang; Chen, Wenguang; Ge, Liangpeng; Li, Xiaoge; Li, Hongli; Li, Shirong; Cao, Chuan

2015-06-01

Epidermal stem cells could contribute to skin repair through the migration of cells from the neighboring uninjured epidermis, infundibulum, hair follicle, or sebaceous gland. However, little is known about the factors responsible for the complex biological processes in wound healing. Herein, we will show that the attracting chemokine, SDF-1/CXCR4, is a major regulator involved in the migration of epidermal stem cells during wound repair. We found that the SDF-1 levels were markedly increased at the wound margins following injury and CXCR4 expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis resulted in a significant reduction in epidermal stem cell migration toward SDF-1 in vitro and delayed wound healing in vivo, while an SDF-1 treatment enhanced epidermal stem cell migration and proliferation and accelerated wound healing. These results provide direct evidence that SDF-1 promotes epidermal stem cell migration, accelerates skin regeneration, and makes the development of new regenerative therapeutic strategies for wound healing possible.

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

PubMed

Peng, Wei; de Bruijn, Henriette S; Farrell, Eric; Sioud, Mouldy; Mashayekhi, Vida; Oliveira, Sabrina; van Dam, Go M; Roodenburg, Jan L N; Witjes, Max J H; Robinson, Dominic J

2018-05-19

The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm -2 (at 5 mW.cm -2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density. Intrinsic biological properties of tumor cell lines also play a role in determining the efficacy of targeted PIT. We have shown that in scc-U2 cells this difference may be caused by differences in the apoptopic pathway. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.

PubMed

Xiao, Mei; An, LiLong; Yang, XueYi; Ge, Xin; Qiao, Hai; Zhao, Ting; Ma, XiaoFei; Fan, JingZhuang; Zhu, MengYang; Dou, ZhongYing

2008-09-01

The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant transplantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1 x 10(9) mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with beta-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet-like clusters, as identified by dithizone staining, which expressed the transcription factor of the insulin and secreted the insulin and C-peptide. Furthermore, the transplantation of mhPSCs-induced pancreatic islets into the subcapsular region of the kidney in streptozotocin-induced diabetic rats could reduce blood glucose levels and prolong the life time.

Role of Epidermal Growth Factor Receptors and Their Ligands in Normal Mammary Epithelial and Breast Cancer Cells

DTIC Science & Technology

1997-07-01

have an estrogen receptor and progesterone receptor negative phenotype, high proliferation rates, poor response to endocrine therapy, and reduced...mitogenic effects of estrogen, progesterone and prolactin in breast cancer cell lines (3), and part of the growth promoting effects of an activated ras...of the cases (51,52), and an inverse relationship with estrogen and progesterone receptors; in such tumors, a poor response to endocrine therapy

Effects of adenosine 5'-monophosphate on epidermal turnover.

PubMed

Furukawa, Fukumi; Kanehara, Shoko; Harano, Fumiki; Shinohara, Shigeo; Kamimura, Junko; Kawabata, Shigekatsu; Igarashi, Sachiyo; Kawamura, Mitsuaki; Yamamoto, Yuki; Miyachi, Yoshiki

2008-10-01

The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

Morphology, ultrastructure, and chemical compounds of the osmeterium of Heraclides thoas (Lepidoptera: Papilionidae).

PubMed

Martínez, Luis Carlos; Plata-Rueda, Angelica; da Silva Neves, Guilherme; Cossolin, Jamile Fernanda; Dos Santos, Marcelo Henrique; Zanuncio, José Cola; Serrão, José Eduardo

2018-05-11

The osmeterium, found in papilionoid larvae, is an eversible organ with an exocrine gland that produces substances in response to the mechanical disturbances caused by natural enemies. The anatomy, histology and ultrastructure of the osmeterium, and the chemical composition of its secretion in Heraclides thoas (Lepidoptera: Papilionidae) were studied. Heraclides thoas larvae have a Y-shaped osmeterium in the thorax. The surface of the osmeterium has a rough cuticle lining cells with papillae and irregular folds, whereas the cells that limited the gland pores are irregular, folded, and devoid of papillae. Two types of cells are found: (i) cuticular epidermal cells on the surface of the tubular arms of the osmeterium and (ii) secretory cells of the ellipsoid gland within the region of the glandular pore. Cuticular epidermal cells show a thick cuticle, with several layers divided into epicuticle and lamellar endocuticle. Secretory cells are polygonal, with extensive folds in the basal plasma membrane that formed extracellular channels. The cytoplasm has mitochondria, ribosomes, and numerous vacuoles, whereas the nucleus is irregular in shape with decondensed chromatin. The chemical composition of the osmeterial secretion comprised (Z)-α-bisabolene (25.4%), α-bisabol (20.6%), β-bisabolene (13.1%), (E)-α-bisabolene 8%), β-pinene (9.91%), longipinene epoxide (8.92%), (Z)-β-farnesene (6.96%), β-caryophyllene (2.05%), farnesol (1.86%), linalyl propionate (1.86%), and 1-octyn-4-ol (1.07%). The morphological features suggest that the cuticular epidermal cells play a major role in the maintenance and protection of the osmeterium, whereas secretory cells are responsible for production of osmeterial secretions.

Targeting cancer stem cell plasticity through modulation of epidermal growth factor and insulin-like growth factor receptor signaling in head and neck squamous cell cancer.

PubMed

Leong, Hui Sun; Chong, Fui Teen; Sew, Pui Hoon; Lau, Dawn P; Wong, Bernice H; Teh, Bin-Tean; Tan, Daniel S W; Iyer, N Gopalakrishna

2014-09-01

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics. ©AlphaMed Press.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

PubMed

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin.

PubMed

Yarani, Reza; Mansouri, Kamran; Mohammadi-Motlagh, Hamid Reza; Bakhtiari, Mitra; Mostafaie, Ali

2013-06-01

Conventional isolation of epidermis from the dermis and disruption of epidermal sheets to liberate the cells, are performed using proteolytic enzymes such as thermolysin or collagenase. Selective population expansion of melanocytes is achieved by suppressing proliferation of keratinocytes and fibroblasts in epidermal cell suspensions, using phorbol esters and cholera toxin. Here, we introduce a new procedure for isolation of epidermal cells, using proteolytic activity of kiwi fruit actinidin, and also an improved growth medium for melanocytes in the presence of leukaemia inhibitory factor (LIF) and forskolin. Dermo-epidermal separation and epidermal sheet cell dispersion were performed using actinidin compared to conventional proteases including collagenase, thermolysin or trypsin. Thereafter, melanocyte culture was performed in two common media and one modified medium to discover optimization for these cells. We found that dermo-epidermal separation and epidermal sheet cell dispersion using kiwi fruit actinidin were considerably better than previously used methods, both from the aspect of less fibroblast and keratinocyte contamination, and of more viable native cells. Also, melanocytes proliferated better in phorbol ester- and cholera toxin-free proliferation medium supplemented with LIF and forskolin. Less contamination and higher numbers of viable cells were actinidin preferential for separation of epidermis and isolation of epidermal cells. Supplementation of LIF and forskolin to new medium increased proliferation potential of melanocytes in comparison to exogenous mitogens. © 2013 Blackwell Publishing Ltd.

Evolution and genetics of root hair stripes in the root epidermis.

PubMed

Dolan, L; Costa, S

2001-03-01

Root hair pattern develops in a number of different ways in angiosperm. Cells in the epidermis of some species undergo asymmetric cell divisions to form a smaller daughter cell from which a hair grows, and a larger cell that forms a non-hair epidermal cell. In other species any cell in the epidermis can form a root hair. Hair cells are arranged in files along the Arabidopsis root, located in the gaps between underlying cortical cell files. Epidermal cells overlying a single cortical cell file develop as non-hair epidermal cells. Genetic analysis has identified a transcription factor cascade required for the formation of this pattern. WEREWOLF (WER) and GLABRA2 (GL2) are required for the formation of non-hair epidermal cells while CAPRICE (CPC) is required for hair cell development. Recent analyses of the pattern of epidermal cells among the angiosperms indicate that this striped pattern of cell organization evolved from non-striped ancestors independently in a number of diverse evolutionary lineages. The genetic basis for the evolution of epidermal pattern in angiosperms may now be examined.

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering overall survival.

Spared pre-irradiated area in pustular lesions induced by icotinib showing decreased expressions of CD1a+ langerhans cells and FGFR2.

PubMed

Zhao, Qiong; Wang, Yi Na; Wang, Bo

2013-02-01

Icotinib hydrochloride, a novel inhibitor of epidermal growth factor receptor tyrosine kinase, has been approved by the State Food and Drug Administration for the treatment of advanced non-small-cell lung cancer. Up to date, cutaneous response to icotinib is largely unknown. Here we report an uncommon lesional phenomenon in a 56-year-old Chinese male with non-small-cell lung cancer, who received icotinib as a second-line treatment. Characteristic papulopustular rash on the chest and back was observed 4 days later. Interestingly, the rash completely spares a pre-irradiated area. The immunohistochemical study in the lesional skin area and spared skin area revealed a significant decrease in CD1a(+) Langerhans cells, Ki-67 as well as FGFR2 in the spared area than in the lesional area. Thus, the present case indicated that loss of the basal layer of proliferative cells and antigen-presenting cells (Langerhans cell), as well as the down-regulation of FGFR2 signaling in the pre-irradiated skin area, may join forces in inhibiting icotinib-associated cutaneous reactions. To our knowledge, this is the first report of both lesional area and lesion-spared area in a Chinese male receiving treatment with a new epidermal growth factor receptor-tyrosine kinase inhibitor (icotinib). The immunohistochemical reactions described here also provide new insight into the pathogenesis of epidermal growth factor receptor-tyrosine kinase inhibitor-related skin toxicities, and the role that other tyrosine kinase receptors (including FGFR) played in non-small-cell lung cancer.

Anti-cancer Effects of Polyphenolic Compounds in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-resistant Non-small Cell Lung Cancer

PubMed Central

Jeong, Hyungmin; Phan, Ai N. H.; Choi, Jong-Whan

2017-01-01

Background: Polyphenolic phytochemicals are natural compounds, easily found in fruits and vegetables. Importantly, polyphenols have been intensively studied as excellent antioxidant activity which contributes to anticancer function of the natural compounds. Lung cancer has been reported to mainly account for cancer-related deaths in the world. Moreover, epidermal growth factor receptor tyrosine kinase inhibitor (TKI) resistance is one of the biggest issues in cancer treatment, especially in nonsmall cell lung cancer (NSCLC). Even though several studies both in preclinical and clinical trials have showed promising therapeutic effects of polyphenolic compounds in anticancer therapy, the function of the natural compounds in TKI-resistant (TKIR) lung cancer remains poorly studied. Objective: The aim of this study is to screen polyphenolic compounds as potential anticancer adjuvants which suppress TKIR lung cancer. Materials and Methods: Colony formation and thiazolyl blue tetrazolium blue assay were performed in the pair-matched TKI-sensitive (TKIS) versus TKIR tumor cell lines to investigate the therapeutic effect of polyphenolic compounds in TKIR NSCLC. Results: Our data show that equol, kaempferol, resveratrol, and ellagic acid exhibit strong anticancer effect in HCC827 panel. Moreover, the inhibitory effect of most of tested polyphenolic compounds was highly selective for TKIR lung cancer cell line H1993 while sparing the TKIS one H2073. Conclusion: This study provides an important screening of potential polyphenolic compounds for drug development to overcome TKI resistance in advanced lung cancer. SUMMARY The study provides an important screening of potential polyphenolic compounds for drug development to overcome tyrosine kinase inhibitor (TKI) resistance in advance lung cancerEquol, kaempferol, resveratrol, and ellagic acid show strong anticancer effect in HCC827 panel, including TKI-sensitive (TKIS) and TKI-resistant clonesThe inhibitory effect of polyphenolic compounds such as equol, kaempferol, resveratrol, ellagic acid, gallic acid, p-Coumaric, and hesperidin is highly selective for TKI-resistant lung cancer cell line H1993 while sparing the TKIS one H2073. Abbreviations used: EGFR: Epidermal growth factor receptor, EMT: Epithelial-to-mesenchymal transition, GTP: Green tea polyphenols, IGF1R: Insulin-like growth factor 1 receptor, MET: Met proto-oncogene, MTT: Thiazolyl blue tetrazolium blue, NSCLC: Non-small cell lung cancer, ROS: Reactive oxygen species, RTK: Receptor tyrosine kinase, STAT3: Signal transducer and activator of transcription 3, TKIR: TKI-resistant, TKIs: Tyrosine kinase inhibitors, TKIS: TKI-sensitive. PMID:29200719

Epithelial morphogenesis of germline-derived pluripotent stem cells on organotypic skin equivalents in vitro.

PubMed

van de Kamp, Julia; Kramann, Rafael; Anraths, Julia; Schöler, Hans R; Ko, Kinarm; Knüchel, Ruth; Zenke, Martin; Neuss, Sabine; Schneider, Rebekka K

2012-03-01

For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

BnEPFL6, an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for filament elongation in Brassica napus.

PubMed

Huang, Yi; Tao, Zhangsheng; Liu, Qiong; Wang, Xinfa; Yu, Jingyin; Liu, Guihua; Wang, Hanzhong

2014-07-01

Inflorescence architecture, pedicel length and stomata patterning in Arabidopsis thaliana are specified by inter-tissue communication mediated by ERECTA and its signaling ligands in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family of secreted cysteine-rich peptides. Here, we identified and characterized BnEPFL6 from Brassica napus. Heterologous expression of this gene under the double enhanced CaMV promoter (D35S) in Arabidopsis resulted in shortened stamen filaments, filaments degradation, and reduced filament cell size that displayed down-regulated expression of AHK2, in which phenotypic variation of ahk2-1 mutant presented highly consistent with that of BnEPFL6 transgenic lines. Especially, the expression level of BnEPFL6 in the shortened filaments of four B. napus male sterile lines (98A, 86A, SA, and Z11A) was similar to that of BnEPFL6 in the transgenic Arabidopsis lines. The activity of pBnEPFL6.2::GUS was intensive in the filaments of transgenic lines. These observations reveal that BnEPFL6 plays an important role in filament elongation and may also affect organ morphology and floral organ specification via a BnEPFL6-mediated cascade.

JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

PubMed

Hassan, Hala; Scheres, Ben; Blilou, Ikram

2010-05-01

In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

PubMed

Berning, Manuel; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

2015-09-01

Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

Conditioned media from a renal cell carcinoma cell line demonstrates the presence of basic fibroblast growth factor.

PubMed

Mydlo, J H; Zajac, J; Macchia, R J

1993-09-01

In a previous report, we demonstrated the isolation and purification of a heparin binding growth factor from human renal carcinoma, and suggested that this growth factor may play a role in the neovascularity and growth of the tumor. In this report, we demonstrate that the growth of the renal cell carcinoma cell line RC29 is stimulated by the addition of exogenous fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha). Also, media conditioned by this cell line was able to stimulate growth of the A431 vulvar tumor cell line, known for its high concentration of EGF receptors, 3T3 fibroblasts, human umbilical vein (HUV) cells and RC29 cells. Using heparin-sepharose chromatography and then SDS polyacrylamide gel electrophoresis (PAGE), we were able to demonstrate several proteins in the conditioned media of the RC29 cell line. Using Western blot analysis, we detected that at least one of the proteins expressed in this conditioned media was FGF and that it belongs to the basic, not acidic, family of fibroblast growth factors. These findings suggest that renal tumors may express growth factors that may play a direct role in maintaining their unrestricted proliferation.

Metalloprobes: Synthesis, Characterization, and Potency of a Novel Gallium(III) Complex in Human Epidermal Carcinoma Cells

PubMed Central

Harpstrite, Scott E.; Prior, Julie; Rath, Nigam P.; Sharma, Vijay

2009-01-01

Multidrug resistance (MDR) mediated by overexpression of the MDR1 gene product, P-glycoprotein (Pgp), represents one of the best characterized barriers to chemotherapeutic treatment in cancer and may be a pivotal factor in progression of Alzheimer’s disease (AD). Thus, agents capable of probing Pgp-mediated transport could be beneficial in biomedical imaging. Herein, we synthesized and structurally characterized a gallium(III) complex of the naphthol-Schiff base ligand (5). The crystal structure revealed octahedral geometry for the metallodrug. Cytotoxicity profiles of 5 were evaluated in KB-3-1 (Pgp−) and KB-8-5 (Pgp+) human epidermal carcinoma cell lines. Compared with an LC50 (the half-maximal cytotoxic concentration) value of 1.93 μM in drug-sensitive (Pgp−) cells, the gallium(III) complex 5 demonstrated an LC50 value > 100 μM in drug-resistant (Pgp+) cells, thus indicating that 5 was recognized by the Pgp as its substrate, thereby extruded from the cells and sequestered away from their cytotoxic targets. Radiolabeled analogues of 5 could be beneficial in noninvasive imaging of Pgp-mediated transport in vivo. PMID:17617464

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

PubMed

Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi

2014-02-01

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.

Antiaging action of retinol: from molecular to clinical.

PubMed

Bellemère, G; Stamatas, G N; Bruère, V; Bertin, C; Issachar, N; Oddos, T

2009-01-01

The antiaging efficacy of retinol (ROL) has been explored mainly clinically in photoprotected skin sites and for high doses of ROL (0.4-1.6%). The objective of the study was to demonstrate the antiaging action of a low and tolerable dose of ROL (0.1%) ex vivo by measuring the expression of cellular retinoic-acid-binding protein II (CRABP2) and heparin-binding epidermal growth factor (HBEGF) by a histological evaluation of the epidermis and in vivo by assessing major aging signs and performing three-dimensional profilometry and digital imaging during a 9-month double-blind placebo-controlled study involving 48 volunteers. Finally, epidermal cell proliferation was evaluated using tryptophan fluorescence spectroscopy. Our results demonstrate that 0.1% ROL induced CRABP2 and HBEGF gene expression and increased keratinocyte proliferation and epidermal thickness. In human volunteers, topical application of a ROL-containing product improved all major aging signs assessed in our study (wrinkles under the eyes, fine lines and tone evenness). Moreover, tryptophan fluorescence increased in the active-agent-treated group and not in the placebo-treated group, indicating that cell proliferation was accelerated in vivo. These data demonstrate that a product containing a low dose (0.1%) of ROL promotes keratinocyte proliferation ex vivo and in vivo, induces epidermal thickening ex vivo and alleviates skin aging signs, without any significant adverse reaction. Copyright 2009 S. Karger AG, Basel.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

PubMed

Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

2014-10-01

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

NASA Astrophysics Data System (ADS)

Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

2016-01-01

The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

Overexpression of cyclin D1 induces the reprogramming of differentiated epidermal cells into stem cell-like cells.

PubMed

Zhao, Along; Yang, Leilei; Ma, Kui; Sun, Mengli; Li, Lei; Huang, Jin; Li, Yang; Zhang, Cuiping; Li, Haihong; Fu, Xiaobing

2016-01-01

It has been reported that Wnt/β-catenin is critical for dedifferentiation of differentiated epidermal cells. Cyclin D1 (CCND1) is a β-catenin target gene. In this study, we provide evidence that overexpression of CCND1 induces reprogramming of epidermal cells into stem cell-like cells. After introducing CCND1 gene into differentiated epidermal cells, we found that the large flat-shaped cells with a small nuclear-cytoplasmic ratio changed into small round-shaped cells with a large nuclear-cytoplasmic ratio. The expressions of CK10, β1-integrin, Oct4 and Nanog in CCND1 induced cells were remarkably higher than those in the control group (P < 0.01). In addition, the induced cells exhibited a high colony-forming ability and a long-term proliferative potential. When the induced cells were implanted into a wound of laboratory animal model, the wound healing was accelerated. These results suggested that overexpression of CCND1 induced the reprogramming of differentiated epidermal cells into stem cell-like cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

Inhibitory effect on activator protein-1, nuclear factor-kappaB, and cell transformation by extracts of strawberries (Fragaria x ananassa Duch.).

PubMed

Wang, Shiow Y; Feng, Rentian; Lu, Yongju; Bowman, Linda; Ding, Min

2005-05-18

The inhibitory effects of strawberry (Fragaria x ananassa Duch.) antioxidant enzymes on tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B (UVB) induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) were studied. The inhibitory effects of strawberry extracts on the proliferation and transformation of human and mouse cancer cells were also evaluated. Strawberries had high activities of glutathione peroxidase, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase. Strawberry extracts inhibited the proliferation of human lung epithelial cancer cell line A549 and decreased TPA-induced neoplastic transformation of JB6 P+ mouse epidermal cells. Pretreatment of JB6 P+ mouse epidermal cells with strawberry extract resulted in the inhibition of both UVB- and TPA-induced AP-1 and NF-kappaB transactivation. Furthermore, strawberry extract also blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs) and UVB-induced phosphorylation of ERKs and JNK kinase in JB6 P+ mouse epidermal cell culture. These results suggest that the ability of strawberries to block UVB- and TPA-induced AP-1 and NF-kappaB activation may be due to their antioxidant properties and their ability to reduce oxidative stress. The oxidative events that regulate AP-1 and NF-kappaB transactivation can be important molecular targets for cancer prevention. The strawberries may be highly effective as a chemopreventive agent that acts by targeting the down-regulation of AP-1 and NF-kappaB activities, blocking MAPK signaling, and suppressing cancer cell proliferation and transformation.

Developmental patterning of the sub-epidermal integument cell layer in Arabidopsis seeds

PubMed Central

Coen, Olivier; Fiume, Elisa; Xu, Wenjia; De Vos, Delphine; Lu, Jing; Pechoux, Christine; Lepiniec, Loïc

2017-01-01

Angiosperm seed development is a paradigm of tissue cross-talk. Proper seed formation requires spatial and temporal coordination of the fertilization products – embryo and endosperm – and the surrounding seed coat maternal tissue. In early Arabidopsis seed development, all seed integuments were thought to respond homogenously to endosperm growth. Here, we show that the sub-epidermal integument cell layer has a unique developmental program. We characterized the cell patterning of the sub-epidermal integument cell layer, which initiates a previously uncharacterized extra cell layer, and identified TRANSPARENT TESTA 16 and SEEDSTICK MADS box transcription factors as master regulators of its polar development and cell architecture. Our data indicate that the differentiation of the sub-epidermal integument cell layer is insensitive to endosperm growth alone and to the repressive mechanism established by FERTILIZATION INDEPENDENT ENDOSPERM and MULTICOPY SUPPRESSOR OF IRA1 Polycomb group proteins. This work demonstrates the different responses of epidermal and sub-epidermal integument cell layers to fertilization. PMID:28348169

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

NASA Astrophysics Data System (ADS)

Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

2016-09-01

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

A sea urchin in vivo model to evaluate Epithelial-Mesenchymal Transition.

PubMed

Romancino, Daniele P; Anello, Letizia; Lavanco, Antonella; Buffa, Valentina; Di Bernardo, Maria; Bongiovanni, Antonella

2017-04-01

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti-EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti-EMT candidates. © 2017 Japanese Society of Developmental Biologists.

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis.

PubMed Central

Melaragno, JE; Mehrotra, B; Coleman, AW

1993-01-01

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon. PMID:12271050

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

PubMed

Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

2012-05-01

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Effect of Ocimum sanctum on Oral Cancer Cell Line: An in vitro Study.

PubMed

Shivpuje, Prachi; Ammanangi, Renuka; Bhat, Kishore; Katti, Sandeep

2015-09-01

Cancer till today remains the leading cause of death in both developed and developing countries. Plants have been beacon of therapeutic sources for curing diseases from times immemorial. Hence, the present study aimed at evaluating the antiproliferative activity of extract of Ocimum sanctum leaves on oral cancer cell line. To evaluate the antiproliferative effect and to analyze dose dependent cytotoxic activity of aqueous extract of O. sanctum leaves on KB mouth cell line. To compare the effectiveness among different variety of O. sanctum. KB cells (Mouth Epidermal Carcinoma Cells) were used for the present study. Aqueous and dry extract of O. sanctum with both dark (Krishna Tulsi) and light (Rama Tulsi) leaves were prepared in the institution. The antiproliferative and cytotoxic activity on KB cell line was evaluated by MTT assay. Statistical analysis with Mann-Whitney U-test and Wilcoxon matched pairs test was carried out. The aqueous extract of O. sanctum of both the leaves exhibited significant cytotoxic effect against oral cancer cell line. Aqueous extract of O. sanctum leaves was effective as an antiproliferative agent which caused apoptosis in oral cancer cell line. Ocimum sanctum herb which is abundantly grown in India can be used for its anticancer properties for treating oral cancer. This will not only be cost-effective but will also have less or no side effects.

Extracellular Matrix as a Regulator of Epidermal Stem Cell Fate.

PubMed

Chermnykh, Elina; Kalabusheva, Ekaterina; Vorotelyak, Ekaterina

2018-03-27

Epidermal stem cells reside within the specific anatomic location, called niche, which is a microenvironment that interacts with stem cells to regulate their fate. Regulation of many important processes, including maintenance of stem cell quiescence, self-renewal, and homeostasis, as well as the regulation of division and differentiation, are common functions of the stem cell niche. As it was shown in multiple studies, extracellular matrix (ECM) contributes a lot to stem cell niches in various tissues, including that of skin. In epidermis, ECM is represented, primarily, by a highly specialized ECM structure, basement membrane (BM), which separates the epidermal and dermal compartments. Epidermal stem cells contact with BM, but when they lose the contact and migrate to the overlying layers, they undergo terminal differentiation. When considering all of these factors, ECM is of fundamental importance in regulating epidermal stem cells maintenance, proper mobilization, and differentiation. Here, we summarize the remarkable progress that has recently been made in the research of ECM role in regulating epidermal stem cell fate, paying special attention to the hair follicle stem cell niche. We show that the destruction of ECM components impairs epidermal stem cell morphogenesis and homeostasis. A deep understanding of ECM molecular structure as well as the development of in vitro system for stem cell maintaining by ECM proteins may bring us to developing new approaches for regenerative medicine.

TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells

PubMed Central

Sutton, Selina K.; Koach, Jessica; Tan, Owen; Liu, Bing; Carter, Daniel R.; Wilmott, James S.; Yosufi, Benafsha; Haydu, Lauren E.; Mann, Graham J.; Thompson, John F.; Long, Georgina V.; Liu, Tao; McArthur, Grant; Zhang, Xu Dong; Scolyer, Richard A.; Cheung, Belamy B.; Marshall, Glenn M.

2014-01-01

High basal or induced expression of the tripartite motif protein, TRIM16, leads to reduce cell growth and migration of neuroblastoma and skin squamous cell carcinoma cells. However, the role of TRIM16 in melanoma is currently unknown. TRIM16 protein levels were markedly reduced in human melanoma cell lines, compared with normal human epidermal melanocytes due to both DNA methylation and reduced protein stability. TRIM16 knockdown strongly increased cell migration in normal human epidermal melanocytes, while TRIM16 overexpression reduced cell migration and proliferation of melanoma cells in an interferon beta 1 (IFNβ1)-dependent manner. Chromatin immunoprecipitation assays revealed TRIM16 directly bound the IFNβ1 gene promoter. Low level TRIM16 expression in 91 melanoma patient samples, strongly correlated with lymph node metastasis, and, predicted poor patient prognosis in a separate cohort of 170 melanoma patients with lymph node metastasis. The BRAF inhibitor, vemurafenib, increased TRIM16 protein levels in melanoma cells in vitro, and induced growth arrest in BRAF-mutant melanoma cells in a TRIM16-dependent manner. High levels of TRIM16 in melanoma tissues from patients treated with Vemurafenib correlated with clinical response. Our data, for the first time, demonstrates TRIM16 is a marker of cell migration and metastasis, and a novel treatment target in melanoma. PMID:25333256

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

PubMed

Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

Gloss, colour and grip: multifunctional epidermal cell shapes in bee- and bird-pollinated flowers.

PubMed

Papiorek, Sarah; Junker, Robert R; Lunau, Klaus

2014-01-01

Flowers bear the function of filters supporting the attraction of pollinators as well as the deterrence of floral antagonists. The effect of epidermal cell shape on the visual display and tactile properties of flowers has been evaluated only recently. In this study we quantitatively measured epidermal cell shape, gloss and spectral reflectance of flowers pollinated by either bees or birds testing three hypotheses: The first two hypotheses imply that bee-pollinated flowers might benefit from rough surfaces on visually-active parts produced by conical epidermal cells, as they may enhance the colour signal of flowers as well as the grip on flowers for bees. In contrast, bird-pollinated flowers might benefit from flat surfaces produced by flat epidermal cells, by avoiding frequent visitation from non-pollinating bees due to a reduced colour signal, as birds do not rely on specific colour parameters while foraging. Moreover, flat petal surfaces in bird-pollinated flowers may hamper grip for bees that do not touch anthers and stigmas while consuming nectar and thus, are considered as nectar thieves. Beside this, the third hypothesis implies that those flower parts which are vulnerable to nectar robbing of bee- as well as bird-pollinated flowers benefit from flat epidermal cells, hampering grip for nectar robbing bees. Our comparative data show in fact that conical epidermal cells are restricted to visually-active parts of bee-pollinated flowers, whereas robbing-sensitive parts of bee-pollinated as well as the entire floral surface of bird-pollinated flowers possess on average flat epidermal cells. However, direct correlations between epidermal cell shape and colour parameters have not been found. Our results together with published experimental studies show that epidermal cell shape as a largely neglected flower trait might act as an important feature in pollinator attraction and avoidance of antagonists, and thus may contribute to the partitioning of flower-visitors.

The organization of human epidermis: functional epidermal units and phi proportionality.

PubMed

Hoath, Steven B; Leahy, D G

2003-12-01

The concept that mammalian epidermis is structurally organized into functional epidermal units has been proposed on the basis of stratum corneum (SC) architecture, proliferation kinetics, melanocyte:keratinocyte ratios (1:36), and, more recently, Langerhans cell: epidermal cell ratios (1:53). This article examines the concept of functional epidermal units in human skin in which the maintenance of phi (1.618034) proportionality provides a central organizing principle. The following empirical measurements were used: 75,346 nucleated epidermal cells per mm2, 1394 Langerhans cells per mm2, 1999 melanocytes per mm2, 16 (SC) layers, 900-microm2 corneocyte surface area, 17,778 corneocytes per mm2, 14-d (SC) turnover time, and 93,124 per mm2 total epidermal cells. Given these empirical data: (1) the number of corneocytes is a mean proportional between the sum of the Langerhans cell + melanocyte populations and the number of epidermal cells, 3393/17,778-17,778/93,124; (2) the ratio of nucleated epidermal cells over corneocytes is phi proportional, 75,346/17,778 approximately phi3; (3) assuming similar 14-d turnover times for the (SC) and Malpighian epidermis, the number of corneocytes results from subtraction of a cellular fraction equal to approximately 2/phi2 x the number of living cells, 75,436 - (2/phi2 x 75,346) approximately 17,778; and (4) if total epidermal turnover time equals (SC) turnover time x the ratio of living/dead cells, then compartmental turnover times are unequal (14 d for (SC) to 45.3 d for nucleated epidermis approximately 1/2phi) and cellular replacement rates are 52.9 corneocytes/69.3 keratinocytes per mm2 per h approximately 2/phi2. These empirically derived equivalences provide logicomathematical support for the presence of functional epidermal units in human skin. Validation of a phi proportional unit architecture in human epidermis will be important for tissue engineering of skin and the design of instruments for skin measurement.

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

PubMed Central

Chatelier, R C; Ashcroft, R G; Lloyd, C J; Nice, E C; Whitehead, R H; Sawyer, W H; Burgess, A W

1986-01-01

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry. PMID:3015587

Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes,more » SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF. - Highlights: • Psoriasis-like skin inflammation increase dermal mast cells. • Keratinocyte produce stem cell factor in psoriasis-like skin inflammation. • Keratinocyte promote mast cell proliferation by stem cell factor dependent manner.« less

Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

PubMed

Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

2017-01-01

Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status.

PubMed

Turin, Ilaria; Delfanti, Sara; Ferulli, Federica; Brugnatelli, Silvia; Tanzi, Matteo; Maestri, Marcello; Cobianchi, Lorenzo; Lisini, Daniela; Luinetti, Ombretta; Paulli, Marco; Perotti, Cesare; Todisco, Elisabetta; Pedrazzoli, Paolo; Montagna, Daniela

2018-05-01

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients' NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.

Epidermal growth factor receptor inhibition by anti-CD147 therapy in cutaneous squamous cell carcinoma.

PubMed

Frederick, John W; Sweeny, Larissa; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L

2016-02-01

Advanced cutaneous squamous cell carcinoma (SCC) is an uncommon and aggressive malignancy. As a result, there is limited understanding of its biology and pathogenesis. CD147 and epidermal growth factor receptor (EGFR) have been identified as oncologically important targets, but their relationship remains undefined in cutaneous SCC. Multiple cutaneous SCC cell lines (Colo-16, SRB-1, and SRB-12), were treated in vitro with a range of chimeric anti-CD147 monoclonal antibody (mAb) (0, 50, 100, and 200 µg/mL) or transfected with a small interfering RNA against CD147 (SiCD147). Cell proliferation, migration (scratch wound healing assay), and protein expression was then assessed. In vivo, Colo-16 flank xenografts were treated anti-CD147 mAb (150 µg i.p. triweekly). After treatment with anti-CD147 (200 µg/mL), there was a significant decrease in proliferation for all cell lines relative to controls (p < .005). In addition, treatment with anti-CD147 (200 µg/mL) resulted in decreased cell migration for all cell lines, with an average of 43% reduction in closure compared to controls (p < .001). Colo-16 SiCD147 expression demonstrated similar reduction in proliferation and wound closure. Anti-CD147 antibody therapy and siRNA mediated reduction in CD147 expression were both found to decrease protein expression of EGFR, which correlated with a reduction in downstream total and phosphorylated protein kinase B (pAKT). Tumor growth in vivo was reduced for both the anti-CD147 treatment group and the SiCD147 group relative to controls. Inhibition and downregulation of CD147 in cutaneous SCC resulted in suppression of the malignant phenotype in vitro and in vivo, which may be mediated in part by an alteration in EGFR expression. As a result, CD147 may serve as a potential therapeutic target for advanced cutaneous SCC. © 2014 Wiley Periodicals, Inc.

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

PubMed Central

Bessette, Darrell C.; Tilch, Erik; Seidens, Tatjana; Quinn, Michael C. J.; Wiegmans, Adrian P.; Shi, Wei; Cocciardi, Sibylle; McCart-Reed, Amy; Saunus, Jodi M.; Simpson, Peter T.; Grimmond, Sean M.; Lakhani, Sunil R.; Khanna, Kum Kum; Waddell, Nic; Al-Ejeh, Fares; Chenevix-Trench, Georgia

2015-01-01

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance. PMID:25969993

21 CFR 358.703 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-04-01

... increased rate of shedding of dead epidermal cells of the scalp. (c) Psoriasis. A condition of the scalp or body characterized by irritation, itching, redness, and extreme excess shedding of dead epidermal cells..., redness, and excess shedding of dead epidermal cells. (e) Selenium sulfide, micronized. Selenium sulfide...

21 CFR 358.703 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-04-01

... increased rate of shedding of dead epidermal cells of the scalp. (c) Psoriasis. A condition of the scalp or body characterized by irritation, itching, redness, and extreme excess shedding of dead epidermal cells..., redness, and excess shedding of dead epidermal cells. (e) Selenium sulfide, micronized. Selenium sulfide...

21 CFR 358.703 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-04-01

... increased rate of shedding of dead epidermal cells of the scalp. (c) Psoriasis. A condition of the scalp or body characterized by irritation, itching, redness, and extreme excess shedding of dead epidermal cells..., redness, and excess shedding of dead epidermal cells. (e) Selenium sulfide, micronized. Selenium sulfide...

21 CFR 358.703 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... increased rate of shedding of dead epidermal cells of the scalp. (c) Psoriasis. A condition of the scalp or body characterized by irritation, itching, redness, and extreme excess shedding of dead epidermal cells..., redness, and excess shedding of dead epidermal cells. (e) Selenium sulfide, micronized. Selenium sulfide...

21 CFR 358.703 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-04-01

... increased rate of shedding of dead epidermal cells of the scalp. (c) Psoriasis. A condition of the scalp or body characterized by irritation, itching, redness, and extreme excess shedding of dead epidermal cells..., redness, and excess shedding of dead epidermal cells. (e) Selenium sulfide, micronized. Selenium sulfide...

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes

PubMed Central

Bernerd, Francoise; Sarasin, Alain; Magnaldo, Thierry

1999-01-01

Galectin-7 is a β-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300–305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis. PMID:10500176

Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

PubMed

Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

NASA Astrophysics Data System (ADS)

Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

2005-04-01

During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

PubMed

Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

2012-12-01

Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao Yongguang; Song Xing; Deng Xiyun

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 couldmore » regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.« less

Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity.

PubMed

Basu, D; Nguyen, T-T K; Montone, K T; Zhang, G; Wang, L-P; Diehl, J A; Rustgi, A K; Lee, J T; Weinstein, G S; Herlyn, M

2010-07-22

Variable drug responses among malignant cells within individual tumors may represent a barrier to their eradication using chemotherapy. Carcinoma cells expressing mesenchymal markers resist conventional and epidermal growth factor receptor (EGFR)-targeted chemotherapy. In this study, we evaluated whether mesenchymal-like sub-populations within human squamous cell carcinomas (SCCs) with predominantly epithelial features contribute to overall therapy resistance. We identified a mesenchymal-like subset expressing low E-cadherin (Ecad-lo) and high vimentin within the upper aerodigestive tract SCCs. This subset was both isolated from the cell lines and was identified in xenografts and primary clinical specimens. The Ecad-lo subset contained more low-turnover cells, correlating with resistance to the conventional chemotherapeutic paclitaxel in vitro. Epidermal growth factor induced less stimulation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways in Ecad-lo cells, which was likely due to lower EGFR expression in this subset and correlated with in vivo resistance to the EGFR-targeted antibody, cetuximab. The Ecad-lo and high E-cadherin subsets were dynamic in phenotype, showing the capacity to repopulate each other from single-cell clones. Taken together, these results provide evidence for a low-turnover, mesenchymal-like sub-population in SCCs with diminished EGFR pathway function and intrinsic resistance to conventional and EGFR-targeted chemotherapies.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells.

PubMed

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells

PubMed Central

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future. PMID:27362942

Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

PubMed

Lim, Kee Siang; Mimura, Kosaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kono, Koji

2018-04-20

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3β inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro

PubMed Central

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-01-01

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC. PMID:29371987

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro.

PubMed

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-12-26

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC.

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

PubMed Central

Lara-Guerra, Humberto; Kawashima, Hiroyuki; Sakai, Ryo; Jayachandran, Gitanjali; Majidi, Mourad; Mehran, Reza; Wang, Jing; Bekele, B. Nebiyou; Baladandayuthapani, Veerabhadran; Yoo, Suk-Young; Wang, Ying; Ying, Jun; Meng, Feng; Ji, Lin; Roth, Jack A.

2015-01-01

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR. PMID:26053020

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock

PubMed Central

Volk, Paige; Moreland, Jessica G.; Dunnwald, Martine

2016-01-01

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production. PMID:27035130

Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

PubMed Central

Tseng, Shih-Heng; Chou, Min-Yuan; Chu, I-Ming

2015-01-01

We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24 hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells. PMID:26056447

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

A STUDY OF THE COMPONENTS OF THE CORNIFIED EPITHELIUM OF HUMAN SKIN

PubMed Central

Matoltsy, A. Gedeon; Balsamo, Constance A.

1955-01-01

Pulverized cornified epithelium of human skin was divided into a "soluble fraction" and a "residue." About half of the "soluble fraction" proved to be soluble epidermal keratin (keratin A); the remainder, dialyzable substances of low molecular weight. The "residue" contained epidermal keratin and resistant cell membranes of cornified cells. Epidermal keratin was found to form an oriented and dense submicroscopic structure in the cornified cells. It showed high resistance toward strong acid and moderately strong alkali solutions as well as concentrated urea. In strong alkali, reducing substances, alkaline urea, and mixtures of reducing substance with alkali, epidermal keratin dissociated and yielded a non-dialyzable derivative of high molecular weight (keratin B) which resembled true proteins. The cell membranes of cornified cells showed higher resistance toward strong alkali and reducing substance than did epidermal keratin. PMID:13242598

Molecular characterization of breast cancer cell lines through multiple omic approaches.

PubMed

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways. These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results.

Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

PubMed Central

Kubo, Takuya; Murakami, Yuichi; Kawahara, Akihiko; Azuma, Koichi; Abe, Hideyuki; Kage, Masayoshi; Yoshinaga, Aki; Tahira, Tomoko; Hayashi, Kenshi; Arao, Tokuzo; Nishio, Kazuto; Rosell, Rafael; Kuwano, Michihiko; Ono, Mayumi

2012-01-01

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance. PMID:22815900

Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves.

PubMed

Kinoshita, Isao; Sanbe, Akiko; Yokomura, E-iti

2008-01-01

Changes in nuclear DNA content and cell size of adaxial and abaxial epidermal pavement cells were investigated using bright light-induced leaf expansion of Phaseolus vulgaris plants. In primary leaves of bean plants grown under high (sunlight) or moderate (ML; photon flux density, 163 micromol m(-2) s(-1)) light, most adaxial epidermal pavement cells had a nucleus with the 4C amount of DNA, whereas most abaxial pavement cells had a 2C nucleus. In contrast, plants grown under low intensity white light (LL; 15 micromol m(-2) s(-1)) for 13 d, when cell proliferation of epidermal pavement cells had already finished, had a 2C nuclear DNA content in most adaxial pavement cells. When these LL-grown plants were transferred to ML, the increase in irradiance raised the frequency of 4C nuclei in adaxial but not in abaxial pavement cells within 4 d. On the other hand, the size of abaxial pavement cells increased by 53% within 4 d of transfer to ML and remained unchanged thereafter, whereas adaxial pavement cells continuously enlarged for 12 d. This suggests that the increase in adaxial cell size after 4 d is supported by the nuclear DNA doubling. The different responses between adaxial and abaxial epidermal cells were not induced by the different light intensity at both surfaces. It was shown that adaxial epidermal cells have a different property than abaxial ones.

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice[W][OA

PubMed Central

Steffens, Bianka; Sauter, Margret

2009-01-01

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H2O2). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ-S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Gα subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H2O2 was nearly completely abolished, indicating that signaling through Gα is essential. Ethylene and H2O2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Gα. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Gα activity is regulated posttranscriptionally. PMID:19656904

Preclinical Evaluation of MET Inhibitor INC-280 With or Without the Epidermal Growth Factor Receptor Inhibitor Erlotinib in Non–Small-Cell Lung Cancer

PubMed Central

Lara, Matthew S.; Holland, William S.; Chinn, Danielle; Burich, Rebekah A.; Lara, Primo N.; Gandara, David R.; Kelly, Karen; Mack, Philip C.

2018-01-01

The MET inhibitor INC-280 restored sensitivity to erlotinib and promoted apoptosis in non–small-cell lung cancer models rendered resistant to erlotinib by hepatocyte growth factor. Background Although the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non–small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation. Methods Four NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9. Results As a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF. Conclusion Although the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a preclinical rationale for an ongoing phase 1 clinical trial of erlotinib plus INC-280 in EGFR-mutated NSCLC. PMID:28038979

Notch Signaling Regulates Late-Stage Epidermal Differentiation and Maintains Postnatal Hair Cycle Homeostasis

PubMed Central

Lin, Hsien-Yi; Kao, Cheng-Heng; Lin, Kurt Ming-Chao; Kaartinen, Vesa; Yang, Liang-Tung

2011-01-01

Background Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis. Methodology and Principal Findings We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes. Significance our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells. PMID:21267458

Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.

PubMed

Cheuk, Stanley; Wikén, Maria; Blomqvist, Lennart; Nylén, Susanne; Talme, Toomas; Ståhle, Mona; Eidsmo, Liv

2014-04-01

Psoriasis is a common and chronic inflammatory skin disease in which T cells play a key role. Effective treatment heals the skin without scarring, but typically psoriasis recurs in previously affected areas. A pathogenic memory within the skin has been proposed, but the nature of such site-specific disease memory is unknown. Tissue-resident memory T (TRM) cells have been ascribed a role in immunity after resolved viral skin infections. Because of their localization in the epidermal compartment of the skin, TRM may contribute to tissue pathology during psoriasis. In this study, we investigated whether resolved psoriasis lesions contain TRM cells with the ability to maintain and potentially drive recurrent disease. Three common and effective therapies, narrowband-UVB treatment and long-term biologic treatment systemically inhibiting TNF-α or IL-12/23 signaling were studied. Epidermal T cells were highly activated in psoriasis and a high proportion of CD8 T cells expressed TRM markers. In resolved psoriasis, a population of cutaneous lymphocyte-associated Ag, CCR6, CD103, and IL-23R expressing epidermal CD8 T cells was highly enriched. Epidermal CD8 T cells expressing the TRM marker CD103 responded to ex vivo stimulation with IL-17A production and epidermal CD4 T cells responded with IL-22 production after as long as 6 y of TNF-α inhibition. Our data suggest that epidermal TRM cells are retained in resolved psoriasis and that these cells are capable of producing cytokines with a critical role in psoriasis pathogenesis. We provide a potential mechanism for a site-specific T cell-driven disease memory in psoriasis.

Chemoprevention of Head and Neck Cancer by Simultaneous Blocking of Epidermal Growth Factor Receptor and Cyclooxygenase-2 Signaling Pathways: Preclinical and Clinical Studies

PubMed Central

Shin, Dong M.; Zhang, Hongzheng; Saba, Nabil; Chen, Amy; Nannapaneni, Sreenivas; Amin, A.R.M. Ruhul; Müller, Susan; Lewis, Melinda; Sica, Gabriel; Kono, Scott; Brandes, Johann C.; Grist, William; Moreno-Williams, Rachel; Beitler, Jonathan J.; Thomas, Sufi M.; Chen, Zhengjia; Shin, Hyung Ju C.; Grandis, Jennifer R.; Khuri, Fadlo R.; Chen, Zhuo Georgia

2013-01-01

Purpose We investigated the efficacy and underlying molecular mechanism of a novel chemopreventive strategy combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with cyclooxygenase-2 inhibitor (COX-2I). Experimental Design We examined the inhibition of tumor cell growth by combined EGFR-TKI (erlotinib) and COX-2I (celecoxib) treatment using head and neck cancer (HNC) cell lines and a preventive xenograft model. We studied the antiangiogenic activity of these agents and examined the affected signaling pathways by immunoblotting analysis in tumor cell lysates and immunohistochemistry (IHC) and enzyme immunoassay (EIA) analyses on the mouse xenograft tissues and blood, respectively. Biomarkers in these signaling pathways were studied by IHC, EIA, and an antibody array analysis in samples collected from participants in a phase I chemoprevention trial of erlotinib and celecoxib. Results The combined treatment inhibited HNC cell growth significantly more potently than either single agent alone in cell line and xenograft models, and resulted in greater inhibition of cell cycle progression at G1 phase than either single drug. The combined treatment modulated the EGFR and mTOR signaling pathways. A phase I chemoprevention trial of combined erlotinib and celecoxib revealed an overall pathologic response rate of 71% at time of data analysis. Analysis of tissue samples from participants consistently showed downregulation of EGFR, pERK and pS6 levels after treatment, which correlated with clinical response. Conclusion Treatment with erlotinib combined with celecoxib offers an effective chemopreventive approach through inhibition of EGFR and mTOR pathways, which may serve as potential biomarkers to monitor the intervention of this combination in the clinic. PMID:23422093

The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis.

PubMed

Kwak, Su-Hwan; Schiefelbein, John

2007-02-01

Cell-type patterning in the Arabidopsis root epidermis is achieved by a network of transcription factors and influenced by a position-dependent mechanism. The SCRAMBLED receptor-like kinase is required for the normal pattern to arise, but its precise role is not understood. Here we describe genetic and molecular studies to define the spatial and temporal role of SCM in epidermal patterning and its relationship to the transcriptional network. Our results suggest that SCM helps unspecified epidermal cells interpret their position in relation to the underlying cortical cells and establish distinct cell identities. Furthermore, SCM loss-of-function and overexpression analyses suggest that SCM influences cell fate through its negative transcriptional regulation of the WEREWOLF MYB gene in epidermal cells at the H position. We also find that SCM function is specifically required for patterning the post-embryonic root epidermis and not for the analogous epidermal cell-type patterning during embryogenesis or hypocotyl development. In addition, we show that two closely related SCM-like genes in Arabidopsis (SRF1 and SRF3) are not required alone or together with SCM for proper epidermal patterning. These findings help define the developmental and mechanistic role of SCM and suggest a new model for its action in root epidermal cell patterning.

Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

PubMed Central

Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

2013-01-01

After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

PubMed

Sánchez, O; Montesino, R; Toledo, J R; Rodríguez, E; Díaz, D; Royle, L; Rudd, P M; Dwek, R A; Gerwig, G J; Kamerling, J P; Harvey, D J; Cremata, J A

2007-08-15

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

First-line treatment of EGFR-mutant non-small-cell lung cancer: the role of erlotinib and other tyrosine kinase inhibitors

PubMed Central

Nguyen, Kim-Son H; Neal, Joel W

2012-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) were initially established as second- or third-line treatment of advanced non-small-cell lung cancer (NSCLC). Subsequent studies, including IPASS, OPTIMAL, and EURTAC, have demonstrated that these TKIs are effective first-line therapeutic options in patients with tumors harboring activating mutations in the EGFR gene. The TKIs are better tolerated than conventional chemotherapy, with frequent yet mild side effects such as rash and diarrhea, and rarely interstitial lung disease. Because most patients on TKIs develop resistance due to a variety of mechanisms, the use of TKIs in the acquired-resistance setting and in the setting of earlier-staged cancers is being extensively studied. Here we review the major trials leading to the established use of EGFR TKIs in NSCLC, followed by discussion of recently completed and ongoing trials using the next-generation EGFR inhibitor afatinib. PMID:23055691

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor.

PubMed

Gilmore, Andrew P; Valentijn, Anthony J; Wang, Pengbo; Ranger, Ann M; Bundred, Nigel; O'Hare, Michael J; Wakeling, Alan; Korsmeyer, Stanley J; Streuli, Charles H

2002-08-02

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.

HER2, MET and FGFR2 oncogenic driver alterations define distinct molecular segments for targeted therapies in gastric carcinoma.

PubMed

Liu, Y J; Shen, D; Yin, X; Gavine, P; Zhang, T; Su, X; Zhan, P; Xu, Y; Lv, J; Qian, J; Liu, C; Sun, Y; Qian, Z; Zhang, J; Gu, Y; Ni, X

2014-03-04

Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs. Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors. Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074. Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

PubMed Central

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A.

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding. PMID:25870605

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

PubMed

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

Epidermal growth factor receptor tyrosine kinase inhibitors: application in non-small cell lung cancer.

PubMed

Thomas, Melodie

2003-12-01

Despite treatment advances over the past decade, long-term survival for patients with non-small cell lung cancer (NSCLC) remains poor, and treatment options available after second-line therapy are limited. Increased understanding of cancer biology has led to the identification of several potential targets for treatment. The epidermal growth factor receptor (EGFR) belongs to a family of plasma membrane receptor tyrosine kinases that controls many important cellular functions, from growth and proliferation to cell death. This receptor is a particularly promising therapeutic target because it often is overexpressed in patients with NSCLC and has been implicated in the pathogenesis as well as the proliferation, invasion, and metastasis of lung cancer and other malignancies. New agents developed to inhibit EGFR function include small-molecule tyrosine kinase inhibitors, monoclonal antibodies to EGFR, and pan-EGFR inhibitors. Completed and ongoing clinical trials have shown that EGFR inhibitors have remarkable efficacy for patients with relapsed NSCLC. Among these, two phase 2 trials have shown that ZD1839 is effective when used as monotherapy. The response rates are comparable with those for docetaxel given in the second-line setting. Another phase 2 trial has shown that OSI-774 is effective in the same setting. Data from phase 3 trials indicate that adding an EGFR tyrosine kinase inhibitor to chemotherapy does not provide an additional survival benefit, as compared with standard chemotherapy alone for first-line treatment of NSCLC. It appears that EGFR tyrosine kinase inhibitors are safe and well tolerated by patients with cancer. Further studies will elucidate how these new agents can best be used for NSCLC and other tumor types.

Epidermal growth factor expression in esophageal adenocarcinoma: a clinically relevant target?

PubMed

Harper, Nicholas; Li, Yan; Farmer, Russell; Martin, Robert C G

2012-05-01

There has been recent widespread enthusiasm in epidermal growth factor (EGFR) as a molecularly active target in esophageal adenocarcinoma (EAC). However, there is limited data on the extent of EGFR expression in EAC. Thus, the aim of this study was to evaluated EGFR, pErk1/2, and total Erk1/2 expression in malignant and benign specimens. Baseline expression of EGFR in the human normal squamous, Barrett's, and EAC cell lines were determined as well as after bile acid treatment and curcumin pretreatment. In addition, EGFR expression was also evaluated in 60 matched normal and malignant EAC resected specimens. The in vitro studies in the Het-1a, BarT, and OE19 cell lines failed to show any measurable expression of EGFR via Western blot technique. The marker serving as the positive control for the study, MnSOD, showed expression in each cell line for all three treatment regimens at approximately 24 kDa EGFR, showing moderate staining in the malignant tumor specimens and low staining in the benign tissue specimens. pErk1/2 showed low staining in the malignant tumor specimens and no staining in the benign tissue specimens. Total Erk1/2 showed high staining in both the malignant tumor specimens and benign tissue specimens. The differences in the mean staining scores for the malignant versus benign tissue specimens for pErk1/2 and total Erk1/2 are not statistically significant (p = 0.0726 and p = 0.7054, respectively). Thus, in conclusion, EGFR expression has been confirmed to be limited to non-existent in EAC and thus its use as a clinically active target is limited at best. Prior to the use of these expensive anti-EGFR therapies, confirmation of overexpression should be verified.

Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT).

PubMed

Chen, Li-Mei; Verity, Nicole J; Chai, Karl X

2009-10-22

The glycosylphosphatidylinositol (GPI)-anchored epithelial extracellular membrane serine protease prostasin (PRSS8) is expressed abundantly in normal epithelia and essential for terminal epithelial differentiation, but down-regulated in human prostate, breast, and gastric cancers and invasive cancer cell lines. Prostasin is involved in the extracellular proteolytic modulation of the epidermal growth factor receptor (EGFR) and is an invasion suppressor. The aim of this study was to evaluate prostasin expression states in the transitional cell carcinomas (TCC) of the human bladder and in human TCC cell lines. Normal human bladder tissues and TCC on a bladder cancer tissue microarray (TMA) were evaluated for prostasin expression by means of immunohistochemistry. A panel of 16 urothelial and TCC cell lines were evaluated for prostasin and E-cadherin expression by western blot and quantitative PCR, and for prostasin gene promoter region CpG methylation by methylation-specific PCR (MSP). Prostasin is expressed in the normal human urothelium and in a normal human urothelial cell line, but is significantly down-regulated in high-grade TCC and lost in 9 (of 15) TCC cell lines. Loss of prostasin expression in the TCC cell lines correlated with loss of or reduced E-cadherin expression, loss of epithelial morphology, and promoter DNA hypermethylation. Prostasin expression could be reactivated by demethylation or inhibition of histone deacetylase. Re-expression of prostasin or a serine protease-inactive variant resulted in transcriptional up-regulation of E-cadherin. Loss of prostasin expression in bladder transitional cell carcinomas is associated with epithelial-mesenchymal transition (EMT), and may have functional implications in tumor invasion and resistance to chemotherapy.

Antitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor

NASA Astrophysics Data System (ADS)

Han, Duksun; Cho, Jin Hyoung; Lee, Ra Ham; Bang, Woong; Park, Kyungho; Kim, Minseok S.; Shim, Jung-Hyun; Chae, Jung-Il; Moon, Se Youn

2017-02-01

Human colorectal cancer cell lines (HT29 and HCT116) were exposed to dielectric barrier discharge (DBD) plasma at atmospheric pressure to investigate the anticancer capacity of the plasma. The dose- and time-dependent effects of DBDP on cell viability, regulation of transcription factor Sp1, cell-cycle analysis, and colony formation were investigated by means of MTS assay, DAPI staining, propidium iodide staining, annexin V-FITC staining, Western blot analysis, RT-PCR analysis, fluorescence microscopy, and anchorage-independent cell transformation assay. By increasing the duration of plasma dose times, significant reductions in the levels of both Sp1 protein and Sp1 mRNA were observed in both cell lines. Also, expression of negative regulators related to the cell cycle (such as p53, p21, and p27) was increased and of the positive regulator cyclin D1 was decreased, indicating that the plasma treatment led to apoptosis and cell-cycle arrest. In addition, the sizes and quantities of colony formation were significantly suppressed even though two cancer promoters, such as TPA and epidermal growth factor, accompanied the plasma treatment. Thus, plasma treatment inhibited cell viability and colony formation by suppressing Sp1, which induced apoptosis and cell-cycle arrest in these two human colorectal cancer cell lines.

Inositol-C2-PAF down-regulates components of the antigen presentation machinery in a 2D-model of epidermal inflammation.

PubMed

Semini, Geo; Hildmann, Annette; Klein, Andreas; Lucka, Lothar; Schön, Margarete; Schön, Michael P; Shmanai, Vadim; Danker, Kerstin

2014-02-01

In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis. Copyright © 2013 Elsevier Inc. All rights reserved.

Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

PubMed

Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

2016-10-01

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. Copyright © 2016 Elsevier B.V. All rights reserved.

Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

First-line therapy for advanced non-small cell lung cancer with activating EGFR mutation: is combined EGFR-TKIs and chemotherapy a better choice?

PubMed

Wang, Shuyun; Gao, Aiqin; Liu, Jie; Sun, Yuping

2018-03-01

As the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation, EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have significantly improved the median progression-free survival (PFS) up to 18.9 months. However, almost all patients eventually develop acquired resistance to EGFR-TKIs, which limits the first-line PFS. To overcome the resistance and improve overall survival, researchers have tried to identify the resistance mechanisms and develop new treatment strategies, among which a combination of EGFR-TKIs and cytotoxic chemotherapy is one of the hotspots. The data from preclinical and clinical studies on combined EGFR-TKIs and chemotherapy have shown very interesting results. Here, we reviewed the available preclinical and clinical studies on first-line EGFR-TKIs-chemotherapy combination in patients with advanced NSCLC harboring activating EGFR mutation, aiming to provide evidences for more potential choices and shed light on clinical treatment.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booth, Brian W., E-mail: brbooth@clemson.edu; Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC 29634; Boulanger, Corinne A.

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signalingmore » pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.« less

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics.

PubMed

Booth, Brian W; Boulanger, Corinne A; Anderson, Lisa H; Jimenez-Rojo, Lucia; Brisken, Cathrin; Smith, Gilbert H

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG(-/-) mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro. Copyright 2009 Elsevier Inc. All rights reserved.

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma.

PubMed

Rainusso, N; Brawley, V S; Ghazi, A; Hicks, M J; Gottschalk, S; Rosen, J M; Ahmed, N

2012-03-01

Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.

An AKT activity threshold regulates androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines.

PubMed

Paliouras, Miltiadis; Diamandis, Eleftherios P

2008-06-01

The androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancer in vitro model. Using the cell line PC3(AR)(2), we stimulated cells with 5-alpha-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)(2) cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose an in vitro androgen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.

Planar cell polarity pathway in vertebrate epidermal development, homeostasis and repair

PubMed Central

Dworkin, Sebastian; Jane, Stephen M

2011-01-01

The planar cell polarity (PCP) pathway plays a critical role in diverse developmental processes that require coordinated cellular movement, including neural tube closure and renal tubulogenesis. Recent studies have demonstrated that this pathway also has emerging relevance to the epidermis, as PCP signaling underpins many aspects of skin biology and pathology, including epidermal development, hair orientation, stem cell division and cancer. Coordinated cellular movement required for epidermal repair in mammals is also regulated by PCP signaling, and in this context, a new PCP gene encoding the developmental transcription factor Grainyhead-like 3 (Grhl3) is critical. This review focuses on the role that PCP signaling plays in the skin across a variety of epidermal functions and highlights perturbations that induce epidermal pathologies. PMID:22041517

Genetic analysis of Ras genes in epidermal development and tumorigenesis

PubMed Central

Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

2013-01-01

Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Epidermal Cell Death in Rice Is Regulated by Ethylene, Gibberellin, and Abscisic Acid

PubMed Central

Steffens, Bianka; Sauter, Margret

2005-01-01

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth. PMID:16169967

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

NASA Astrophysics Data System (ADS)

Lucas, Leanne J.; Chen, Xiaoke K.; Smith, Aaron J.; Korbelik, Mladen; Zeng, Haishan; Lee, Patrick W. K.; Hewitt, Kevin Cecil

2015-01-01

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

Inactivated Wnt signaling in resveratrol-treated epidermal squamous cancer cells and its biological implication

PubMed Central

Liu, Zhi-Li; Li, Hong; Liu, Jia; Wu, Mo-Li; Chen, Xiao-Yan; Liu, Li-Hong; Wang, Qian

2017-01-01

Squamous cell carcinoma (SCC) is the most common epidermal malignancy, and Wnt/β-catenin signaling is frequently activated in SCC. Resveratrol prevents rodent epidermal carcinogenesis, while its effect on human epidermal cancer remains unknown. To address this issue, the impact of resveratrol on the growth and Wnt signaling of skin SCC Colo16 cells were investigated at the cellular and molecular biology levels by flow cytometry, immunocytochemistry, reverse transcription-polymerase chain reaction, western blotting and β-catenin-specific small interfering RNA (siRNA) transfection. Resveratrol (100 µM) suppressed cell growth and induced apoptosis in Colo16 cells. Wnt2 and its downstream genes were downregulated, which was accompanied by increased expression of the Wnt signaling inhibitor Axin2. Transfection with a β-catenin-specific siRNA did not affect cell growth but enhanced the resveratrol susceptibility of Colo16 transfectants. The present results suggest the inhibitory effects of resveratrol on epidermal SCCs and inactivation of Wnt signaling as one of the resveratrol-caused molecular events in Colo16 cells. β-catenin oriented siRNA is insufficient to induce cell crisis, implicating the presence of more critical cancer-associated element(s) as the target in Colo16 cells. PMID:28781663

Modeling the Morphogenesis of Epidermal Tissues on the Surface of a 3D Last

NASA Astrophysics Data System (ADS)

McCleery, W. Tyler; Crews, Sarah M.; Mashburn, David N.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

2014-03-01

Embryogenesis in the fruit fly Drosophila melanogaster is coordinated by the interaction of cells in adjacent tissues. For some events of embryogenesis, e.g., dorsal closure, two-dimensional models have been sufficient to elucidate the relevant cell and tissue mechanics. Here, we describe a new three-dimensional cell-level finite element model for investigating germ band retraction - a morphogenetic event where one epidermal tissue, the germ band, initially wraps around the posterior end of the ellipsoidal embryo. This tissue then retracts with a mechanical assist from contraction of cells in a second epidermal tissue, the amnioserosa. To speed simulation run times and focus on the relevant tissues, we only model epidermal tissue interactions. Epidermal cells are defined as polygons constrained to lie on the surface of the ellipsoidal last, but have adjustable parameters such as edge tensions and cell pressures. Tissue movements are simulated by balancing these dynamic cell-level forces with viscous resistance and allowing cells to exchange neighbors. Our choice of modeling parameters is informed by in vivo measurements of cell-level forces using laser microsurgery. We use this model to investigate the multicellular stress fields in normal and aberrant development.

Functions of Vγ4 T Cells and Dendritic Epidermal T Cells on Skin Wound Healing

PubMed Central

Li, Yashu; Wu, Jun; Luo, Gaoxing; He, Weifeng

2018-01-01

Wound healing is a complex and dynamic process that progresses through the distinct phases of hemostasis, inflammation, proliferation, and remodeling. Both inflammation and re-epithelialization, in which skin γδ T cells are heavily involved, are required for efficient skin wound healing. Dendritic epidermal T cells (DETCs), which reside in murine epidermis, are activated to secrete epidermal cell growth factors, such as IGF-1 and KGF-1/2, to promote re-epithelialization after skin injury. Epidermal IL-15 is not only required for DETC homeostasis in the intact epidermis but it also facilitates the activation and IGF-1 production of DETC after skin injury. Further, the epidermal expression of IL-15 and IGF-1 constitutes a feedback regulatory loop to promote wound repair. Dermis-resident Vγ4 T cells infiltrate into the epidermis at the wound edges through the CCR6-CCL20 pathway after skin injury and provide a major source of IL-17A, which enhances the production of IL-1β and IL-23 in the epidermis to form a positive feedback loop for the initiation and amplification of local inflammation at the early stages of wound healing. IL-1β and IL-23 suppress the production of IGF-1 by DETCs and, therefore, impede wound healing. A functional loop may exist among Vγ4 T cells, epidermal cells, and DETCs to regulate wound repair.

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana.

PubMed

Pietra, Stefano; Lang, Patricia; Grebe, Markus

2015-03-01

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non-hair cells and represents a model system for studying the control of cell-fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell-fate stabilization. Our work opens the door for future studies addressing SAB-dependent functions of the cytoskeleton during root epidermal patterning. © 2014 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.

Roles of CONSTITUTIVE PHOTOMORPHOGENIC 10 in Arabidopsis stomata development

PubMed Central

Delgado, Dolores; Ballesteros, Isabel; Mena, Montaña; Fenoll, Carmen

2012-01-01

Stomata are epidermal bi-celled structures that differentiate within special cell lineages initiated by a subset of protodermal cells. Recently, we showed that the Arabidopsis photomorphogenic repressor COP10 controls specific cell-lineage and cell-signaling developmental mechanisms in stomatal lineages. Loss-of-function cop10-1 mutant cotyledons and leaves produced (in the light and in the dark) abundant stomatal clusters, but nonlineage epidermal cells were not affected. Here we examine COP10 role in hypocotyls, cylindrical organs displaying a distinct epidermal organization with alternate files of protruding and non-protruding cells, with the latter producing a limited number of stomata. COP10 prevents stomatal clusters and restricts stomata production in hypocotyls; these roles are specific to lineage cells as in cotyledons, since COP10 loss of function does not elicit stomatal fate in nonlineage cells; COP10 also sustains the directional cell expansion of all hypocotyl epidermal cell types, and seems necessary for the differentiation between protruding and non-protruding cell files. PMID:22836493

Activity of insulin growth factors and shrimp neurosecretory organ extracts on a lepidopteran cell line.

PubMed

Hatt, P J; Liebon, C; Morinière, M; Oberlander, H; Porcheron, P

1997-01-01

Ecdysteroids, or molting hormones, have been proven to be key differentiation regulators for epidermal cells in the postembryonic development of arthropods. Regulators of cell proliferation, however, remain largely unknown. To date, no diffusible insect peptidic growth factors have been characterized. Molecules structurally related to insulin have been discovered in insects, as in other eucaryotes. We developed in vitro tests for the preliminary characterization of potential growth factors in arthropods by adapting the procedures designed to detect such factors in vertebrates to an insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth. We verified the ability of these tests to measure the proliferation of IAL-PID2 cells. We tested mammalian insulin and insulin-like growth factors (IGF-I, IGF-II). Following an arrest of cell proliferation by serum deprivation, IGF-I and IGF-II caused partial resumption of the cell cycle, evidenced by DNA synthesis. In contrast, the addition of 20-hydroxyecdysone arrested the proliferation of the IAL-PID2 cells. The cell line was then used in a test for functional characterization of potential growth factors originating from the penaeid shrimp, Penaeus vannamei. Crude extracts of neurosecretory and nervous tissues, eyestalks, and ventral neural chain compensated for serum deprivation and stimulated completion of mitosis. Arch.

Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

PubMed

Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

2017-05-01

Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling*

PubMed Central

Kim, Jong Hyun; Wang, Aibing; Conti, Mary Anne; Adelstein, Robert S.

2012-01-01

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways. PMID:22718763

Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

PubMed Central

TU, DOM-GENE; YU, YUN; LEE, CHE-HSIN; KUO, YU-LIANG; LU, YIN-CHE; TU, CHI-WEN; CHANG, WEN-WEI

2016-01-01

Hinokitiol, alternatively known as β-thujaplicin, is a tropolone-associated natural compound with antimicrobial, anti-inflammatory and antitumor activity. Breast cancer stem/progenitor cells (BCSCs) are a subpopulation of breast cancer cells associated with tumor initiation, chemoresistance and metastatic behavior, and may be enriched by mammosphere cultivation. Previous studies have demonstrated that BCSCs exhibit vasculogenic mimicry (VM) activity via the epidermal growth factor receptor (EGFR) signaling pathway. The present study investigated the anti-VM activity of hinokitiol in BCSCs. At a concentration below the half maximal inhibitory concentration, hinokitiol inhibited VM formation of mammosphere cells derived from two human breast cancer cell lines. Hinokitiol was additionally indicated to downregulate EGFR protein expression in mammosphere-forming BCSCs without affecting the expression of messenger RNA. The protein stability of EGFR in BCSCs was also decreased by hinokitiol. The EGFR protein expression and VM formation capability of hinokitiol-treated BCSCs were restored by co-treatment with MG132, a proteasome inhibitor. In conclusion, the present study indicated that hinokitiol may inhibit the VM activity of BCSCs through stimulating proteasome-mediated EGFR degradation. Hinokitiol may act as an anti-VM agent, and may be useful for the development of novel breast cancer therapeutic agents. PMID:27073579

Inhibitors of the epidermal growth factor receptor in apple juice extract.

PubMed

Kern, Melanie; Tjaden, Zeina; Ngiewih, Yufanyi; Puppel, Nicole; Will, Frank; Dietrich, Helmut; Pahlke, Gudrun; Marko, Doris

2005-04-01

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.

A theoretical approach to the relationship between wettability and surface microstructures of epidermal cells and structured cuticles of flower petals

PubMed Central

Taneda, Haruhiko; Watanabe-Taneda, Ayako; Chhetry, Rita; Ikeda, Hiroshi

2015-01-01

Background and Aims The epidermal surface of a flower petal is composed of convex cells covered with a structured cuticle, and the roughness of the surface is related to the wettability of the petal. If the surface remains wet for an excessive amount of time the attractiveness of the petal to floral visitors may be impaired, and adhesion of pathogens may be promoted. However, it remains unclear how the epidermal cells and structured cuticle contribute to surface wettability of a petal. Methods By considering the additive effects of the epidermal cells and structured cuticle on petal wettability, a thermodynamic model was developed to predict the wetting mode and contact angle of a water droplet at a minimum free energy. Quantitative relationships between petal wettability and the geometries of the epidermal cells and the structured cuticle were then estimated. Measurements of contact angles and anatomical traits of petals were made on seven herbaceous species commonly found in alpine habitats in eastern Nepal, and the measured wettability values were compared with those predicted by the model using the measured geometries of the epidermal cells and structured cuticles. Key Results The model indicated that surface wettability depends on the height and interval between cuticular steps, and on a height-to-width ratio for epidermal cells if a thick hydrophobic cuticle layer covers the surface. For a petal epidermis consisting of lenticular cells, a repellent surface results when the cuticular step height is greater than 0·85 µm and the height-to-width ratio of the epidermal cells is greater than 0·3. For an epidermis consisting of papillate cells, a height-to-width ratio of greater than 1·1 produces a repellent surface. In contrast, if the surface is covered with a thin cuticle layer, the petal is highly wettable (hydrophilic) irrespective of the roughness of the surface. These predictions were supported by the measurements of petal wettability made on flowers of alpine species. Conclusions The results indicate that surface roughness caused by epidermal cells and a structured cuticle produces a wide range of petal wettability, and that this can be successfully modelled using a thermodynamic approach. PMID:25851137

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells

PubMed Central

Gu, Ha Ra; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-01-01

Background/Aims Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Methods Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Results Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Conclusions Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future. PMID:25834802

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells.

PubMed

Gu, Ha Ra; Park, Su Cheol; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-03-01

Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.

Dasatinib and Doxorubicin Treatment of Sarcoma Initiating Cells: A Possible New Treatment Strategy.

PubMed

Aggerholm-Pedersen, Ninna; Demuth, Christina; Safwat, Akmal; Meldgaard, Peter; Kassem, Moustapha; Sandahl Sorensen, Boe

2016-01-01

Background. One of the major challenges affecting sarcoma treatment outcome, particularly that of metastatic disease, is resistance to chemotherapy. Cancer-initiating cells are considered a major contributor to this resistance. Methods. An immortalised nontransformed human stromal (mesenchymal) stem cell line hMSC-TERT4 and a transformed cell line hMSC-TERT20-CE8, known to form sarcoma-like tumours when implanted in immune-deficient mice, were used as models. Receptor tyrosine kinase (RTK) activation was analysed by RTK arrays and cellular viability after tyrosine kinases inhibitor (TKI) treatment with or without doxorubicin was assessed by MTS assay. Results. Initial results showed that the hMSC-TERT4 was more doxorubicin-sensitive while hMSC-TERT20-CE8 was less doxorubicin-sensitive evidenced by monitoring cell viability in the presence of doxorubicin at different doses. The epidermal growth factor receptor (EGFR) was activated in both cell lines. However hMSC-TERT20-CE8 exhibited significantly higher expression of the EGFR ligands. EGFR inhibitors such as erlotinib and afatinib alone or in combination with doxorubicin failed to further decrease cell viability of hMSC-TERT20-CE8. However, inhibition with the TKI dasatinib in combination with doxorubicin decreased cell viability of the hMSC-TERT20-CE8 cell line. Conclusion. Our results demonstrate that dasatinib, but not EGFR-directed treatment, can decrease cell viability of stromal cancer stem cells less sensitive to doxorubicin.

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

PubMed

Wang, Yun-Liang; Dong, Feng-Lin; Yang, Jian; Li, Zhi; Zhi, Qiao-Ming; Zhao, Xin; Yang, Yong; Li, De-Chun; Shen, Xiao-Chun; Zhou, Jin

2015-01-01

Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

PubMed

Heindl, Stefan; Eggenstein, Evelyn; Keller, Simone; Kneissl, Julia; Keller, Gisela; Mutze, Kathrin; Rauser, Sandra; Gasteiger, Georg; Drexler, Ingo; Hapfelmeier, Alexander; Höfler, Heinz; Luber, Birgit

2012-05-01

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

Dynamics of keratinocytes in vivo using HO labeling: a sensitive marker of epidermal proliferation state.

PubMed

Hsieh, Elaine A; Chai, Christine M; de Lumen, Benito O; Neese, Richard A; Hellerstein, Marc K

2004-09-01

A heavy water ((2)H(2)O) labeling method recently developed to measure cell proliferation in vivo is applied here to the measurement of murine epidermal cell turnover and to investigate conditions in which keratinocyte proliferation is either inhibited or stimulated. The technique is based on incorporation of (2)H(2)O into the deoxyribose moiety of deoxyribonucleotides in dividing cells. Label incorporation and die-away studies in cells isolated from C57BL/6J mouse epidermis revealed the replacement rate to be 34%-44% per wk (half-life of 1.6-2 wk). The kinetics provided evidence of a non-proliferative subpopulation of cells (10%-15% of the total) within the epidermis. Topical administration of 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate for 3 wk increased epidermal cell proliferation by 55% in SENCAR mice. Topical addition of lunasin, an anti-mitotic agent from soy, decreased epidermal cell proliferation modestly though significantly (16% given alone, 9% given with carcinogens). Caloric restriction (by 33% of energy intake) for 4 wk decreased the epidermal cell proliferation rate by 45% in C57BL/6J mice. In summary, epidermal cell proliferation can be measured in vivo using (2)H(2)O labeling in normal, hyper- and hypo-proliferative conditions. Potential applications of this inherently safe method in humans might include studies of psoriasis, wound healing, chemopreventive agents, and caloric intake.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

PubMed

Guzmán-Uribe, Daniela; Alvarado-Estrada, Keila Neri; Pierdant-Pérez, Mauricio; Torres-Álvarez, Bertha; Sánchez-Aguilar, Jesus Martin; Rosales-Ibáñez, Raúl

2017-01-01

The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Emerging Phytochemicals for the Prevention and Treatment of Head and Neck Cancer.

PubMed

Katiyar, Santosh K

2016-11-24

Despite the development of more advanced medical therapies, cancer management remains a problem. Head and neck squamous cell carcinoma (HNSCC) is a particularly challenging malignancy and requires more effective treatment strategies and a reduction in the debilitating morbidities associated with the therapies. Phytochemicals have long been used in ancient systems of medicine, and non-toxic phytochemicals are being considered as new options for the effective management of cancer. Here, we discuss the growth inhibitory and anti-cell migratory actions of proanthocyanidins from grape seeds (GSPs), polyphenols in green tea and honokiol, derived from the Magnolia species. Studies of these phytochemicals using human HNSCC cell lines from different sub-sites have demonstrated significant protective effects against HNSCC in both in vitro and in vivo models. Treatment of human HNSCC cell lines with GSPs, (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic component of green tea or honokiol reduced cell viability and induced apoptosis. These effects have been associated with inhibitory effects of the phytochemicals on the epidermal growth factor receptor (EGFR), and cell cycle regulatory proteins, as well as other major tumor-associated pathways. Similarly, the cell migration capacity of HNSCC cell lines was inhibited. Thus, GSPs, honokiol and EGCG appear to be promising bioactive phytochemicals for the management of head and neck cancer.

Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.

PubMed

Lin, Y; Schiefelbein, J

2001-10-01

A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

Steroids are required for epidermal cell fate establishment in Arabidopsis roots.

PubMed

Kuppusamy, Kavitha T; Chen, Andrew Y; Nemhauser, Jennifer L

2009-05-12

The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate.

Steroids are required for epidermal cell fate establishment in Arabidopsis roots

PubMed Central

Kuppusamy, Kavitha T.; Chen, Andrew Y.; Nemhauser, Jennifer L.

2009-01-01

The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate. PMID:19416891

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

PubMed Central

GUZMÁN-URIBE, Daniela; ALVARADO-ESTRADA, Keila Neri; PIERDANT-PÉREZ, Mauricio; TORRES-ÁLVAREZ, Bertha; SÁNCHEZ-AGUILAR, Jesus Martin; ROSALES-IBÁÑEZ, Raúl

2017-01-01

Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues. PMID:28403359

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

PubMed

Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

2016-06-01

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

Epidermal Notch signalling: differentiation, cancer and adhesion.

PubMed

Watt, Fiona M; Estrach, Soline; Ambler, Carrie A

2008-04-01

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

Study of lung-metastasized prostate cancer cell line chemotaxis to epidermal growth factor with a BIOMEMS device

NASA Astrophysics Data System (ADS)

Tata, Uday; Rao, Smitha M. N.; Sharma, Akash; Pabba, Krishna; Pokhrel, Kushal; Adhikari, Bandita; Lin, Victor K.; Chiao, J.-C.

2012-09-01

Understanding the effects of different growth factors on cancer metastasis will enable researchers to develop effective post-surgery therapeutic strategies to stop the spread of cancer. Conventional Boyden chamber assays to evaluate cell motility in metastasis studies require high volumes of reagents and are impractical for high-throughput analysis. A microfluidic device was designed for arrayed assaying of prostate cancer cell migration towards different growth factors. The device was created with polydimethylsiloxane (PDMS) and featured two wells connected by 10 micro channels. One well was for cell seeding and the other well for specific growth factors. Each channel has a width of 20 μm, a length of 1 mm and a depth of 10 μm. The device was placed on a culture dish and primed with growth media. Lung-metastasized cells in suspension of RPMI 1640 media1 supplemented with 2% of fetal bovine serum (FBS) were seeded in the cell wells. Cell culture media with epidermal growth factor (EGF) of 25, 50, 75, 100 and 125 ng ml-1 concentrations were individually added in the respective growth factor wells. A 5-day time-lapsed study of cell migration towards the chemoattractant was performed. The average numbers of cells per device in the microchannels were obtained for each attractant condition. The results indicated migration of cells increased from 50 to 100 ng ml-1 of EGF and significantly decreased at 125 ng ml-1 of EGF, as compared to control.

A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells.

PubMed

Lee, Hyunseung; Kim, Soo Jung; Jung, Kyung Hee; Son, Mi Kwon; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

2013-08-01

Lung cancer is the leading cause of cancer-related mortality in the world, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases. Since more than 60% of NSCLC cases express the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors are used to treat NSCLC. However, due to the acquired resistance associated with EGFR-targeted therapy, other strategies for the treatment of NSCLC are urgently needed. Therefore, we investigated the anticancer effects of a novel phosphatidylinositol 3-kinase α (PI3Kα) inhibitor, HS-173, in human NSCLC cell lines. HS-173 demonstrated anti-proliferative effects in NSCLC cells and effectively inhibited the PI3K signaling pathway in a dose‑dependent manner. In addition, it induced cell cycle arrest at G2/M phase as well as apoptosis. Taken together, our results demonstrate that HS-173 exhibits anticancer activities, including the induction of apoptosis, by blocking the PI3K/Akt/mTOR pathway in human NSCLC cell lines. We, therefore, suggest that this novel drug could potentially be used for targeted NSCLC therapy.

Human eccrine sweat gland cells turn into melanin-uptaking keratinocytes in dermo-epidermal skin substitutes.

PubMed

Böttcher-Haberzeth, Sophie; Biedermann, Thomas; Pontiggia, Luca; Braziulis, Erik; Schiestl, Clemens; Hendriks, Bart; Eichhoff, Ossia M; Widmer, Daniel S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2013-02-01

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

Biologic properties of the thymocyte-activating factor (CETAF) produced by a rabbit corneal cell line (SIRC).

PubMed

Grabner, G; Luger, T A; Luger, B M; Smolin, G; Oh, J O

1983-05-01

Rabbit corneal epithelial cell cultures produce a cytokine (CETAF) that greatly enhances the proliferation of C3H/HeJ mouse thymocytes. The rabbit corneal cell line SIRC was used to generate CETAF activity in the culture supernatant. CETAF was then partially purified by Sephacryl S-200 gel filtration, where peaks of activity eluted in a molecular weight range of 95,000-55,000 (CETAF I) and 30,000-15,000 (CETAF II). Similar to the epidermal cell-derived thymocyte-activating factor (ETAF), CETAF (I and II) stimulated the growth of a human dermal fibroblast line (CRL 1445) in a dose-dependent manner, but failed to enhance the proliferation of an Interleukin 2 (IL 2)-dependent T-cell line (CT 6). Although CETAF did not exhibit any IL 2 activity, it clearly enhanced the IL 2 production by C3H/HeJ mouse splenocytes stimulated with suboptimal doses of lectins. Crude SIRC supernatants as well as the partially purified CETAF preparations showed a marked inhibition of polymorphonuclear neutrophil migration at high concentrations, but were significantly chemotactic when diluted samples were tested. CETAF release by SIRC cells was increased by stimulation with mitomycin C, phorbolmyristate acetate, hydroxyurea, silica, lipopolysaccaride B, and when the cells were cultured under serum-free conditions. These observations suggest that corneal epithelial cells may not only interact with the immune system in a way similar to keratinocytes, but may also stimulate corneal stromal cell through the production of CETAF.

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

PubMed

Cacci, E; Villa, A; Parmar, M; Cavallaro, M; Mandahl, N; Lindvall, O; Martinez-Serrano, A; Kokaia, Z

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

Histone deacetylases inhibitor Trichostatin A ameliorates DNFB-induced allergic contact dermatitis and reduces epidermal Langerhans cells in mice

PubMed Central

Shi, Yu-Ling; Gu, Jun; Park, Jun-Yang; Xu, Ying-Ping; Yu, Fu-Shin; Zhou, Li; Mi, Qing-Sheng

2012-01-01

Background Histone deacetylases (HDACs) influence chromatin organization, representing a key epigenetic regulatory mechanism in cells. Trichostatin A (TSA), a potent HDAC inhibitor, has anti-tumor and anti-inflammatory effects. Allergic contact dermatitis (ACD) is a T-cell-mediated inflammatory reaction in skin and is regulated by epidermal Langerhans cells (LCs). Objective The aim of this study was to investigate if TSA treatment prevents 2,4-dinitrofluorobenzene (DNFB)-induced ACD in mice and regulates epidermal LCs and other immune cells during ACD development. Methods ACD was induced by sensitizing and challenging with DNFB topically. Mice were treated intraperitoneally with TSA or vehicle DMSO as a control every other day before and during induction of ACD. The ear swelling response was measured and skin biopsies from sensitized skin areas were obtained for histology. Epidermal cells, thymus, spleens and skin draining lymph nodes were collected for immune staining. Results TSA treatment ameliorated skin lesion severity of DNFB-induced ACD. The percentages of epidermal LCs and splenic DCs as well as LC maturation were significantly reduced in TSA-treated mice. However, TSA treatment did not significantly affect the homeostasis of conventional CD4+ and CD8+ T cells, Foxp3+CD4+ regulatory T cells, iNKT cells, and γδ T cells in thymus, spleen and draining lymph nodes (dLNs). Furthermore, there were no significant differences in IL-4 and IFN-γ-producing T cells and iNKT cells between TSA- and DMSO-treated mice. Conclusion Our findings suggest that TSA may ameliorate ACD through the regulation of epidermal LCs and HDACs could serve as potential therapeutic targets for ACD and other LCs-related skin diseases. PMID:22999682

Gold nanoparticles-conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt-mediated pathway in breast cancer cell lines (MCF-7 and MDA-MB-231).

PubMed

Balakrishnan, Solaimuthu; Mukherjee, Sudip; Das, Sourav; Bhat, Firdous Ahmad; Raja Singh, Paulraj; Patra, Chitta Ranjan; Arunakaran, Jagadeesan

2017-06-01

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles-conjugated quercetin (AuNPs-Qu-5) in MCF-7 and MDA-MB-231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs-Qu-5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V-FITC staining were performed. AuNPs and AuNPs-Qu-5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs-Qu-5 exhibited lower IC 50 value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs-Qu-5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)-mediated PI3K/Akt/GSK-3β signalling by immunoblotting and immunocytochemistry. The pro-apoptotic proteins (Bax, Caspase-3) were found to be up regulated and anti-apoptotic protein (Bcl-2) was down regulated on treatment with AuNPs-Qu-5. Additionally, AuNPs-Qu-5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK-3β. In conclusion, administration of AuNPs-Qu-5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs-Qu-5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Production and emission of volatile compounds by petal cells.

PubMed

Baudino, Sylvie; Caissard, Jean-Claude; Bergougnoux, Véronique; Jullien, Frédéric; Magnard, Jean-Louis; Scalliet, Gabriel; Cock, J Mark; Hugueney, Philippe

2007-11-01

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa x hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission.

Production and Emission of Volatile Compounds by Petal Cells

PubMed Central

Caissard, Jean-Claude; Bergougnoux, Véronique; Jullien, Frédéric; Magnard, Jean-Louis; Scalliet, Gabriel; Cock, J Mark; Hugueney, Philippe

2007-01-01

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa × hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission. PMID:19704548

The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice.

PubMed

Troy, Tammy-Claire; Li, Yuhua; O'Malley, Lauren; Turksen, Kursad

2007-02-01

The importance of the epidermal permeability barrier (EPB) in protecting the mammalian species against harmful UV irradiation, microorganism invasion and water loss is well recognized, as is the role of calcium (Ca(2+)) in keratinocyte differentiation, cell-cell contact and the EPB. In a previous study, we reported that the overexpression of the Ca(2+)-sensing receptor (CaSR) in the undifferentiated basal cells of the epidermis induced a modified epidermal differentiation program including an accelerated EPB formation in transgenic mice, suggesting a role for CaSR signaling in the differentiation of embryonic epidermal cells during development. We now describe the expression profile of claudins (Cldns) and keratin markers in the accelerated EPB formation of K14-CaSR transgenic mice during development as compared to the wild type from E12.5 to newborn stages. Our data show that the transgenic epidermis undergoes an advanced epidermal differentiation program as compared to the wild type as evidenced morphologically as well as by the expression of K14, K1, loricrin, Cldn6, Cldn18 and Cldn11. In addition, we report for the first time the sequential expression of Cldns in epidermal development and describe that the localization of some Cldns change within the epidermis as it matures. Furthermore, we demonstrate that Cldn6 is expressed very early in epidermal morphogenesis, followed by Cldn18, Cldn11 and Cldn1.

Erythropoietin Augments Survival of Glioma Cells After Radiation and Temozolomide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hassouna, Imam; Sperling, Swetlana; Kim, Ella

2008-11-01

Purpose: Despite beneficial effects of irradiation/chemotherapy on survival of glioblastoma (GBM) patients, collateral damage to intact neural tissue leads to 'radiochemobrain' and reduced quality of life in survivors. For prophylactic neuroprotection, erythropoietin (EPO) is a promising candidate, provided that concerns regarding potential tumor promoting effects are alleviated. Methods and Materials: Human GBM-derived cell lines U87, G44, G112, and the gliosarcoma-derived line G28 were treated with EPO, with and without combinations of irradiation or temozolomide (TMZ). Responsiveness of glioma cells to EPO was measured by cell migration from spheroids, cell proliferation, and clonogenic survival. Implantation of U87 cells into brains ofmore » nude mice, followed 5 days later by EPO treatment (5,000 U/kg intraperitoneal every other day for 2 weeks) should reveal effects of EPO on tumor growth in vivo. Reverse transcriptase-polymerase chain reaction was performed for EPOR, HIF-1{alpha}, and epidermal growth factor receptor (EGFR)vIII in cell lines and 22 human GBM specimens. Results: EPO did not modulate basal glioma cell migration and stimulated proliferation in only one of four cell lines. Importantly, EPO did not enhance tumor growth in mouse brains. Preincubation of glioma cells with EPO for 3 h, followed by irradiation and TMZ for another 24 h, resulted in protection against chemoradiation-induced cytotoxicity in three cell lines. Conversely, EPO induced a dose-dependent decrease in survival of G28 gliosarcoma cells. In GBM specimens, expression of HIF-1{alpha} correlated positively with expression of EPOR and EGFRvIII. EPOR and EGFRvIII expression did not correlate. Conclusions: EPO is unlikely to appreciably influence basal glioma growth. However, concomitant use of EPO with irradiation/chemotherapy in GBM patients is not advisable.« less

Laminin-5γ-2 (LAMC2) Is Highly Expressed in Anaplastic Thyroid Carcinoma and Is Associated With Tumor Progression, Migration, and Invasion by Modulating Signaling of EGFR

PubMed Central

Kanojia, Deepika; Okamoto, Ryoko; Jain, Saket; Madan, Vikas; Chien, Wenwen; Sampath, Abhishek; Ding, Ling-Wen; Xuan, Meng; Said, Jonathan W.; Doan, Ngan B.; Liu, Li-Zhen; Yang, Henry; Gery, Sigal; Braunstein, Glenn D.; Koeffler, H. Phillip

2014-01-01

Context: Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy having no effective treatment. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein involved in cell migration and tumor invasion and might represent an ideal target for the development of novel therapeutic approaches for ATC. Objective: The objective of the investigation was to study the role of LAMC2 in ATC tumorigenesis. Design: LAMC2 expression was evaluated by RT-PCR, Western blotting, and immunohistochemistry in tumor specimens, adjacent noncancerous tissues, and cell lines. The short hairpin RNA (shRNA) approach was used to investigate the effect of LAMC2 knockdown on the tumorigenesis of ATC. Results: LAMC2 was highly expressed in ATC samples and cell lines compared with normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed the migration, invasion, and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered the expression of genes associated with migration, invasion, proliferation, and survival. Immunoprecipitation studies showed that LAMC2 bound to epidermal growth factor receptor (EGFR) in the ATC cells. Silencing LAMC2 partially blocked epidermal growth factor-mediated activation of EGFR and its downstream pathway. Interestingly, cetuximab (an EGFR blocking antibody) or EGFR small interfering RNA additively enhanced the antiproliferative activity of the LAMC2 knockdown ATC cells compared with the control cells. Conclusions: To our knowledge, this is the first report investigating the effect of LAMC2 on cell growth, cell cycle, migration, invasion, and EGFR signaling in ATC cells, suggesting that LAMC2 may be a potential therapeutic target for the treatment of ATC. PMID:24170107

Vγ4 T Cells Inhibit the Pro-healing Functions of Dendritic Epidermal T Cells to Delay Skin Wound Closure Through IL-17A

PubMed Central

Li, Yashu; Wang, Yangping; Zhou, Lina; Liu, Meixi; Liang, Guangping; Yan, Rongshuai; Jiang, Yufeng; Hao, Jianlei; Zhang, Xiaorong; Hu, Xiaohong; Huang, Yong; Wang, Rupeng; Yin, Zhinan; Wu, Jun; Luo, Gaoxing; He, Weifeng

2018-01-01

Dendritic epidermal T cells (DETCs) and dermal Vγ4 T cells engage in wound re-epithelialization and skin inflammation. However, it remains unknown whether a functional link between Vγ4 T cell pro-inflammation and DETC pro-healing exists to affect the outcome of skin wound closure. Here, we revealed that Vγ4 T cell-derived IL-17A inhibited IGF-1 production by DETCs to delay skin wound healing. Epidermal IL-1β and IL-23 were required for Vγ4 T cells to suppress IGF-1 production by DETCs after skin injury. Moreover, we clarified that IL-1β rather than IL-23 played a more important role in inhibiting IGF-1 production by DETCs in an NF-κB-dependent manner. Together, these findings suggested a mechanistic link between Vγ4 T cell-derived IL-17A, epidermal IL-1β/IL-23, DETC-derived IGF-1, and wound-healing responses in the skin. PMID:29483920

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.

PubMed

Fotopoulos, N; Wernike, D; Chen, Y; Makil, N; Marte, A; Piekny, A

2013-11-01

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells

PubMed Central

Dutta, Abhik; Pincha, Neha; Rana, Isha; Ghosh, Subhasri; Witherden, Deborah; Kandyba, Eve; MacLeod, Amanda; Kobielak, Krzysztof; Havran, Wendy L

2017-01-01

The cutaneous wound-healing program is a product of a complex interplay among diverse cell types within the skin. One fundamental process that is mediated by these reciprocal interactions is the mobilization of local stem cell pools to promote tissue regeneration and repair. Using the ablation of epidermal caspase-8 as a model of wound healing in Mus musculus, we analyzed the signaling components responsible for epithelial stem cell proliferation. We found that IL-1α and IL-7 secreted from keratinocytes work in tandem to expand the activated population of resident epidermal γδT-cells. A downstream effect of activated γδT-cells is the preferential proliferation of hair follicle stem cells. By contrast, IL-1α-dependent stimulation of dermal fibroblasts optimally stimulates epidermal stem cell proliferation. These findings provide new mechanistic insights into the regulation and function of epidermal cell–immune cell interactions and into how components that are classically associated with inflammation can differentially influence distinct stem cell niches within a tissue. PMID:29199946

Cellular pattern formation by SCRAMBLED, a leucine-rich repeat receptor-like kinase in Arabidopsis.

PubMed

Kwak, Su-Hwan; Schiefelbein, John

2008-02-01

The appropriate specification of distinct cell types is important for generating the proper tissues and bodies of multicellular organisms. In the root epidermis of Arabidopsis, cell fate determination is accomplished by a transcriptional regulatory circuit that is influenced by positional signaling. A leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), has been shown to be responsible for the position-dependent aspect of this epidermal pattern. In a recent report, we find that SCM affects the transcriptional regulatory network by down-regulating the WEREWOLF (WER) MYB gene expression in a set of epidermal cells located in a specific position. We also find that SCM and the SCM-related SRF1 and SRF3 are not required for embryonic epidermal patterning and that SRF1 and SRF3 do not act redundantly with SCM. This suggests that distinct positional signaling mechanisms exist for embryonic and post-embryonic epidermal patterning. In this addendum, we discuss the implications of our recent findings and extend our working model for epidermal cell pattering.

Cellular pattern formation by SCRAMBLED, a leucine-rich repeat receptor-like kinase in Arabidopsis

PubMed Central

Kwak, Su-Hwan

2008-01-01

The appropriate specification of distinct cell types is important for generating the proper tissues and bodies of multicellular organisms. In the root epidermis of Arabidopsis, cell fate determination is accomplished by a transcriptional regulatory circuit that is influenced by positional signaling. A leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), has been shown to be responsible for the position-dependent aspect of this epidermal pattern. In a recent report, we find that SCM affects the transcriptional regulatory network by down-regulating the WEREWOLF (WER) MYB gene expression in a set of epidermal cells located in a specific position. We also find that SCM and the SCM-related SRF1 and SRF3 are not required for embryonic epidermal patterning and that SRF1 and SRF3 do not act redundantly with SCM. This suggests that distinct positional signaling mechanisms exist for embryonic and post-embryonic epidermal patterning. In this addendum, we discuss the implications of our recent findings and extend our working model for epidermal cell pattering. PMID:19704725

Anthocyanins potentiate the activity of trastuzumab in human epidermal growth factor receptor 2-positive breast cancer cells in vitro and in vivo.

PubMed

Liu, Weihua; Xu, Jinmei; Liu, Yilun; Yu, Xiaoping; Tang, Xi; Wang, Zhi; Li, Xin

2014-10-01

Human epidermal growth factor receptor 2 (HER2) has been found to be overexpressed in ~25% of invasive breast cancer and is significantly associated with a poor prognosis in breast cancer patients. The anthocyanins cyanidin-3-glucoside (C3G) and peonidin-3-glucoside have been identified as potential drugs for the therapy of HER2‑positive breast cancer. They have been used as supplements in targeted therapeutics and chemotherapeutics in Asia, however, the underlying mechanism remains to be elucidated. The aim of the present study was to investigate the synergism between C3G and trastuzumab (Trast). To address this question, the response to C3G, Trast and a combination of the two drugs, in three representative HER2‑positive cell lines was evaluated. The combination treatments induced apoptosis, inhibited cell growth and affected HER2 and its downstream signaling pathway in MDA‑MB‑453, BT474 and HCC1569 cells, and the effects were synergistic. The combination of 3CG and Trast inhibited tumor growth in an in vivo xenograft model. The data from the present study suggested that C3G exhibits potent antitumor activity when combined with Trast under the investigated conditions.

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

PubMed

Kim, Hee-Jun; Min, Ahrum; Im, Seock-Ah; Jang, Hyemin; Lee, Kyung Hun; Lau, Alan; Lee, Miso; Kim, Seongyeong; Yang, Yaewon; Kim, Jungeun; Kim, Tae Yong; Oh, Do-Youn; Brown, Jeffrey; O'Connor, Mark J; Bang, Yung-Jue

2017-01-01

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC 50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment. © 2016 UICC.

The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly1

PubMed Central

Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Costa, Fabrizio; Aharoni, Asaph

2015-01-01

The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning. PMID:26443676

Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

PubMed

Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

2009-01-01

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation.

PubMed

Osoegawa, Atsushi; Hashimoto, Takafumi; Takumi, Yohei; Abe, Miyuki; Yamada, Tomonori; Kobayashi, Ryoji; Miyawaki, Michiyo; Takeuchi, Hideya; Okamoto, Tatsuro; Sugio, Kenji

2018-03-28

Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Plant meiosis: the means to 1N.

PubMed

Bhatt, A M; Canales, C; Dickinson, H G

2001-03-01

Meiosis is pivotal in the life history of plants. In addition to providing an opportunity for genetic reassortment, it marks the transition from diploid sporophyte to haploid gametophyte. Recent molecular data suggest that, like animals, plants possess a common set of genes (also conserved in eukaryotic microorganisms) responsible for meiotic recombination and chromosome segregation. However, unlike animals, plant meiocytes do not differentiate from a pool of primordial germ cells, but rather arise de novo from a germline formed from sub-epidermal cells in the anthers and ovules. Mutants defective in the specification of these reproductive cell lines and disrupted in different aspects of the meiotic process are beginning to reveal many features unique to plant meiosis.

Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes

PubMed Central

Ouellette, Marie-Hélène

2016-01-01

The antagonism between the GTPases Rac1 and RhoA controls cell-to-cell heterogeneity in isogenic populations of cells in vitro and epithelial morphogenesis in vivo. Its involvement in the regulation of cell-to-cell heterogeneity during epidermal morphogenesis has, however, never been addressed. We used a quantitative cell imaging approach to characterize epidermal morphogenesis at a single-cell level during early elongation of Caenorhabditis elegans embryos. This study reveals that a Rac1-like pathway, involving the Rac/Cdc42 guanine-exchange factor β-PIX/PIX-1 and effector PAK1/PAK-1, and a RhoA-like pathway, involving ROCK/LET-502, control the remodeling of apical junctions and the formation of basolateral protrusions in distinct subsets of hypodermal cells. In these contexts, protrusions adopt lamellipodia or an amoeboid morphology. We propose that lamella formation may reduce tension building at cell–cell junctions during morphogenesis. Cell-autonomous antagonism between these pathways enables cells to switch between Rac1- and RhoA-like morphogenetic programs. This study identifies the first case of cell-to-cell heterogeneity controlled by Rac1/RhoA antagonism during epidermal morphogenesis. PMID:27821782

Cellular, ultrastructural and molecular analyses of epidermal cell development in the planarian Schmidtea mediterranea.

PubMed

Cheng, Li-Chun; Tu, Kimberly C; Seidel, Chris W; Robb, Sofia M C; Guo, Fengli; Sánchez Alvarado, Alejandro

2018-01-15

The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles. Copyright © 2017 Elsevier Inc. All rights reserved.

Pemetrexed in previously treated non-small-cell lung cancer.

PubMed

Rosell, Rafael; Sanchez, Jose Miguel; Taron, Miquel; Moran, Teresa; Ciuraqui, Beatriz; Canela, Mercedes; Felip, Enriqueta; Massuti, Bartomeu; Camps, Carlos

2004-07-01

Several decades of chemotherapy trials in non-small-cell lung cancer (NSCLC) have clearly shown a survival benefit for chemotherapy over best supportive care. However, only short-lived responses are attained, with an average of four cycles of chemotherapy, before tumor progression is observed. Second-line chemotherapy has been demonstrated to improve outcome, with docetaxel (Taxotere) as the predominant cytotoxic drug. A recent randomized trial in second-line NSCLC indicated that the novel drug pemetrexed (Alimta) attained the same response, time to progression, and survival as docetaxel. This finding ushers in a new age in second-line treatment that can be further invigorated by the addition of targeted agents. Accumulated evidence indicates that overexpression of epidermal growth factor receptor and HER2/neu, which occurs frequently in NSCLC, leads to the deregulation of PI3K and MAPK, activating Akt and enhancing chemoresistance. Future clinical trials in NSCLC will include tailored and multitargeted therapy and pemetrexed represents a significant step forward in this direction.

Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re‐epithelialization

PubMed Central

Garcin, Clare L.; Ansell, David M.; Headon, Denis J.; Paus, Ralf

2016-01-01

Abstract The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re‐establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15+ve bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo‐epidermis. However, the identity of the first‐responding cells, and in particular whether this process involves a direct contribution of K15+ve bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin‐mediated partial ablation of K15+ve bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377–1385 PMID:26756547

Epidermal Cadm1 expression promotes autoimmune alopecia via enhanced T cell adhesion and cytotoxicity.

PubMed

Giangreco, Adam; Hoste, Esther; Takai, Yoshimi; Rosewell, Ian; Watt, Fiona M

2012-02-01

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.

3D Culture Represents Apoptosis Induced by Trastuzumab Better than 2D Monolayer Culture.

PubMed

Tatara, Takashi; Mukohara, Toru; Tanaka, Rina; Shimono, Yohei; Funakoshi, Yohei; Imamura, Yoshinori; Toyoda, Masanori; Kiyota, Naomi; Hirai, Midori; Kakeji, Yoshihiro; Minami, Hironobu

2018-05-01

Our hypothesis was that three-dimensional (3D) culture better represents differential in vivo responses to trastuzumab between PIK3CA-wild-type (wt) and mutant (mt) cell lines than does two-dimensional (2D) culture. Apoptosis and cell signaling proteins were evaluated in response to trastuzumab with and without BKM120, a pan-phosphatidylinositol 3-kinase (PI3K) inhibitor, using western blot analysis of four breast cancer cell lines with human epidermal growth factor receptor 2 (HER2) amplification. Increased expression of cleaved poly (ADP-ribose) polymerase (PARP) was observed only in 3D-cultured PIK3CA-wt lines in response to trastuzumab, but not in 2D-cultured PIK3CA-wt or PIK3CA-mt lines. Decrease in the ratio of phosphorylated (p-)AKT to AKT in response to trastuzumab was more profound in PIK3CA-wt cells than in PIK3CA-mt cells in 3D culture, while the difference between PIK3CA genotypes was less apparent in 2D culture. Treatment with BKM120 and trastuzumab resulted in a stronger increase in cleaved PARP than either treatment alone. 3D Culture appears to better represent trastuzumab-induced apoptosis and resistance to trastuzumab associated with PIK3CA mutation. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Determination of HER2 amplification status in breast cancer cells using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Bi, Xiaohong; Rexer, Brent; Arteaga, Carlos L.; Guo, Mingsheng; Li, Ming; Mahadevan-Jansen, Anita

2010-02-01

The overexpression of HER2 (human epidermal growth factor receptor 2) in breast cancer is associated with increased disease recurrence and worse prognosis. Current diagnosis of HER2 positive breast cancer is time consuming with an estimated 20% inaccuracy. Raman spectroscopy is a proven method for pathological diagnosis based on the molecular composition of tissues. This study aimed to determine the feasibility of Raman spectroscopy to differentially identify the amplification of HER2 in cells. Three cell lines including BT474 (HER2 overexpressing breast cancer cell), MCF-10A (human breast epithelial cell), and MCF-10A with overexpressing HER2, were investigated using a bench top confocal Raman system. A diagnostic algorithm based on generalized linear model (GLM) with elastic-net penalties was established to discriminate 318 spectra collected from the cells, and to identify the spectra regions that differentiate the cell lines. The algorithm was able to differentially identify BT474 breast cancer cells with an overall sensitivity of 100% and specificity of 99%. The results demonstrate the capability of Raman spectroscopy to determine HER2 status in cells. Raman spectroscopy shows promise for application in the diagnosis of HER2 positive breast cancer in clinical practice.

Carbon Dioxide Metabolism in Leaf Epidermal Tissue 1

PubMed Central

Willmer, C. M.; Pallas, J. E.; Black, C. C.

1973-01-01

A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose-1,5-diphosphate carboxylase, malic enzyme, and NAD+ and NADP+ malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD+ and NADP+ malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO2 fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism. Images PMID:16658581

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis

PubMed Central

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-01-01

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications. PMID:28629179

p63 regulates glutaminase 2 expression

PubMed Central

Giacobbe, Arianna; Bongiorno-Borbone, Lucilla; Bernassola, Francesca; Terrinoni, Alessandro; Markert, Elke Katrin; Levine, Arnold J.; Feng, Zhaohui; Agostini, Massimilano; Zolla, Lello; Agrò, Alessandro Finazzi; Notterman, Daniel A.; Melino, Gerry; Peschiaroli, Angelo

2013-01-01

The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes. PMID:23574722

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

PubMed

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-06-19

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities.

PubMed

Troy, Tammy-Claire; Turksen, Kursad

2007-06-01

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo.

PubMed

Morita, Toshiyuki; Tsuchiya, Akiko; Sugimoto, Masazumi

2011-09-01

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

PubMed

Douchkov, Dimitar; Lueck, Stefanie; Hensel, Goetz; Kumlehn, Jochen; Rajaraman, Jeyaraman; Johrde, Annika; Doblin, Monika S; Beahan, Cherie T; Kopischke, Michaela; Fuchs, René; Lipka, Volker; Niks, Rients E; Bulone, Vincent; Chowdhury, Jamil; Little, Alan; Burton, Rachel A; Bacic, Antony; Fincher, Geoffrey B; Schweizer, Patrick

2016-10-01

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

PubMed

Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C; Ambler, Carrie A

2016-04-21

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization

PubMed Central

Nunan, Robert; Campbell, Jessica; Mori, Ryoichi; Pitulescu, Mara E.; Jiang, Wen G.; Harding, Keith G.; Adams, Ralf H.; Nobes, Catherine D.; Martin, Paul

2015-01-01

Summary For a skin wound to successfully heal, the cut epidermal-edge cells have to migrate forward at the interface between scab and healthy granulation tissue. Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back. Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells. Additionally, this signaling leads to the shutdown of actomyosin stress fibers in these same epidermal cells, which may act to release tension within the wound monolayer. If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man. PMID:26549443

Engineered Modular Recombinant Transporters: Application of New Platform for Targeted Radiotherapeutic Agents to {alpha}-Particle Emitting {sup 211}At

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenkranz, Andrey A.; Department of Biophysics, Biological Faculty, Moscow State University, Moscow; Vaidyanathan, Ganesan

2008-09-01

Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting {sup 211}At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). Methods and Materials: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[{sup 211}At]astato-5-guanidinomethylbenzoate (SAGMB), its {sup 125}I analogue SGMIB, or with {sup 131}I using Iodogen.more » Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. Results: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [{sup 211}At]astatide for all three cell lines. Conclusion: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted {alpha}-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this {sup 211}At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.« less

Immunoelectron microscopic localisation of keratin and luminal epithelial antigens in normal and neoplastic urothelium.

PubMed

Wilson, P D; Nathrath, W B; Trejdosiewicz, L K

1982-01-01

Immunoelectron microscope cytochemistry was carried out on 2% paraformaldehyde fixed, 50 mu sections of normal urothelium and bladder carcinoma cells in culture using antisera raised in rabbits to human 40-63 000 MW epidermal "broad spectrum" keratin and calf urothelial "luminal epithelial antigen" (aLEA) Both the unconjugated and indirect immunoperoxidase-DAB techniques were used before routine embedding. The localisation of both keratin and luminal epithelial antigen (LEA) was similar in normal and neoplastic cells and reaction product was associated not only with tonofilaments but also lining membrane vesicles and on fine filaments in the cytoplasmic ground substance.

Mixed vitiligo of Blaschko lines: a newly discovered presentation of vitiligo responsive to combination treatment.

PubMed

Kovacevic, Maja; Stanimirovic, Andrija; Vucic, Majda; Goren, Andy; Situm, Mirna; Lukinovic Skudar, Vesna; Lotti, Torello

2016-07-01

Vitiligo, depigmenting disorder of the skin and mucous membranes, affects up to 1% of the population worldwide. It is classified into four major types: segmental, non-segmental, mixed, and unclassified type. Non-segmental vitiligo refers to non-dermatomal distribution of lesions, while dermatomal distribution of lesions is present in patients with segmental vitiligo. Segmental vitiligo can also follow Blaschko lines - pathways of epidermal cell migration and proliferation during the development of the fetus. Here, we present patient with segmental and non-segmental vitiligo following Blaschko lines with excellent therapeutic response to combined therapy. Prior to our report, a case of segmental and non-segmental vitiligo followed by Blaschko lines was never described, therefore we suggest the term "mixed vitiligo of Blaschko lines" to describe this entity. This is also a rare case in which 90% repigmentation was achieved in patient with segmental and nonsegmental vitiligo following Blaschko lines in only 2 months of combined therapy. © 2016 Wiley Periodicals, Inc.

Osimertinib: A third-generation tyrosine kinase inhibitor for treatment of epidermal growth factor receptor-mutated non-small cell lung cancer with the acquired Thr790Met mutation.

PubMed

Bollinger, Meredith K; Agnew, Amanda S; Mascara, Gerard P

2017-01-01

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for the treatment of metastatic EGFR T790M mutation-positive non-small cell lung cancer (NSCLC) in patients failing previous TKI therapy. The T790M mutation is an acquired resistance mechanism found in over half of patients with NSCLC progressing on first-generation TKIs. First- and second-generation TKIs do not inhibit the T790M mutation at clinically relevant concentrations. Osimertinib is selective for mutated forms of EGFR, including the TKI-sensitizing mutations L858R and exon 19 deletions, as well as the acquired T790M resistance mutation. In a trial comparing osimertinib to platinum doublet therapy among patients with the T790M mutation progressing on first-line TKI therapy, median progression-free survival was significantly longer in patients receiving osimertinib. Osimertinib has a favorable safety profile compared to platinum-doublet chemotherapy. Common adverse events include diarrhea, skin rash, dry skin, and paronychia; however, because it spares wild-type EGFR, these toxicities appear to occur with less frequency and severity compared to other TKIs. Serious, but rare, adverse events include pneumonitis, interstitial lung disease-like events, QT interval prolongation, and reduced ejection fraction. Osimertinib has the unique ability to distribute readily into brain tissue compared with other TKIs, giving it a potential role in the treatment and/or prevention of CNS metastases; future studies are warranted in this area. An ongoing study evaluating osimertinib versus first-generation TKIs as first-line treatment for patients with EGFR mutation-positive NSCLC may help to define the role of osimertinib as front-line therapy.

Targeting epidermal growth factor receptor in the treatment of non-small-cell lung cancer.

PubMed

Kotsakis, Athanasios; Georgoulias, Vassilis

2010-10-01

The management of non-small-cell lung cancer (NSCLC) has undergone a paradigm shift in the last decade, with the survival advantage demonstrated by the incorporation of anti-epidermal growth factor receptor (EGFR) agents to the standard treatment of advanced/metastatic NSCLC. We review the existing data regarding the distinct anti-EGFR agents in the NSCLC treatment and the potential role of the investigated biomarkers in the clinical outcome. Tyrosine kinase inhibitors have been used in first-line, second-line and more settings with extremely good results in a subgroup of patients. Cetuximab remains the only anti-EGFR monoclonal antibody to show survival benefit when combined with a cytotoxic agent in the front-line setting. Anti-EGFR treatment is associated with a dramatic clinical benefit in a subgroup of patients, emphasizing the importance of customizing treatment. Several biomarkers have been investigated for their predictive or prognostic value. Validation of identification of biomarkers remains a focus of intense research that may ultimately guide therapeutic decision making, as none of these is considered ideal to discriminate responding from non-responding patients. However, the current evidence of the EGFR mutation analysis from a recent randomised trial suggests that EGFR mutation analysis is quite a good predictive marker for responsiveness to anti-EGFR TKIs. Moreover, the identification of surrogate markers to indicate optimal activity of the anti-EGFR agent is also needed. This review article provides data from large clinical trials using anti-EGFR agents and correlates these results with the tested biomarkers. EGFR inhibition has shown very encouraging results and has improved the outcome of the NSCLC treatment. However, a plateau of significant clinical benefit seems to have been reached and we believe that the time to move away from the traditional treatment approach to more individualizing therapies has come.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okano, Junko, E-mail: jokano@belle.shiga-med.ac.jp; Kojima, Hideto; Katagi, Miwako

Bone marrow-derived cells (BMDCs) can migrate into the various organs in the mice irradiated by ionizing radiation (IR). However, it may not be the case in the skin. While IR is used for bone marrow (BM) transplantation, studying with the epidermal sheets demonstrated that the BMDC recruitment is extraordinarily rare in epidermis in the mouse. Herein, using the chimera mice with BM from green fluorescent protein (GFP) transgenic mice, we simply examined if BMDCs migrate into any layers in the total skin, as opposed to the epidermal sheets, in response to IR. Interestingly, we identified the presence of GFP-positive (GFP{supmore » +}) cells in the epidermis-dermis junction in the total skin sections although the epidermal cell sheets failed to have any GFP cells. To examine a possibility that the cells in the junction could be mechanically dissociated during separating epidermal sheets, we then salvaged such dissociated cells and examined its characteristics. Surprisingly, some GFP{sup +} cells were found in the salvaged cells, indicating that these cells could be derived from BM. In addition, such BMDCs were also associated with inflammation in the junction. In conclusion, BMDCs can migrate to and reside in the epidermis-dermis junction after IR. - Highlights: • Bone marrow-derived cells (BMDCs) migrate in the epidermis due to ionizing radiation (IR). • BMDCs dissociate from the epidermis-dermis junction in preparing epidermal sheets. • The doses of IR determine the location and the number of migrating BMDCs in the skin.« less

Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization.

PubMed

Garcin, Clare L; Ansell, David M; Headon, Denis J; Paus, Ralf; Hardman, Matthew J

2016-05-01

The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re-establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15(+ve) bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo-epidermis. However, the identity of the first-responding cells, and in particular whether this process involves a direct contribution of K15(+ve) bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin-mediated partial ablation of K15(+ve) bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377-1385. © 2016 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

A near-infrared Fourier transform Raman spectroscopy of epidermal keratinocytes: changes in the protein?DNA structure following malignant transformation

NASA Astrophysics Data System (ADS)

Gao, Xiaoling; Butler, Ian S.; Kremer, Richard

2005-01-01

We report here the use of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy to analyze normal human epidermal keratinocytes prior to and following malignant transformation. Our analysis indicates specific Raman spectral differences between immortalized (HPK1A) and malignant ras transformed (HPK1A- ras) cells. In addition, striking spectral differences are seen in the DNA isolated from these cells and particularly in the 843/810 cm -1 ratio with values of 1.6 ± 0.13 in HPK1A cells and 0.68 ± 0.09 in HPK1A- ras cells (mean ± S.D., n = 12, P < 0.001) indicating specific alterations in the backbone conformation markers following malignant transformation. Subsequently, we analysed the effect of a strong inhibitor of keratinocyte growth, the Vitamin D analog EB1089, on the Raman spectra of intact cells and on the 843/810 cm -1 ratio in the DNA isolated from both cell lines. Specific changes were observed in intact cells in the 1300-750 cm -1 region. Furthermore, the 843/810cm -1 ratio of isolated DNA from HPK1A cells was not affected by EB1089 but significantly increased in DNA isolated from HPK1A-ras cells so much that it became closer to the value observed for HPK1A cells (1.07 ± 0.10). Our data suggest that Raman analysis of DNA and in particular the 843/810cm -1 ratio can provide useful indices of malignant transformation and efficacy of anticancer agents.

Generation of human cortical neurons from a new immortal fetal neural stem cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cacci, E.; Villa, A.; Parmar, M.

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markersmore » like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.« less

Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases.

PubMed

Isozaki, Hideko; Ichihara, Eiki; Takigawa, Nagio; Ohashi, Kadoaki; Ochi, Nobuaki; Yasugi, Masayuki; Ninomiya, Takashi; Yamane, Hiromichi; Hotta, Katsuyuki; Sakai, Katsuya; Matsumoto, Kunio; Hosokawa, Shinobu; Bessho, Akihiro; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

2016-03-15

Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. ©2015 American Association for Cancer Research.

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mill, Christopher P.; Auburn University Harrison School of Pharmacy, Auburn, AL 36849-5501; Gettinger, Kathleen L.

2011-02-15

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility,more » and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1{beta}. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.« less

A blood biomarker for monitoring response to anti-EGFR therapy.

PubMed

Hughes, Nicholas P; Xu, Lingyun; Nielsen, Carsten H; Chang, Edwin; Hori, Sharon S; Natarajan, Arutselvan; Lee, Samantha; Kjær, Andreas; Kani, Kian; Wang, Shan X; Mallick, Parag; Gambhir, Sanjiv Sam

2018-04-13

To monitor therapies targeted to epidermal growth factor receptors (EGFR) in non-small cell lung cancer (NSCLC), we investigated Peroxiredoxin 6 (PRDX6) as a biomarker of response to anti-EGFR agents. We studied cells that are sensitive (H3255, HCC827) or resistant (H1975, H460) to gefitinib. PRDX6 was examined with either gefitinib or vehicle treatment using enzyme-linked immunosorbent assays. We created xenograft models from one sensitive (HCC827) and one resistant cell line (H1975) and monitored serum PRDX6 levels during treatment. PRDX6 levels in cell media from sensitive cell lines increased significantly after gefitinib treatment vs. vehicle, whereas there was no significant difference for resistant lines. PRDX6 accumulation over time correlated positively with gefitinib sensitivity. Serum PRDX6 levels in gefitinib-sensitive xenograft models increased markedly during the first 24 hours of treatment and then decreased dramatically during the following 48 hours. Differences in serum PRDX6 levels between vehicle and gefitinib-treated animals could not be explained by differences in tumor burden. Our results show that changes in serum PRDX6 during the course of gefitinib treatment of xenograft models provide insight into tumor response and such an approach offers several advantages over imaging-based strategies for monitoring response to anti-EGFR agents.

Anti-Pigmentary Effect of (-)-4-Hydroxysattabacin from the Marine-Derived Bacterium Bacillus sp.

PubMed

Kim, Kyuri; Leutou, Alain S; Jeong, Haein; Kim, Dayoung; Seong, Chi Nam; Nam, Sang-Jip; Lim, Kyung-Min

2017-05-13

Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT-PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue.

Anti-Pigmentary Effect of (-)-4-Hydroxysattabacin from the Marine-Derived Bacterium Bacillus sp.

PubMed Central

Kim, Kyuri; Leutou, Alain S.; Jeong, Haein; Kim, Dayoung; Seong, Chi Nam; Nam, Sang-Jip; Lim, Kyung-Min

2017-01-01

Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT–PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue. PMID:28505073

Comparative anti-psoriatic efficacy studies of clobetasol loaded chitin nanogel and marketed cream.

PubMed

Panonnummal, Rajitha; Jayakumar, R; Sabitha, M

2017-01-01

In the present study chitin nanogel loaded with anti-psoriatic drug clobetasol was developed (CLCNG) for its topical delivery in psoriasis. CLCNG had the particle size of 132±14nm, with gel like consistency, stability in refrigerator, having higher drug release properties at acidic pH. CLCNG exhibited significant toxicity towards HaCaT and THP-1cell lines by MTT assay. The uptake of nanogel by HaCaT cell lines was confirmed by fluorescent microscopy. CLCNG at 0.35mg/ml exhibited significant anti-inflammatory activity with an average of 65% and 70% inhibition in COX and LOX activities expressed in THP-1 cells. In vitro skin permeation studies revealed the increased transdermal flux with fragmented stratum corneum and loosened epidermal layers in CLCNG treated samples, compared with control drug solution. The in vivo anti-psoriatic studies done on imiquimod model confirmed the potential benefits of the nanogel for the topical delivery of clobetasol in psoriasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Epidermal growth in the bottlenose dolphin, Tursiops truncatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hicks, B.D.; St. Aubin, D.J.; Geraci, J.R.

1985-07-01

Epidermal growth in two mature female bottlenose dolphins, Tursiops truncatus, was investigated by following the movement of a cohort of tritiated thymidine-labeled epidermal cells for 59 days. The majority of the cells migrated in a cluster which was estimated to reach the skin surface in 73 days. The authors calculate that the outermost cell layer is sloughed 12 times per day. Turnover time and sloughing rate are estimated to be 1.7 times longer and 8.5 times faster than the respective values for epidermal cell kinetics in humans. This apparent inconsistency of slow transit time and rapid sloughing rate is reconciledmore » by the convoluted structure of the stratum germinativum in the dolphin which results in a ratio of germinatival to superficial cells of 876:1. The stratum germinativum of dolphin epidermis appears to lack morphologically distinct, spatially segregated subpopulations of anchoring and stem cells. Dolphin epidermis has a large capacity for cell population, relatively long turnover time, and rapid sloughing rate. The adaptive advantages of these characteristics are discussed.« less

Orbital metastasis secondary to pulmonary adenocarcinoma treated with gefitinib: a case report.

PubMed

Koma, Yasuko; Goto, Keiko; Yoshida, Chihiro; Kimura, Kengo; Matsumoto, Yusuke; Koyama, Midori; Nakashima, Nariyasu; Masuya, Daiki; Matsuoka, Hirofumi; Yoshimatsu, Harukazu; Azumi, Atsushi; Suzuki, Yujiro

2012-10-18

Orbital metastases of lung cancer are rare. However, because the number of patients diagnosed with lung cancer is increasing, the probability that a physician will see a patient with an orbital metastasis is also increasing. Unfortunately, the clinical course and response of these patients to cytotoxic chemotherapy are generally poor and keeping a patient's quality of vision is difficult. In recent years, gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, has brightened the outlook for patients with advanced non-small cell lung cancer, especially for those who carry epidermal growth factor receptor-activating mutations. A 62-year-old Japanese man presented with swelling of the eyelid margin and ptosis of his right eye. A physical examination revealed double vision in his right eye and an alteration in elevator muscle mobility. A magnetic resonance image demonstrated a right intra-orbital mass (18 × 16mm). Screening examinations were carried out because this mass was suspected to be a metastasis from another organ. Chest computed tomography revealed a 42 × 37mm mass shadow on the left side of the hilum with mediastinal lymph node metastases. Adenocarcinoma with an epidermal growth factor receptor gene mutation (exon 19 deletion L747-E749; A750P) was detected in a transbronchial biopsy specimen; the patient was diagnosed with stage IV (T2N2M1) non-small cell lung cancer.Gefitinib (250mg/day) was chosen as first-line chemotherapy because there was no pre-existing interstitial shadow. After two months of treatment, the patient's right eye opened completely and follow-up magnetic resonance imaging revealed a marked reduction of the intra-orbital mass to 14 × 13mm. Three months after treatment initiation, a follow-up computed tomography showed a marked reduction in the size of the primary lesion to 23 × 20mm. The patient is continuing gefitinib treatment without any adverse effects noted on computed tomography, physical, or laboratory examination. We report the case of a patient with an orbital non-small cell lung cancer metastasis with epidermal growth factor receptor-activating mutations. This metastasis, as well as the primary lesion, showed a marked response to the molecular targeting drug gefitinib, and the patient's vision was kept without an invasive procedure. Gefitinib may be a good first choice for patients with orbital non-small cell lung cancer metastasis harboring epidermal growth factor receptor-activating mutations.

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

PubMed Central

Adhikary, Gautam; Grun, Dan; Kerr, Candace; Balasubramanian, Sivaprakasam; Rorke, Ellen A.; Vemuri, Mohan; Boucher, Shayne; Bickenbach, Jackie R.; Hornyak, Thomas; Xu, Wen; Fisher, Matthew L.; Eckert, Richard L.

2013-01-01

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation. PMID:24376802

Hemangiosarcoma and its cancer stem cell subpopulation are effectively killed by a toxin targeted through epidermal growth factor and urokinase receptors.

PubMed

Schappa, Jill T; Frantz, Aric M; Gorden, Brandi H; Dickerson, Erin B; Vallera, Daniel A; Modiano, Jaime F

2013-10-15

Targeted toxins have the potential to overcome intrinsic or acquired resistance of cancer cells to conventional cytotoxic agents. Here, we hypothesized that EGFuPA-toxin, a bispecific ligand-targeted toxin (BLT) consisting of a deimmunized Pseudomonas exotoxin (PE) conjugated to epidermal growth factor and urokinase, would efficiently target and kill cells derived from canine hemangiosarcoma (HSA), a highly chemotherapy resistant tumor, as well as cultured hemangiospheres, used as a surrogate for cancer stem cells (CSC). EGFuPA-toxin showed cytotoxicity in four HSA cell lines (Emma, Frog, DD-1 and SB) at a concentration of ≤100 nM, and the cytotoxicity was dependent on specific ligand-receptor interactions. Monospecific targeted toxins also killed these chemoresistant cells; in this case, a "threshold" level of EGFR expression appeared to be required to make cells sensitive to the monospecific EGF-toxin, but not to the monospecific uPA-toxin. The IC₅₀ of CSCs was higher by approximately two orders of magnitude as compared to non-CSCs, but these cells were still sensitive to EGFuPA-toxin at nanomolar (i.e., pharmacologically relevant) concentrations, and when targeted by EGFuPA-toxin, resulted in death of the entire cell population. Taken together, our results support the use of these toxins to treat chemoresistant tumors such as sarcomas, including those that conform to the CSC model. Our results also support the use of companion animals with cancer for further translational development of these cytotoxic molecules. Copyright © 2013 UICC.

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo.

PubMed

Zhou, Z N; Sharma, V P; Beaty, B T; Roh-Johnson, M; Peterson, E A; Van Rooijen, N; Kenny, P A; Wiley, H S; Condeelis, J S; Segall, J E

2014-07-17

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Blumeria graminis interactions with barley conditioned by different single R genes demonstrate a temporal and spatial relationship between stomatal dysfunction and cell death.

PubMed

Prats, Elena; Gay, Alan P; Roberts, Peter C; Thomas, Barry J; Sanderson, Ruth; Paveley, Neil; Lyngkjaer, Michael F; Carver, Tim L W; Mur, Luis A J

2010-01-01

Hypersensitive response (HR) against Blumeria graminis f. sp. hordei infection in barley (Hordeum vulgare) was associated with stomata "lock-up" leading to increased leaf water conductance (g(l)). Unique spatio-temporal patterns of HR formation occurred in barley with Mla1, Mla3, or MlLa R genes challenged with B. graminis f. sp. hordei. With Mla1, a rapid HR, limited to epidermal cells, arrested fungal growth before colonies initiated secondary attacks. With Mla3, mesophyll HR preceded that in epidermal cells whose initial survival supported secondary infections. With MlLa, mesophyll survived and not all attacked epidermal cells died immediately, allowing colony growth and secondary infection until arrested. Isolines with Mla1, Mla3, or MlLa genes inoculated with B. graminis f. sp. hordei ranging from 1 to 100 conidia mm(2) showed abnormally high g(l) during dark periods whose timing and extent correlated with those of each HR. Each isoline showed increased dark g(l) with the nonpathogen B. graminis f. sp. avenae which caused a single epidermal cell HR. Guard cell autofluorescence was seen only after drying of epidermal strips and closure of stomata suggesting that locked open stomata were viable. The data link stomatal lock-up to HR associated cell death and has implications for strategies for selecting disease resistant genotypes.

Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

PubMed

Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

2012-01-01

Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients. Copyright © 2011 John Wiley & Sons, Ltd.

Epidermal Homeostasis and Radiation Responses in a Multiscale Tissue Modeling Framework

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2013-01-01

The surface of skin is lined with several thin layers of epithelial cells that are maintained throughout life time by a small population of stem cells. High dose radiation exposures could injure and deplete the underlying proliferative cells and induce cutaneous radiation syndrome. In this work we propose a multiscale computational model for skin epidermal dynamics that links phenomena occurring at the subcellular, cellular, and tissue levels of organization, to simulate the experimental data of the radiation response of swine epidermis, which is closely similar to human epidermis. Incorporating experimentally measured histological and cell kinetic parameters, we obtain results of population kinetics and proliferation indexes comparable to observations in unirradiated and acutely irradiated swine experiments. At the sub-cellular level, several recently published Wnt signaling controlled cell-cycle models are applied and the roles of key components and parameters are analyzed. Based on our simulation results, we demonstrate that a moderate increase of proliferation rate for the survival proliferative cells is sufficient to fully repopulate the area denuded by high dose radiation, as long as the integrity of underlying basement membrane is maintained. Our work highlights the importance of considering proliferation kinetics as well as the spatial organization of tissues when conducting in vivo investigations of radiation responses. This integrated model allow us to test the validity of several basic biological rules at the cellular level and sub-cellular mechanisms by qualitatively comparing simulation results with published research, and enhance our understanding of the pathophysiological effects of ionizing radiation on skin.

Differentiated epidermal outgrowths in the planarian Dugesia gonocephala: a model for studying cell renewal and patterning in single-layered epithelial tissue.

PubMed

Chandebois, R

1985-01-01

Large deep wounds on the ventral side of a flatworm (Planaria) will not heal. Instead, the damage to the parenchyma in the wound's roof will result in a differentiated swelling in the dorsal epidermis, above the wound which will eventually disappear with the disintegration of the underlying damaged tissue and a ventrodorsal hole appears in place of the wound. The dorsal epidermal outgrowth is formed by a number of excrescences, the development of which involves four successive stages. Their analysis suggests that epidermal cells are continuously produced by their own stem cells which remain unnoticed because their nuclei are hardly stainable. The daughter cells differentiate without information from either the underlying tissues or the basal epithelial membrane. During the first stage of this differentiation the cells become ciliated and motile, with some embryonic features. They then produce rhabdites and take up a columnar shape as they may become attached to the basal membrane. After wound setting the production of epidermal cells increases and the overcrowding of the basal membrane results in (1) detachment of stem cells and motile ciliated cells from the basal tissues, i.e. outgrowths; (2) stretching of columnar cells at the base of the outgrowths. When in the process of tissue disintegration the basal membrane of the epithelium also disappears, the cells remain in a single-layered epithelial configuration and retain their original polarity. These results are at variance with the generally accepted hypothesis that, in planarians, epidermal cells originate from the parenchyma and the epidermis is not an autonomous tissue.

Egr-5 is a post-mitotic regulator of planarian epidermal differentiation

PubMed Central

Tu, Kimberly C; Cheng, Li-Chun; TK Vu, Hanh; Lange, Jeffrey J; McKinney, Sean A; Seidel, Chris W; Sánchez Alvarado, Alejandro

2015-01-01

Neoblasts are an abundant, heterogeneous population of adult stem cells (ASCs) that facilitate the maintenance of planarian tissues and organs, providing a powerful system to study ASC self-renewal and differentiation dynamics. It is unknown how the collective output of neoblasts transit through differentiation pathways to produce specific cell types. The planarian epidermis is a simple tissue that undergoes rapid turnover. We found that as epidermal progeny differentiate, they progress through multiple spatiotemporal transition states with distinct gene expression profiles. We also identified a conserved early growth response family transcription factor, egr-5, that is essential for epidermal differentiation. Disruption of epidermal integrity by egr-5 RNAi triggers a global stress response that induces the proliferation of neoblasts and the concomitant expansion of not only epidermal, but also multiple progenitor cell populations. Our results further establish the planarian epidermis as a novel paradigm to uncover the molecular mechanisms regulating ASC specification in vivo. DOI: http://dx.doi.org/10.7554/eLife.10501.001 PMID:26457503

MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab.

PubMed

Zhang, Kai-Liang; Zhou, Xuan; Han, Lei; Chen, Lu-Yue; Chen, Ling-Chao; Shi, Zhen-Dong; Yang, Ming; Ren, Yu; Yang, Jing-Xuan; Frank, Thomas S; Zhang, Chuan-Bao; Zhang, Jun-Xia; Pu, Pei-Yu; Zhang, Jian-Ning; Jiang, Tao; Wagner, Eric J; Li, Min; Kang, Chun-Sheng

2014-03-20

Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.

Phthalocyanine-Peptide Conjugates for Epidermal Growth Factor Receptor Targeting1

PubMed Central

Ongarora, Benson G.; Fontenot, Krystal R.; Hu, Xiaoke; Sehgal, Inder; Satyanarayana-Jois, Seetharama D.; Vicente, M. Graça H.

2012-01-01

Four phthalocyanine (Pc)-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) were synthesized and evaluated in vitro using four cell lines: human carcinoma A431 and HEp2, human colorectal HT-29, and kidney Vero (negative control) cells. Two peptide ligands for EGFR were investigated: EGFR-L1 and -L2, bearing 6 and 13 amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical (Autodock) and experimental (SPR) investigations. The Pc-EGFR-L1 conjugates 5a and 5b efficiently targeted EGFR and were internalized, in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2 conjugates 4b and 6a poorly targeted EGFR maybe due to their low aqueous solubility. All conjugates were non-toxic (IC50 > 100 µM) to HT-29 cells, both in the dark and upon light activation (1 J/cm2). Intravenous (iv) administration of conjugate 5b into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a near-IR fluorescence signal at ca. 700 nm, 24 h after administration. Our studies show that Pc-EGFR-L1 conjugates are promising near-IR fluorescent contrast agents for CRC, and potentially other EGFR over-expressing cancers. PMID:22468711

Establishment of paclitaxel-resistant breast cancer cell line and nude mice models, and underlying multidrug resistance mechanisms in vitro and in vivo.

PubMed

Chen, Si-Ying; Hu, Sa-Sa; Dong, Qian; Cai, Jiang-Xia; Zhang, Wei-Peng; Sun, Jin-Yao; Wang, Tao-Tao; Xie, Jiao; He, Hai-Rong; Xing, Jian-Feng; Lu, Jun; Dong, Ya-Lin

2013-01-01

Breast cancer is a common malignant tumor which affects health of women and multidrug resistance (MDR) is one of the main factors leading to failure of chemotherapy. This study was conducted to establish paclitaxel-resistant breast cancer cell line and nude mice models to explore underlying mechanisms of MDR. The breast cancer drug-sensitive cell line MCF-7 (MCF-7/S) was exposed in stepwise escalating paclitaxel (TAX) to induce a resistant cell line MCF-7/TAX. Cell sensitivity to drugs and growth curves were measured by MTT assay. Changes of cell morphology and ultrastructure were examined by optical and electron microscopy. The cell cycle distribution was determined by flow cytometry. Furthermore, expression of proteins related to breast cancer occurrence and MDR was tested by immunocytochemistry. In Vivo, nude mice were injected with MCF-7/S and MCF-7/TAX cells and weights and tumor sizes were observed after paclitaxel treatment. In addition, proteins involved breast cancer and MDR were detected by immunohistochemistry. Compared to MCF-7/S, MCF-7/TAX cells had a higher resistance to paclitaxel, cross-resistance and prolonged doubling time. Moreover, MCF-7/TAX showed obvious alterations of ultrastructure. Estrogen receptor (ER) expression was low in drug resistant cells and tumors while expression of human epidermal growth factor receptor 2 (HER2) and Ki-67 was up-regulated. P-glycoprotein (P-gp), lung resistance-related protein (LRP) and glutathione-S-transferase-π (GST-π) involved in the MDR phenotype of resistant cells and tumors were all overexpressed. The underlying MDR mechanism of breast cancer may involve increased expression of P-gp, LRP and GST-π.

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

PubMed Central

Johnson, Laura A.; Davis, Jeremy L.; Zheng, Zhili; Woolard, Kevin D.; Reap, Elizabeth A.; Feldman, Steven A.; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A.; Rosenberg, Steven A.

2012-01-01

Abstract No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application. PMID:22780919

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

PubMed

Morgan, Richard A; Johnson, Laura A; Davis, Jeremy L; Zheng, Zhili; Woolard, Kevin D; Reap, Elizabeth A; Feldman, Steven A; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A; Rosenberg, Steven A

2012-10-01

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

PubMed Central

Larsen, Sarah L.; Laenkholm, Anne-Vibeke; Duun-Henriksen, Anne Katrine; Bak, Martin; Lykkesfeldt, Anne E.; Kirkegaard, Tove

2015-01-01

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen. PMID:25706943

Benzo(a)pyrene quinones increase cell proliferation, generate reactive oxygen species, and transactivate the epidermal growth factor receptor in breast epithelial cells.

PubMed

Burdick, Andrew D; Davis, John W; Liu, Ke Jian; Hudson, Laurie G; Shi, Honglian; Monske, Michael L; Burchiel, Scott W

2003-11-15

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. We have reported previously that BaP can mimic growth factor signaling and increase cell proliferation in primary human mammary epithelial cells and the human mammary epithelial cell line MCF-10A. BaP-quinones (BPQs) are important metabolites of BaP that have been associated with the production of reactive oxygen species. Using a model of epidermal growth factor (EGF) withdrawal in MCF-10A, we hypothesized that production of reactive oxygen species by BPQs could lead to the activation of the EGF receptor (EGFR). Here, we demonstrate through electron paramagnetic resonance spectroscopy and flow cytometry that 1,6-BPQ and 3,6-BPQ produce superoxide anion and hydrogen peroxide in MCF-10A cells. Furthermore, we show that BPQs increase EGFR, Akt, and extracellular signal-regulated kinase activity, leading to increased cell number in the absence of EGF. The BPQ-induced EGFR activity and associated cell proliferation were attenuated by the EGFR inhibitor AG1478, as well as by the antioxidant N-acetyl cysteine. Overexpression of catalase, but not Cu/Zn superoxide dismutase, reduced the extent of BPQ-dependent increased cell number and EGFR pathway activation. Moreover, the direct treatment of MCF-10A cells with hydrogen peroxide enhanced EGFR, Akt, and extracellular-regulated kinase phosphorylation that could be similarly inhibited by AG1478, N-acetyl cysteine, and catalase. Taken together, these data indicate that BPQs, through the generation of hydrogen peroxide, activate the EGFR in MCF-10A cells, leading to increased cell number under EGF-deficient conditions.

Adding a Piece to the Leaf Epidermal Cell Shape Puzzle.

PubMed

von Wangenheim, Daniel; Wells, Darren M; Bennett, Malcolm J

2017-11-06

The jigsaw puzzle-shaped pavement cells in the leaf epidermis collectively function as a load-bearing tissue that controls organ growth. In this issue of Developmental Cell, Majda et al. (2017) shed light on how the jigsaw shape can arise from localized variations in wall stiffness between adjacent epidermal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Transient over-expression of barley BAX Inhibitor-1 weakens oxidative defence and MLA12-mediated resistance to Blumeria graminis f.sp. hordei.

PubMed

Eichmann, Ruth; Dechert, Cornelia; Kogel, Karl-Heinz; Hückelhoven, Ralph

2006-11-01

SUMMARY BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 (HvBI-1) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X(L) in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X(L) partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo-mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12-barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.

Morphometric analysis of epidermal differentiation in primary roots of Zea mays

NASA Technical Reports Server (NTRS)

Moore, R.; Smith, H. S.

1990-01-01

Epidermal differentiation in primary roots of Zea mays was divided into six cell types based on cellular shape and cytoplasmic appearance. These six cell types are: 1) apical protoderm, located at the tip of the root pole and characterized by periclinally flattened cells; 2) cuboidal protoderm, located approximately 230 microns from the root pole and characterized by cuboidal cells; 3) tabular epidermis, located approximately 450 microns from the root pole and characterized by anticlinally flattened cells; 4) cuboidal epidermis, located approximately 900 microns from the root pole and characterized by cuboidal cells having numerous small vacuoles; 5) vacuolate cuboidal epidermis, located approximately 1,500 microns from the root pole and characterized by cuboidal cells containing several large vacuoles; and 6) columnar epidermis, located approximately 2,200 microns from the root pole (i.e., at the beginning of the zone of elongation) and characterized by elongated cells. We also used stereology to quantify the cellular changes associated with epidermal differentiation. The quiescent center and the apical protoderm have significantly different ultrastructures. The relative volume of dictyosomes increases dramatically during the early stages of epidermal differentiation. This increase correlates inversely with the amount of coverage provided by the root cap and mucilage.

Tamoxifen resistance: from cell culture experiments towards novel biomarkers.

PubMed

Nass, Norbert; Kalinski, Thomas

2015-03-01

Tamoxifen is still the most frequently used antiestrogen for the treatment of patients with premenopausal, estrogen receptor positive breast cancer. However, in 20-30% of these cases, tamoxifen therapy fails due to an existing or developing resistance. The prediction of tamoxifen resistance by appropriate biomarker analysis and the development of novel therapies for tamoxifen resistance in premenopausal breast cancer is, therefore, an important goal of ongoing research. Tamoxifen resistance is associated with altered estrogen receptor expression especially on the plasma membrane, including the alternative G-protein coupled receptor GPR-30 (GPER) and estrogen receptor splice products, such as ERα36. Tamoxifen resistant cells often use alternative pathways to promote proliferation in the absence of genomic estrogen signaling. These pathways involve the epidermal growth factor EGF, the inflammation associated transcription factor NF-κB- and the IGF-1 pathway. Tamoxifen resistant mamma carcinoma cell lines are useful models to understand tamoxifen resistance in-vitro and to search for prognostic or predictive biomarkers. Furthermore, such cell lines can be used to identify potential targets for therapy. Copyright © 2015 Elsevier GmbH. All rights reserved.

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

PubMed Central

Dong, Jia; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K.N.; Knobeloch, Daniel; Gerlach, Jörg C.; Zeilinger, Katrin

2008-01-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes. PMID:19003182

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

PubMed

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

PubMed

Yamaura, Takumi; Ezaki, Junji; Okabe, Naoyuki; Takagi, Hironori; Ozaki, Yuki; Inoue, Takuya; Watanabe, Yuzuru; Fukuhara, Mitsuro; Muto, Satoshi; Matsumura, Yuki; Hasegawa, Takeo; Hoshino, Mika; Osugi, Jun; Shio, Yutaka; Waguri, Satoshi; Tamura, Hirosumi; Imai, Jun-Ichi; Ito, Emi; Yanagisawa, Yuka; Honma, Reiko; Watanabe, Shinya; Suzuki, Hiroyuki

2018-02-01

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor ( EGFR ) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B ( FAM83B ) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR . Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR . Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR .

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.

PubMed

Taron, Miguel; Ichinose, Yukito; Rosell, Rafael; Mok, Tony; Massuti, Bartomeu; Zamora, Lurdes; Mate, Jose Luis; Manegold, Christian; Ono, Mayumi; Queralt, Cristina; Jahan, Thierry; Sanchez, Jose Javier; Sanchez-Ronco, Maria; Hsue, Victor; Jablons, David; Sanchez, Jose Miguel; Moran, Teresa

2005-08-15

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non-small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non-small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity.

PubMed

Jayachandran, Aparna; Shrestha, Ritu; Dhungel, Bijay; Huang, I-Tao; Vasconcelos, Marianna Yumi Kawashima; Morrison, Brian J; Ramlogan-Steel, Charmaine A; Steel, Jason C

2017-09-26

To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors. Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes

PubMed Central

Uda, Junki; Kubo, Hirokazu; Nakajima, Yuka; Goto, Arisa; Akaki, Junji; Yoshida, Ikuyo; Matsuoka, Nobuya; Hayakawa, Takao

2016-01-01

Aloe has been used as a folk medicine because it has several important therapeutic properties. These include wound and burn healing, and Aloe is now used in a variety of commercially available topical medications for wound healing and skin care. However, its effects on epidermal keratinocytes remain largely unclear. Our data indicated that both Aloe vera gel (AVG) and Cape aloe extract (CAE) significantly improved wound healing in human primary epidermal keratinocytes (HPEKs) and a human skin equivalent model. In addition, flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with AVG and CAE. These increases may contribute to cell migration and wound healing. Treatment with Aloe also resulted in significant changes in cell-cycle progression and in increases in cell number. Aloe increased gene expression of differentiation markers in HPEKs, suggesting roles for AVG and CAE in the improvement of keratinocyte function. Furthermore, human skin epidermal equivalents developed from HPEKs with medium containing Aloe were thicker than control equivalents, indicating the effectiveness of Aloe on enhancing epidermal development. Based on these results, both AVG and CAE have benefits in wound healing and in treatment of rough skin. PMID:27736988

UV fluorescence excitation spectroscopy as a non-invasive predictor of epidermal proliferation and clinical performance of cosmetic formulations

NASA Astrophysics Data System (ADS)

Maidhof, Robert; Liebel, Frank; Hwang, Cheng; Ruvolo, Eduardo; Lyga, John

2017-02-01

The epidermis is the outermost layer of skin and is composed of cells primarily containing keratin. It consists of about ten layers of living cells (keratinocytes) and ten layers of dead cells (corneocytes). These cells are continually shed from the outside and replaced from the inside in a process called desquamation which is controlled by two biological events - proliferation and differentiation. One method to non-invasively study biological changes in the skin is using fluorescence excitation spectroscopy. Several characteristic excitation-emission peaks occur in skin that have been related to the epidermal and dermal composition. The magnitude of the peak that occurs at 295nm excitation (F295) has been linked to changes in skin proliferation, cell turnover, epidermal thickening, and skin aging. We hypothesize that changes in this fluorescent signal could be used to assess the potential activity of cosmetic anti-aging compounds to deliver a benefit to skin. Previous work with retinol and glycolic acid, two commonly used actives that effect epidermal proliferation and exfoliation, has demonstrated an increase in F295 (attributed to tryptophan excitation fluorescence). In this study we present the results of a placebo controlled study that aims to correlate changes in F295 with biological performance (epidermal thickening and Ki67 expression).

Histone deacetylation, as opposed to promoter methylation, results in epigenetic BIM silencing and resistance to EGFR TKI in NSCLC.

PubMed

Zhao, Mingchuan; Zhang, Yishi; Li, Jiayu; Li, Xuefei; Cheng, Ningning; Wang, Qi; Cai, Weijing; Zhao, Chao; He, Yayi; Chang, Jianhua; Zhou, Caicun

2018-01-01

Drug resistance remains a major challenge in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. Bcl-2-like protein 11 (BIM), a B-cell lymphoma 2 family pro-apoptotic protein, is a prime target for specific anti-cancer therapeutics. However, the epigenetic regulation of BIM in non-small cell lung cancer (NSCLC) cell lines and patients with NSCLC in association with EGFR-TKI resistance requires investigation. Methylation-specific PCR (MSP), pyrosequencing, and nested quantitative (q)-MSP were conducted to explore the methylation status of BIM in NSCLC cell lines. In addition, the methylation profile of BIM in patients with NSCLC was assessed by nested q-MSP using circulating free DNA. Cell lines, treated with methylation inhibitor 5-Aza-2'-deoxycytidine (AZA) or histone deacetylation inhibitor trichostatin A (TSA) prior to gefitinib treatment, were examined for BIM gene expression and resistance to gefitinib. All cell lines used in the present study presented with hypo-methylated BIM . Treatment with AZA had no effect on BIM RNA expression in PC9 cells or the gefitinib-resistant cell lines PC9/R and PC9/G2, nor did it reverse their resistance to gefitinib. In contrast, TSA treatment produced the opposite result. In the present study, 25 (78.1%) patients with hypo-methylated BIM and 7 patients (21.9%) with partial or hyper-methylated BIM were identified. The clinicopathological data revealed a random hypo-methylated BIM distribution amongst patients with NSCLC. In the overall study group and EGFR mutant group, hypo-methylated BIM carriers presented with no significant differences in progression free survival compared with patients with partial or hyper-methylated BIM . All cell lines in the present study and the majority of patients with NSCLC carried hypo-methylated BIM . Histone deacetylation, as opposed to promoter methylation, may contribute to the epigenetic silencing of BIM and lead to EGFR TKI resistance in NSCLC.

pH-Responsive Artemisinin Derivatives and Lipid Nanoparticle Formulations Inhibit Growth of Breast Cancer Cells In Vitro and Induce Down-Regulation of HER Family Members

PubMed Central

Zhang, Yitong J.; Gallis, Byron; Taya, Michio; Wang, Shusheng; Ho, Rodney J. Y.; Sasaki, Tomikazu

2013-01-01

Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH's that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives. PMID:23516601

MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo.

PubMed

Sahoo, Anupama; Lee, Bongyong; Boniface, Katia; Seneschal, Julien; Sahoo, Sanjaya K; Seki, Tatsuya; Wang, Chunyan; Das, Soumen; Han, Xianlin; Steppie, Michael; Seal, Sudipta; Taieb, Alain; Perera, Ranjan J

2017-09-01

Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Biochemistry of epidermal stem cells.

PubMed

Eckert, Richard L; Adhikary, Gautam; Balasubramanian, Sivaprakasam; Rorke, Ellen A; Vemuri, Mohan C; Boucher, Shayne E; Bickenbach, Jackie R; Kerr, Candace

2013-02-01

The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Genetic Ablation of CCAAT/Enhancer Binding Protein α in Epidermis Reveals Its Role in Suppression of Epithelial Tumorigenesis

PubMed Central

Loomis, Kari D.; Zhu, Songyun; Yoon, Kyungsil; Johnson, Peter F.; Smart, Robert C.

2013-01-01

CCAAT/enhancer binding protein y (C/EBPα) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPα is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPα mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPα occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPα as an epithelial tumor suppressor is lacking due to the absence of C/EBPα mutations in epithelial tumors and the lethal effect of C/EBPα deletion in mouse model systems. To examine the function of C/EBPα in epithelial tumor development, an epidermal-specific C/EBPα knockout mouse was generated. The epidermal-specific C/EBPα knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPα is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPα knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPα displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPα contributes to deregulation of tumor cell proliferation. C/EBPα had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPα blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPα is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPα is a suppressor of epithelial tumorigenesis. PMID:17638888

A novel DLX3-PKC integrated signaling network drives keratinocyte differentiation.

PubMed

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-Wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-04-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3-PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.

A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

PubMed Central

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-01-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis. PMID:28186503

Cytotoxic Effect of Nano-SiO2 in Human Breast Cancer Cells via Modulation of EGFR Signaling Cascades.

PubMed

Jeon, Donghwan; Kim, Hyungjoo; Nam, Keesoo; Oh, Sunhwa; Son, Seog-Ho; Shin, Incheol

2017-11-01

Silica nanoparticles (nano-SiO 2 ) are widely used in many industrial areas and there is much controversy surrounding cytotoxic effects of such nanoparticles. In order to determine the toxicity and possible molecular mechanisms involved, we conducted several tests with two breast cancer cell lines, MDA-MB-231 and Hs578T. After exposure to nano-SiO 2 , growth, apoptosis, motility of breast cancer cells were monitored. In addition, modulation of signal transduction induced by nano-SiO 2 was detected through western blot analysis. Treatment of nano-SiO 2 repressed the growth of breast cancer cell lines. It also increased apoptosis and reduced cell motility. Moreover, exposure to nano-SiO 2 significantly disturbed the dimerization of epidermal growth factor receptor (EGFR), followed by down-regulation of its downstream cellular sarcoma kinase (c-SRC) and signal transducer and activator of transcription 3 (STAT3) signaling cascades. Nano-SiO 2 has a cytotoxic effect on MDA-MB-231 and Hs578T breast cancer cells via modulation of EGFR signaling cascades. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Transcript Profiling Identifies Gene Cohorts Controlled by Each Signal Regulating Trans-Differentiation of Epidermal Cells of Vicia faba Cotyledons to a Transfer Cell Phenotype

PubMed Central

Zhang, Hui-Ming; Wheeler, Simon L.; Xia, Xue; Colyvas, Kim; Offler, Christina E.; Patrick, John W.

2017-01-01

Transfer cells (TCs) support high rates of membrane transport of nutrients conferred by a plasma membrane area amplified by lining a wall labyrinth comprised of an uniform wall layer (UWL) upon which intricate wall ingrowth (WI) papillae are deposited. A signal cascade of auxin, ethylene, extracellular hydrogen peroxide (H2O2) and cytosolic Ca2+ regulates wall labyrinth assembly. To identify gene cohorts regulated by each signal, a RNA- sequencing study was undertaken using Vicia faba cotyledons. When cotyledons are placed in culture, their adaxial epidermal cells spontaneously undergo trans-differentiation to epidermal TCs (ETCs). Expressed genes encoding proteins central to wall labyrinth formation (signaling, intracellular organization, cell wall) and TC function of nutrient transport were assembled. Transcriptional profiles identified 9,742 annotated ETC-specific differentially expressed genes (DEGs; Log2fold change > 1; FDR p ≤ 0.05) of which 1,371 belonged to signaling (50%), intracellular organization (27%), cell wall (15%) and nutrient transporters (9%) functional categories. Expression levels of 941 ETC-specific DEGs were found to be sensitive to the known signals regulating ETC trans-differentiation. Significantly, signals acting alone, or in various combinations, impacted similar numbers of ETC-specific DEGs across the four functional gene categories. Amongst the signals acting alone, H2O2 exerted most influence affecting expression levels of 56% of the ETC-specific DEGs followed by Ca2+ (21%), auxin (18%) and ethylene (5%). The dominance by H2O2 was evident across all functional categories, but became more attenuated once trans-differentiation transitioned into WI papillae formation. Amongst the eleven signal combinations, H2O2/Ca2+ elicited the greatest impact across all functional categories accounting for 20% of the ETC-specific DEG cohort. The relative influence of the other signals acting alone, or in various combinations, varied across the four functional categories and two phases of wall labyrinth construction. These transcriptome data provide a powerful information platform from which to examine signal transduction pathways and how these regulate expression of genes encoding proteins engaged in intracellular organization, cell wall construction and nutrient transport. PMID:29234338

Transcript Profiling Identifies Gene Cohorts Controlled by Each Signal Regulating Trans-Differentiation of Epidermal Cells of Vicia faba Cotyledons to a Transfer Cell Phenotype.

PubMed

Zhang, Hui-Ming; Wheeler, Simon L; Xia, Xue; Colyvas, Kim; Offler, Christina E; Patrick, John W

2017-01-01

Transfer cells (TCs) support high rates of membrane transport of nutrients conferred by a plasma membrane area amplified by lining a wall labyrinth comprised of an uniform wall layer (UWL) upon which intricate wall ingrowth (WI) papillae are deposited. A signal cascade of auxin, ethylene, extracellular hydrogen peroxide (H 2 O 2 ) and cytosolic Ca 2+ regulates wall labyrinth assembly. To identify gene cohorts regulated by each signal, a RNA- sequencing study was undertaken using Vicia faba cotyledons. When cotyledons are placed in culture, their adaxial epidermal cells spontaneously undergo trans -differentiation to epidermal TCs (ETCs). Expressed genes encoding proteins central to wall labyrinth formation (signaling, intracellular organization, cell wall) and TC function of nutrient transport were assembled. Transcriptional profiles identified 9,742 annotated ETC-specific differentially expressed genes (DEGs; Log 2 fold change > 1; FDR p ≤ 0.05) of which 1,371 belonged to signaling (50%), intracellular organization (27%), cell wall (15%) and nutrient transporters (9%) functional categories. Expression levels of 941 ETC-specific DEGs were found to be sensitive to the known signals regulating ETC trans -differentiation. Significantly, signals acting alone, or in various combinations, impacted similar numbers of ETC-specific DEGs across the four functional gene categories. Amongst the signals acting alone, H 2 O 2 exerted most influence affecting expression levels of 56% of the ETC-specific DEGs followed by Ca 2+ (21%), auxin (18%) and ethylene (5%). The dominance by H 2 O 2 was evident across all functional categories, but became more attenuated once trans -differentiation transitioned into WI papillae formation. Amongst the eleven signal combinations, H 2 O 2 /Ca 2+ elicited the greatest impact across all functional categories accounting for 20% of the ETC-specific DEG cohort. The relative influence of the other signals acting alone, or in various combinations, varied across the four functional categories and two phases of wall labyrinth construction. These transcriptome data provide a powerful information platform from which to examine signal transduction pathways and how these regulate expression of genes encoding proteins engaged in intracellular organization, cell wall construction and nutrient transport.

Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

PubMed Central

2012-01-01

Background Adenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development. PMID:23020757

WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

PubMed

Lee, M M; Schiefelbein, J

1999-11-24

The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

Role of epidermal stem cells in repair of partial-thickness burn injury after using Moist Exposed Burn Ointment (MEBO(®)) histological and immunohistochemical study.

PubMed

El-Hadidy, M R; El-Hadidy, A R; Bhaa, A; Asker, S A; Mazroa, S A

2014-04-01

Moist Exposed Burn Ointment (MEBO(®)) is widely used topical agent applied on skin burn. This study investigated the effect of MEBO topical application on activation and proliferation of epidermal stem cells through the immunohistochemical localization of cytokeratin 19 (CK19) as a known marker expressed in epidermal stem cells. Biopsies from normal skin and burn wounds were taken from 21 patients with partial thickness burn 1, 4, 7, 14, 21, and 28 days after treatment with MEBO. Tissue sections were prepared for histological study and for CK19 immunohistochemical localization. In control skin, only few cells showed a positive CK19 immune-reaction. Burned skin showed necrosis of full thickness epidermis that extended to dermis. Gradual regeneration of skin accompanied with an enhancement in CK19 immune-reactivity was noted 4, 7, 14 and 21 days after treatment with MEBO. On day 28, a complete regeneration of skin was observed with a return of CK19 immune-reactivity to the basal pattern again. In conclusion, the enhancement of epidermal stem cell marker CK19 after treatment of partial thickness burn injuries with MEBO suggested the role of MEBO in promoting epidermal stem cell activation and proliferation during burn wound healing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carrasco-Garcia, Estefania; Saceda, Miguel; Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cellmore » lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.« less

A human thyroid cancer cell line, DH-14-3, newly established from poorly differentiated thyroid carcinoma.

PubMed

Teshima, Jin; Doi, Hideyuki; Fujimori, Keisei; Watanabe, Michio; Nakajima, Noriaki; Nakano, Tomoyuki; Takahashi, Yoshio; Ohuchi, Noriaki; Satomi, Susumu

2013-06-01

Poorly differentiated thyroid carcinoma (PDTC) is a newly recognized histological type of malignant thyroid tumor, accounting for about 2 - 13% of all thyroid carcinomas. PDTC is considered as a morphologically and biologically intermediate stage between well-differentiated thyroid carcinoma and anaplastic thyroid carcinoma. PDTC preferentially manifests bone metastases. We here established a cell line from a resected tumor specimen from a 70-year-old male patient with PDTC who presented with multiple bone metastases. This new thyroid tumor cell line was designated as DH-14-3 and was subsequently grown in culture for several years. DH-14-3 cells express thyroglobulin in the cytoplasm and thyroid transcription factor-1 in the nuclei, both proteins of which are specific markers for the thyroid gland. Importantly, triiodothyronine (T3) was detected in the cultured medium of DH-14-3 cells, in which, however, thyroxine (T4) was undetectable. Moreover, DH-14-3 cells secreted interleukin-8, transforming growth factor-β1, vascular endothelial growth factor, matrix metalloproteinase-1 and parathyroid hormone-related protein, all of which may be responsible for the aggressiveness or bone metastasis of PDTC. Thus, the production of these proteins may reflect the metastatic potential of this cell line. DH-14-3 cells also express CXC chemokine receptor-4 and epidermal growth factor receptor, and carry a missense mutation in the p53 tumor suppressor gene. In fact, transplantation of DH-14-3 cells into the back of nude mice resulted in the formation of tumors, thereby confirming the capability of tumorigenesis. DH-14-3 cells may be useful for investigating the biological features of PDTC and will contribute to the therapeutic study of thyroid cancer.

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

PubMed

Ekblad, Lars; Lindgren, Gustaf; Persson, Emma; Kjellén, Elisabeth; Wennerberg, Johan

2013-01-25

Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro. Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors. One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3. In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development

PubMed Central

Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U; Wick, S

2014-01-01

It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Apoptosis as a Mechanism for Keratinocyte Death in Canine Toxic Epidermal Necrolysis.

PubMed

Banovic, F; Dunston, S; Linder, K E; Rakich, P; Olivry, T

2017-03-01

In humans and dogs, toxic epidermal necrolysis (TEN) is a life-threatening dermatosis characterized by sudden epidermal death resulting in extensive skin detachment. There is little information on the pathogenesis of keratinocyte cell death in canine TEN. We studied the occurrence of apoptosis in skin lesions of dogs with TEN to determine if apoptosis contributes to the pathogenesis of this disease. Immunostaining with antibodies to activated caspase-3 and the terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling technique revealed positive apoptotic keratinocytes in basal and suprabasal epidermal compartments in 17 biopsy specimens collected from 3 dogs with TEN and 16 from 3 dogs with erythema multiforme (EM). There was no significant difference in the number of positively stained epidermal cells between TEN and EM. These results suggest that apoptosis of epidermal keratinocytes and lymphocytic satellitosis represent one of the early steps in the pathogenesis of canine TEN, as in the human disease counterpart.

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Chang Yeob; Hien, Tran Thi; Lim, Sung Chul

2011-06-24

Highlights: {yields} Pin1 expression is enhanced by low energy UVA irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. {yields} UVA irradiation increases activator protein-1 activity and cyclin D1 in a Pin1-dependent manner. {yields} UVA potentiates EGF-inducible, anchorage-independent growth of epidermal cells, and this is suppressed by Pin1 inhibition or by anti-oxidant. -- Abstract: Ultraviolet A (UVA) radiation ({lambda} = 320-400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, wemore » demonstrated that Pin1 expression was enhanced by low energy UVA (300-900 mJ/cm{sup 2}) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.« less

Biochemistry of epidermal stem cells☆

PubMed Central

Eckert, Richard L.; Adhikary, Gautam; Balasubramanian, Sivaprakasam; Rorke, Ellen A.; Vemuri, Mohan C.; Boucher, Shayne E.; Bickenbach, Jackie R.; Kerr, Candace

2014-01-01

Background The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. Scope of review A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. Major conclusions An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. General significance Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells. PMID:22820019

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells.

PubMed

Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

2016-04-01

Plant leaf epidermal cells exhibit a jigsaw puzzle-like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.

Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis.

PubMed

Salerno, Simona; Messina, Antonietta; Giordano, Francesca; Bader, Augustinus; Drioli, Enrico; De Bartolo, Loredana

2017-02-01

Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT-PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Interaction Between GFP-Labeled Diazotrophic Endophytic Bacterium Herbaspirillum seropedicae RAM10 and Pineapple Plantlets ‘VitóRia’

PubMed Central

Estrela Borges Baldotto, Lílian; Lopes Olivares, Fábio; Bressan-Smith, Ricardo

2011-01-01

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets ‘Vitória’ were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae. PMID:24031612

Structural Interaction Between GFP-Labeled Diazotrophic Endophytic Bacterium Herbaspirillum seropedicae RAM10 and Pineapple Plantlets 'VitóRia'.

PubMed

Estrela Borges Baldotto, Lílian; Lopes Olivares, Fábio; Bressan-Smith, Ricardo

2011-01-01

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

microRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor.

PubMed

Tao, Jun; Wu, Deyao; Xu, Bin; Qian, Weichun; Li, Pengchao; Lu, Qiang; Yin, Changjun; Zhang, Wei

2012-06-01

It has been shown that regulation of EGFR expression in prostate cancer cells is mostly at the transcriptional level. microRNA-133 (miR-133) has long been recognized as a muscle-specific miRNA which may regulate myoblast differentiation and participate in many myogenic diseases. Recently, it has been reported that miR-133 is also involved in other tumors, such as bladder cancer, esophageal cancer and may regulate cell motility in these cancer cells. In the present study, we examined the expression and effects of miR-133 in two hormone-insensitive prostate cancer cell lines. The expression of miR-133a and miR-133b were analyzed by quantitative RT-PCR. After transfection of miR-133a and miR-133b, cell viability assay, luciferase assay, western blot analysis, cell migration and invasion assay were conducted in Du145 and PC3 cells. In this study, we showed that miR‑133a and miR-133b are expressed at the detection limit in two hormone-insensitive prostate cancer cell lines, PC3 and DU145. Ectopic expression of miR-133 inhibited cell proliferation, migration and invasion in these cells. We also provide the first evidence that miR-133 may target EGFR. Our study provided the first glimpse of the functional role of miR-133 in two hormone-independent prostate cancer cell lines. These results may add to our knowledge on the molecular basis of prostate cancer progression.

γδ T cells in homeostasis and host defence of epithelial barrier tissues.

PubMed

Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

2017-12-01

Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

Characterization of an entomopathogenic fungi target integument protein, Bombyx mori single domain von Willebrand factor type C, in the silkworm, Bombyx mori.

PubMed

Han, F; Lu, A; Yuan, Y; Huang, W; Beerntsen, B T; Huang, J; Ling, E

2017-06-01

The insect cuticle works as the first line of defence to protect insects from pathogenic infections and water evaporation. However, the old cuticle must be shed in order to enter the next developmental stage. During each ecdysis, moulting fluids are produced and secreted into the area among the old and new cuticles. In a previous study, the protein Bombyx mori single domain von Willebrand factor type C (BmSVWC; BGIBMGA011399) was identified in the moulting fluids of Bo. mori and demonstrated to regulate ecdysis. In this study we show that in Bo. mori larvae, BmSVWC primarily locates to the integument (epidermal cells and cuticle), wing discs and head. During the moulting stage, BmSVWC is released into the moulting fluids, and is then produced again by epidermal cells after ecdysis. Fungal infection was shown to decrease the amount of BmSVWC in the cuticle, which indicates that BmSVWC is a target protein of entomopathogenic fungi. Thus, BmSVWC is mainly involved in maintaining the integrity of the integument structure, which serves to protect insects from physical damage and pathogenic infection. © 2017 The Royal Entomological Society.

Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity.

PubMed

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C B

2009-11-01

The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established.

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model.

PubMed

Matsumoto, Reiko; Sugimoto, Masazumi

2007-02-01

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.

Real-time visualization of macromolecule uptake by epidermal Langerhans cells in living animals.

PubMed

Frugé, Rachel E; Krout, Colleen; Lu, Ran; Matsushima, Hironori; Takashima, Akira

2012-03-01

As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC(+) epidermal cells became detectable 12-24 hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC(+) epidermal cells expressed Langerin, and >95% of Langerin(+) epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX probes were locally injected into IAβ-enhanced green fluorescent protein (EGFP) knock-in mice and Langerin-EGFP-diphtheria toxin receptor mice--three dimensional rotation images showed close association of most of the internalized DX probes with major histocompatibility complex (MHC) class II molecules, but not with Langerin molecules. These observations support the current view that LCs constantly sample surrounding materials, including harmful and innocuous antigens, at the environmental interface. Our data also validate the potential utility of the newly developed imaging approach to monitor LC function in wild-type animals.

Epidermal growth factor-like growth factors prevent apoptosis of alcohol-exposed human placental cytotrophoblast cells.

PubMed

Wolff, Garen S; Chiang, Po Jen; Smith, Susan M; Romero, Roberto; Armant, D Randall

2007-07-01

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.

Induction of neuronal phenotypes from NG2+ glial progenitors by inhibiting epidermal growth factor receptor in mouse spinal cord injury.

PubMed

Ju, Peijun; Zhang, Si; Yeap, Yeeshan; Feng, Zhiwei

2012-11-01

Besides neural stem cells, some glial cells, such as GFAP+ cells, radial glia, and oligodendrocyte progenitor cells can produce neuronal cells. Attractively, NG2+ glial progenitors exhibit lineage plasticity, and they rapidly proliferate and differentiate in response to central nervous system (CNS) injuries. These attributes of NG2+ glial progenitors make them a promising source of neurons. However, the potential of neuronal regeneration from NG2+ glial progenitors in CNS pathologies remains to be investigated. In this study, we showed that antagonizing epidermal growth factor receptor (EGFR) function with EGFR inhibitor caused a significant number of proliferative NG2+ glial progenitors to acquire neuronal phenotypes in contusive spinal cord injury (SCI), which presumably led to an accumulation of newly generated neurons and contributed to the improved neural behavioral performance of animals. In addition, the neuronal differentiation of glial progenitors induced by EGFR inhibitor was further confirmed with two different cell lines either in vitro or through ex vivo transplantation experiment. The inhibition of EGFR signaling pathway under the gliogenic conditions could induce these cells to acquire neuronal phenotypes. Furthermore, we find that the Ras-ERK axis played a key role in neuronal differentiation of NG2+ glial progenitors upon EGFR inhibition. Taken together, our studies suggest that the EGFR inhibitor could promote neurogenesis post SCI, mainly from the NG2+ glial progenitors. These findings support the possibility of evoking endogenous neuronal replacement from NG2+ glial progenitors and suggest that EGFR inhibition may be beneficial to CNS trauma. Copyright © 2012 Wiley Periodicals, Inc.

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth.

PubMed

Li, H; Li, J; Su, Y; Fan, Y; Guo, X; Li, L; Su, X; Rong, R; Ying, J; Mo, X; Liu, K; Zhang, Z; Yang, F; Jiang, G; Wang, J; Zhang, Y; Ma, D; Tao, Q; Han, W

2014-06-12

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

NASA Astrophysics Data System (ADS)

Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

2012-06-01

The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

PubMed

Rasmussen, Cathy A; Allen-Hoffmann, B Lynn

2012-04-01

For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss. Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites. Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures. Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application. This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.

PubMed

Arwert, Esther N; Lal, Rohit; Quist, Sven; Rosewell, Ian; van Rooijen, Nico; Watt, Fiona M

2010-11-16

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

Epidermal lipid in several cetacean species: ultrastructural observations.

PubMed

Pfeiffer, C J; Jones, F M

1993-09-01

The ultrastructure of the skin of four cetacean species, bottlenose dolphin (Tursiops truncatus) long-finned pilot whale (Globicephala melaena), humpback whale (Megaptera novaeangliae), and fin whale (Balaenoptera physalus) was investigated with particular reference to epidermal lipid. It has already been established that massive lipid reservoirs exist in whales, that the biochemical structures of cetacean lipids are unique, and that unusual intracellular lipid droplets appear in the epidermis. We report here some novel findings on scanning electron microscopic morphology of epidermal lipid, and on its ultrastructural morphology in general and specialized integumentary sites, including species not previously investigated. The intracellular epidermal lipid droplets were more extensive than lamellar body-derived intercellular lipid which is within the interstices of stratum externum cells. The intracellular droplets were spherical, highly variable in size ranging from 0.24 micron to 3.0 microns in diameter, appeared singly or were aggregated in cytoplasmic cavitations, and often were closely associated with epidermal cell nuclei. Evidence for exocytosis of the intracellular droplets was not observed. Significant numbers of intracellular lipid droplets are not observed in the epidermis of terrestrial mammals, so their presence is one of several aquatic specializations of the cetacean integument. Its full significance remains obscure, but it is more probably associated with epidermal cell metabolism than with secretion of lipid.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

PubMed

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

2017-09-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1 , are translationally regulated by SMED-PABPC2 . Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles.

PubMed

Matrisian, L M; Planck, S R; Magun, B E

1984-03-10

We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea

PubMed Central

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Dilipkumar, Shilpa; Gulyani, Akash; Raghavan, Srikala

2017-01-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. PMID:28807897

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

PubMed

Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

2015-08-01

Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors.

PubMed

Malakootian, Mahshid; Mirzadeh Azad, Fatemeh; Fouani, Youssef; Taheri Bajgan, Elham; Saberi, Hooshang; Mowla, Seyed Javad

2018-06-01

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

Synergistic apoptosis in head and neck squamous cell carcinoma cells by co-inhibition of insulin-like growth factor-1 receptor signaling and compensatory signaling pathways.

PubMed

Axelrod, Mark J; Mendez, Rolando E; Khalil, Ashraf; Leimgruber, Stephanie S; Sharlow, Elizabeth R; Capaldo, Brian; Conaway, Mark; Gioeli, Daniel G; Weber, Michael J; Jameson, Mark J

2015-12-01

In head and neck squamous cell carcinoma (HNSCC), resistance to single-agent targeted therapy may be overcome by co-targeting of compensatory signaling pathways. A targeted drug screen with 120 combinations was used on 9 HNSCC cell lines. Multiple novel drug combinations demonstrated synergistic growth inhibition. Combining the insulin-like growth factor-1 receptor (IGF-1R) inhibitor, BMS754807, with either the human epidermal growth factor receptor (HER)-family inhibitor, BMS599626, or the Src-family kinase inhibitor, dasatinib, resulted in substantial synergy and growth inhibition. Depending on the cell line, these combinations induced synergistic or additive apoptosis; when synergistic apoptosis was observed, AKT phosphorylation was inhibited to a greater extent than either drug alone. Conversely, when additive apoptosis occurred, AKT phosphorylation was not reduced by the drug combination. Combined IGF-1R/HER family and IGF-1R/Src family inhibition may have therapeutic potential in HNSCC. AKT may be a node of convergence between IGF-1R signaling and pathways that compensate for IGF-1R inhibition. © 2015 Wiley Periodicals, Inc.

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

PubMed Central

Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

2016-01-01

Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo. PMID:27054467

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Z. N.; Sharma, V. P.; Beaty, B. T.

2014-10-13

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and inmore » particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.« less

Identification of Candidate Transcriptional Regulators of Epidermal Transfer Cell Development in Vicia faba Cotyledons

PubMed Central

Arun-Chinnappa, Kiruba S.; McCurdy, David W.

2016-01-01

Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal–enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal–enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. PMID:27252730

Relationship between the ability of sunscreens containing 2-ethylhexyl-4'-methoxycinnamate to protect against UVR-induced inflammation, depletion of epidermal Langerhans (Ia+) cells and suppression of alloactivating capacity of murine skin in vivo.

PubMed

Walker, S L; Morris, J; Chu, A C; Young, A R

1994-01-01

The UVB sunscreen 2-ethylhexyl-4'-methoxycinnamate was evaluated in hairless albino mouse skin for its ability to inhibit UVR-induced (i) oedema, (ii) epidermal Langerhans cell (Ia+) depletion and (iii) suppression of the alloactivating capacity of epidermal cells (mixed epidermal cell-lymphocyte reaction, MECLR). The sunscreen, prepared at 9% in ethanol or a cosmetic lotion, was applied prior to UVB/UVA irradiation. In some experiments there was a second application halfway through the irradiation. Single applications in both vehicles gave varying degrees of protection from oedema and Langerhans cell depletion but afforded no protection from suppression of MECLR. When the sunscreens were applied twice there was improved protection from oedema and Langerhans cell depletion and complete protection was afforded from suppression of MECLR. There was a clear linear relationship between Langerhans cell numbers and oedema with and without sunscreen application. The relationship between Langerhans cell numbers and MECLR was more complex. These data confirm published discrepancies between protection from oedema (a model for human erythema) and endpoints with immunological significance, but show that 2-ethylhexyl-4'-methoxycinnamate can afford complete immunoprotection, although protection is dependent on the application rate and vehicle.

Delineation of Matriptase Protein Expression by Enzymatic Gene Trapping Suggests Diverging Roles in Barrier Function, Hair Formation, and Squamous Cell Carcinogenesis

PubMed Central

List, Karin; Szabo, Roman; Molinolo, Alfredo; Nielsen, Boye Schnack; Bugge, Thomas H.

2006-01-01

The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma. PMID:16651618

Delineation of matriptase protein expression by enzymatic gene trapping suggests diverging roles in barrier function, hair formation, and squamous cell carcinogenesis.

PubMed

List, Karin; Szabo, Roman; Molinolo, Alfredo; Nielsen, Boye Schnack; Bugge, Thomas H

2006-05-01

The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma.

Molecular Targeted Drugs and Biomarkers in NSCLC, the Evolving Role of Individualized Therapy

PubMed Central

Domvri, Kalliopi; Zarogoulidis, Paul; Darwiche, Kaid; Browning, Robert F.; Li, Qiang; Turner, J. Francis; Kioumis, Ioannis; Spyratos, Dionysios; Porpodis, Konstantinos; Papaiwannou, Antonis; Tsiouda, Theodora; Freitag, Lutz; Zarogoulidis, Konstantinos

2013-01-01

Lung cancer first line treatment has been directed from the non-specific cytotoxic doublet chemotherapy to the molecular targeted. The major limitation of the targeted therapies still remains the small number of patients positive to gene mutations. Furthermore, the differentiation between second line and maintenance therapy has not been fully clarified and differs in the clinical practice between cancer centers. The authors present a segregation between maintenance treatment and second line and present a possible definition for the term “maintenance” treatment. In addition, cancer cell evolution induces mutations and therefore either targeted therapies or non-specific chemotherapy drugs in many patients become ineffective. In the present work pathways such as epidermal growth factor, anaplastic lymphoma kinase, met proto-oncogene and PI3K are extensively presented and correlated with current chemotherapy treatment. Future, perspectives for targeted treatment are presented based on the current publications and ongoing clinical trials. PMID:24312144

The dual PI3K/mTOR inhibitor PKI-587 enhances sensitivity to cetuximab in EGFR-resistant human head and neck cancer models

PubMed Central

D'Amato, V; Rosa, R; D'Amato, C; Formisano, L; Marciano, R; Nappi, L; Raimondo, L; Di Mauro, C; Servetto, A; Fusciello, C; Veneziani, B M; De Placido, S; Bianco, R

2014-01-01

Background: Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. Methods: Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. Results: Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. Conclusions: Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness. PMID:24823695

The dual PI3K/mTOR inhibitor PKI-587 enhances sensitivity to cetuximab in EGFR-resistant human head and neck cancer models.

PubMed

D'Amato, V; Rosa, R; D'Amato, C; Formisano, L; Marciano, R; Nappi, L; Raimondo, L; Di Mauro, C; Servetto, A; Fusciello, C; Veneziani, B M; De Placido, S; Bianco, R

2014-06-10

Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

PubMed

Ohnishi, Yuichi; Yasui, Hiroki; Kakudo, Kenji; Nozaki, Masami

2016-11-01

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerberick, G.F.; Ryan, C.A.; Von Bargen, E.C.

Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis responsemore » compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent.« less

Cellular Migration and Invasion Uncoupled: Increased Migration Is Not an Inexorable Consequence of Epithelial-to-Mesenchymal Transition

PubMed Central

Schaeffer, Daneen; Somarelli, Jason A.; Hanna, Gabi; Palmer, Gregory M.

2014-01-01

Metastatic dissemination requires carcinoma cells to detach from the primary tumor and invade through the basement membrane. To acquire these characteristics, epithelial tumor cells undergo epithelial-to-mesenchymal transitions (EMT), whereby cells lose polarity and E-cadherin-mediated cell-cell adhesion. Post-EMT cells have also been shown, or assumed, to be more migratory; however, there have been contradictory reports on an immortalized human mammary epithelial cell line (HMLE) that underwent EMT. In the context of carcinoma-associated EMT, it is not yet clear whether the change in migration and invasion must be positively correlated during EMT or whether enhanced migration is a necessary consequence of having undergone EMT. Here, we report that pre-EMT rat prostate cancer (PC) and HMLE cells are more migratory than their post-EMT counterparts. To determine a mechanism for increased epithelial cell migration, gene expression analysis was performed and revealed an increase in epidermal growth factor receptor (EGFR) expression in pre-EMT cells. Indeed, inhibition of EGFR in PC epithelial cells slowed migration. Importantly, while post-EMT PC and HMLE cell lines are less migratory, both remain invasive in vitro and, for PC cells, in vivo. Our study demonstrates that enhanced migration is not a phenotypic requirement of EMT, and migration and invasion can be uncoupled during carcinoma-associated EMT. PMID:25002532

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

PubMed Central

WATANABE, YUSAKU; YOSHIMURA, KIYOSHI; YOSHIKAWA, KOICHI; TSUNEDOMI, RYOICHI; SHINDO, YOSHITARO; MATSUKUMA, SOU; MAEDA, NORIKO; KANEKIYO, SHINSUKE; SUZUKI, NOBUAKI; KURAMASU, ATSUO; SONODA, KOUHEI; TAMADA, KOJI; KOBAYASHI, SEI; SAYA, HIDEYUKI; HAZAMA, SHOICHI; OKA, MASAAKI

2014-01-01

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy. PMID:25118635

EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

PubMed Central

Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

2014-01-01

Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.

PubMed

Kumar, S S; Tomita, Y; Wrin, J; Bruhn, M; Swalling, A; Mohammed, M; Price, T J; Hardingham, J E

2017-06-01

Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

PubMed

Konda, John D; Olivero, Martina; Musiani, Daniele; Lamba, Simona; Di Renzo, Maria F

2017-06-01

The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Anti-wrinkle effects of Sargassum muticum ethyl acetate fraction on ultraviolet B-irradiated hairless mouse skin and mechanistic evaluation in the human HaCaT keratinocyte cell line

PubMed Central

Song, Jae Hyoung; Piao, Mei Jing; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Yoon, Weon Jong; Ko, Mi Hee; Lee, Nam Ho; Lee, Mi Young; Chae, Sungwook; Hyun, Jin Won

2016-01-01

The present study investigated the photoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)-induced skin damage and photoaging in a mouse model. HR-1 strain hairless male mice were divided into three groups: An untreated control group, a UVB-irradiated vehicle group and a UVB-irradiated SME group. The UVB-irradiated mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml water per day) and then exposed to radiation at a dose of 60–120 mJ/cm2. Wrinkle formation and skin damage were evaluated by analysis of skin replicas, epidermal thickness and collagen fiber integrity in the dermal connective tissue. The mechanism underlying the action of SME was also investigated in the human HaCaT keratinocyte cell line following exposure of the cells to UVB at a dose of 30 mJ/cm2. The protein expression levels and activity of matrix metalloproteinase-1 (MMP-1), and the binding of activator protein-1 (AP-1) to the MMP-1 promoter were assessed in the HaCaT cells using western blot analysis, an MMP-1 fluorescent assay and a chromatin immune-precipitation assay, respectively. The results showed that the mean length and depth of the wrinkles in the UVB-exposed hairless mice were significantly improved by oral administration of SME, which also prevented the increase in epidermal thickness triggered by UVB irradiation. Furthermore, a marked increase in collagen bundle formation was observed in the UVB-treated mice with SME administration. SME pretreatment also significantly inhibited the UVB-induced upregulation in the expression and activity of MMP-1 in the cultured HaCaT keratinocytes, and the UVB-enhanced association of AP-1 with the MMP-1 promoter. These results suggested that SME may be useful as an anti-photoaging resource for the skin. PMID:27573915

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

PubMed

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting. Copyright © 2015 Elsevier GmbH. All rights reserved.

Characterization of hair-follicle side population cells in mouse epidermis and skin tumors

PubMed Central

Kim, Sun Hye; Sistrunk, Christopher; Miliani de Marval, Paula L.; Rodriguez-Puebla, Marcelo L.

2017-01-01

A subset of cells, termed side-population (SP), which have the ability to efflux Hoeschst 33342, have previously been demonstrated to act as a potential method to isolate stem cells. Numerous stem/progenitor cells have been localized in different regions of the mouse hair follicle (HF). The present study identified a SP in the mouse HF expressing the ABCG2 transporter and MTS24 surface marker. These cells are restricted to the upper isthmus of the HF and have previously been described as progenitor cells. Consistent with their SP characteristic, they demonstrated elevated expression of ABCG2 transporter, which participates in the dye efflux. Analysis of tumor epidermal cell lines revealed a correlation between the number of SP keratinocytes and the grade of malignancy, suggesting that the SP may play a role in malignant progression. Consistent with this idea, the present study observed an increased number of cells expressing ABCG2 and MTS24 in chemically induced skin tumors and skin tumor cell lines. This SP does not express the CD34 surface marker detected in the multipotent stem cells of the bulge region of the HF, which have been defined as tumor initiation cells. The present study concluded that a SP with properties of progenitor cells is localized in the upper isthmus of the HF and is important in mouse skin tumor progression. PMID:29181098

Automated measurement of epidermal thickness from optical coherence tomography images using line region growing

NASA Astrophysics Data System (ADS)

Delacruz, Jomer; Weissman, Jesse; Gossage, Kirk

2010-02-01

Optical Coherence Tomography (OCT) is a non-invasive imaging modality that acquires cross sectional images of tissue in-vivo. It accelerates skin diagnosis by eliminating invasive biopsy and laborious histology in the process. Dermatologists have widely used it for looking at morphology of skin diseases such as psoriasis, dermatitis, basal cell carcinoma etc. Skin scientists have also successfully used it for looking at differences in epidermal thickness and its underlying structure with respect to age, body sites, ethnicity, gender, and other related factors. Similar to other in-vivo imaging systems, OCT images suffer from a high degree of speckle and noise content, which hinders examination of tissue structures. Most of the previous work in OCT segmentation of skin was done manually. This compromised the quality of the results by limiting the analyses to a few frames per area. In this paper, we discuss a region growing method for automatic identification of the upper and lower boundaries of the epidermis in living human skin tissue. This image analysis method utilizes images obtained from a frequency-domain OCT. This system is high-resolution and high-speed, and thus capable of capturing volumetric images of the skin in short time. The three-dimensional (3D) data provides additional information that is used in the segmentation process to help compensate for the inherent noise in the images. This method not only provides a better estimation of the epidermal thickness, but also generates a 3D surface map of the epidermal-dermal junction, from which underlying topography can be visualized and further quantified.

Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chitcholtan, Kenny, E-mail: kenny.chitcholtan@otago.ac.nz; Asselin, Eric, E-mail: Eric.Asselin@uqtr.ca; Parent, Sophie, E-mail: Sophie.Parent@uqtr.ca

2013-01-01

Three-dimensional (3D) in vitro models have an invaluable role in understanding the behaviour of tumour cells in a well defined microenvironment. This is because some aspects of tumour characteristics cannot be fully recapitulated in a cell monolayer (2D). In the present study, we compared growth patterns, expression of signalling molecules, and metabolism-associated proteins of endometrial cancer cell lines in 3D and 2D cell cultures. Cancer cells formed spherical structures in 3D reconstituted basement membrane (3D rBM), and the morphological appearance was cell line dependent. Cell differentiation was observed after 8 days in the 3D rBM. There was reduced proliferation, detectedmore » by less expression of PCNA in 3D rBM than in 2D cell monolayers. The addition of exogenous epidermal growth factor (EGF) to cancer cells induced phosphorylation of EGFR and Akt in both cell culture conditions. The uptake of glucose was selectively altered in the 3D rBM, but there was a lack of association with Glut-1 expression. The secretion of vascular endothelial growth factor (VEGF) and prostaglandin E{sub 2} (PGE{sub 2}) was selectively altered in 3D rBM, and it was cell line dependent. Our data demonstrated that 3D rBM as an in vitro model can influence proliferation and metabolism of endometrial cancer cell behaviour compared to 2D cell monolayer. Changes are specific to individual cell types. The use of 3D rBM is, therefore, important in the in vitro study of targeted anticancer therapies.« less

Blockade of epidermal growth factor receptor signaling leads to inhibition of renal cell carcinoma growth in the bone of nude mice.

PubMed

Weber, Kristy L; Doucet, Michele; Price, Janet E; Baker, Cheryl; Kim, Sun Jin; Fidler, Isaiah J

2003-06-01

Renal cell carcinoma (RCC) frequently produces metastases to the musculoskeletal system that are a major source of morbidity in the form of pain, immobilization, fractures, neurological compromise, and a decreased ability to perform activities of daily living. Patients with metastatic RCC therefore have a dismal prognosis because there is no effective adjuvant treatment for this disease. Because the epidermal growth factor receptor (EGF-R) signaling cascade is important in the growth and metastasis of RCC, its blockade has been hypothesized to inhibit tumor growth and hence prevent resultant bone destruction. We determined whether blockade of EGF-R by the tyrosine kinase inhibitor PKI 166 inhibited the growth of RCC in bone. We use a novel cell line, RBM1-IT4, established from a human RCC bone metastasis. Protein and mRNA expression of the ligands and receptors was assessed by Western and Northern blots. The stimulation of RBM1-IT4 cells with epidermal growth factor or transforming growth factor alpha resulted in increased cellular proliferation and tyrosine kinase autophosphorylation. PKI 166 prevented these effects. First, RBM1-IT4 cells were implanted into the tibia of nude mice, where they established lytic, progressively growing lesions, after which the mice were treated with PKI 166 alone or in combination with paclitaxel (Taxol). Immunohistochemical analysis revealed that tumor cells and tumor-associated endothelial cells in control mice expressed activated EGF-R. Treatment of mice with PKI 166 alone or in combination with Taxol produced a significant decrease in the incidence and size of bone lesions as compared with the results in control or Taxol-treated mice (P < 0.001). Treatment with PKI 166 also decreased the expression of phosphorylated EGF-R by tumor cells and tumor-associated endothelial cells, and this was even more pronounced with PKI 166 plus Taxol treatment. The PKI 166 plus Taxol combination produced apoptosis of tumor cells and tumor-associated endothelial cells. Tumor cell proliferation, shown by proliferating cell nuclear antigen positivity, was decreased in all treatment groups. In addition, the integrity of the bone was maintained in mice treated with PKI 166 or PKI 166 plus Taxol, whereas massive bone destruction was seen in control and Taxol-treated mice. These results suggest that blockade of EGF-R signaling inhibits growth of RCC in the bone by its effect on tumor cells and tumor-associated endothelial cells.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

PubMed

Hickinson, D Mark; Marshall, Gayle B; Beran, Garry J; Varella-Garcia, Marileila; Mills, Elizabeth A; South, Marie C; Cassidy, Andrew M; Acheson, Kerry L; McWalter, Gael; McCormack, Rose M; Bunn, Paul A; French, Tim; Graham, Alex; Holloway, Brian R; Hirsch, Fred R; Speake, Georgina

2009-06-01

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI(50)] < 1 microM), three had intermediate sensitivity (GI(50) 1-7 microM), and six were resistant (GI(50) > 7 microM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming growth factor, alpha (TGF-alpha); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-alpha, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer.

PubMed

Goodwin, C M; Rossanese, O W; Olejniczak, E T; Fesik, S W

2015-12-01

Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC50. ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However, a more comprehensive index composed of Mcl-1, Bcl-xL, Bim, Bak and Noxa protein or mRNA expression correlates well with Mcl-1 sensitivity in TNBC and can also predict Mcl-1 dependency in non-small cell lung cancer cell lines.

Sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in males, smokers, and non-adenocarcinoma lung cancer in patients with EGFR mutations.

PubMed

Zeng, Zhu; Chen, Hua-Jun; Yan, Hong-Hong; Yang, Jin-Ji; Zhang, Xu-Chao; Wu, Yi-Long

2013-09-27

The demographical/clinical characteristics of being Asian, having an adenocarcinoma, being female, and being a "never-smoker" are regarded as favorable predictors for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) efficacy in non-small cell lung cancer (NSCLC) with unknown EGFR gene status. In this study, we examined the effects of the supposedly unfavorable clinical variables in EGFR-mutant patients. In total, 159 EGFR-mutant NSCLC patients' clinical features were correlated with progression-free survival (PFS), response rate (RR), and overall survival (OS). Multivariate analysis of clinical characteristics was performed using the Cox and logistic regression methods. There were 90 females (56.6%), 112 never-smokers (70.4%), and 153 patients with adenocarcinomas (96.2%). All patients were treated with EGFR-TKI, and 52.8% received TKI in a first-line setting. The median PFS of patients receiving first-line TKI was similar, regardless of gender (males vs females: 9.1 vs 9.7 months, p=0.793), smoking status (never-smokers vs smokers: 9.9 vs 9.1 months, p=0.570), or histology (adenocarcinoma vs non-adenocarcinoma: 9.7 vs 9.2 months, p=0.644). OS curves of first-line TKI-treated patients were also not associated with gender (p=0.722), smoking status (p=0.579), or histology (p=0.480). Similar results of PFS and OS were obtained for patients who received TKI beyond first-line. Multivariate analysis indicated that none of these clinical factors was an independent predictor of survival. The supposedly 'favorable' clinical factors of female gender, non-smoking status, and adenocarcinoma were not independent predictive factors for PFS or OS in this population of EGFR-mutant NSCLC patients.

Mast Cells Regulate Epidermal Barrier Function and the Development of Allergic Skin Inflammation.

PubMed

Sehra, Sarita; Serezani, Ana P M; Ocaña, Jesus A; Travers, Jeffrey B; Kaplan, Mark H

2016-07-01

Atopic dermatitis is a chronic inflammatory skin disease characterized by infiltration of eosinophils, T helper cells, and mast cells. The role of mast cells in atopic dermatitis is not completely understood. To define the effects of mast cells on skin biology, we observed that mast cells regulate the homeostatic expression of epidermal differentiation complex and other skin genes. Decreased epidermal differentiation complex gene expression in mice that genetically lack mast cells (Kit(W-sh/W-sh) mice) is associated with increased uptake of protein antigens painted on the skin by dendritic cells (DCs) compared with similarly treated wild-type mice, suggesting a protective role for mast cells in exposure to nominal environmental allergens. To test this further, we crossed Kit(W-sh/W-sh) mice with signal transducer and activator of transcription 6 (i.e., Stat6) VT transgenic mice that develop spontaneous atopic dermatitis-like disease that is dependent on T helper cell 2 cytokines and is associated with high serum concentrations of IgE. We observed that Stat6VT × Kit(W-sh/W-sh) mice developed more frequent and more severe allergic skin inflammation than Stat6VT transgenic mice that had mast cells. Together, these studies suggest that mast cells regulate epidermal barrier function and have a potential protective role in the development of atopic dermatitis-like disease. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells.

PubMed

Badarinath, Krithika; Dutta, Abhik; Hegde, Akshay; Pincha, Neha; Gund, Rupali; Jamora, Colin

2018-06-13

The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells.

Recent Management of Patients with Advanced Epidermal Growth Factor Receptor Mutation Non-small Cell Lung Cancer: Role of Afatinib and Lesson Learned for Developing Countries.

PubMed

Amin, Zulkifli; Jayalie, Vito Filbert; Rajabto, Wulyo

2017-01-01

Lung cancer is a devastating disease with a high incidence, mortality and morbidity rate, especially in developing countries. Conventional treatment with cytotoxic chemotherapy has some limitations attributed to chemoresistance and toxicity. Recent advances have shown that first generation Tyrosine Kinase Inhibitor (TKI), Gefitinib and Erlotinib, and the newest available second generation Tyrosine Kinase Inhibitor (TKI), Afatinib, have the potential to be an option in the management of patients with epidermal growth factor receptor/ EGFR mutation positive advanced/ metastatic non-small cell lung cancer. Afatinib works by binding to EGFR irreversibly, thus inactivating the tyrosine kinase receptor. Some studies demostrated that Afatinib first-line may result in longer progression free survival (PFS) and better disease control, and as an alternative for patients who intolerance to Gefitinib or Erlotinib. In Indonesia, the era of National Health Insurance has been implemented and National Health Insurance has covered treatment for cancer, including first generation TKIs, Gefitinib dan erlotinib, for patients with EGFR mutation positive advanced/ metastatic non-small cell lung cancer at Cipto Mangunkusumo National Hospital. Afatinib, as one of the newest available second generation TKI, may be given free of charge too as an alternative if the National Health Insurance will be covered in the future. Further research is needed to know the efficacy and adverse effects that may occur in patients from developing countries.

Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

PubMed

Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

2016-01-01

In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.

Pharmacological evaluation of phytochemicals from South Indian Black Turmeric (Curcuma caesia Roxb.) to target cancer apoptosis.

PubMed

Mukunthan, K S; Satyan, R S; Patel, T N

2017-09-14

Curcuma caesia Roxb. (Black turmeric), a perennial herb of the family Zingiberaceae is indigenous to India. C. caesia is used as a spice, food preservative and coloring agent commonly in the Indian subcontinent. Functional parametric pharmacological evaluations like drug ability and toxicity profile of this endangered species is poorly documented. In our present study, among all the extracts of dried C. caesia rhizome viz- hexane, ethyl acetate, methanol and water tested for free radical scavenging capacity by total antioxidant activity (TAO) method, Hexane Rhizome Extract (HRE) was found to possess remarkable activity (1200mg ascorbic acid equivalent/100g). In MTT assay across three cancer cell lines and a control cell line, HRE exhibited a dose-dependent inhibition only in cancer cells, with notable activity in HepG2 cell lines (IC50: 0976µg/mL). Further, western blotting and flow cytometry experiments proved that HRE induces cell arrest at G2/M phase along with cellular apoptosis as suggestive by multiple-point mitochondrial mediated intrinsic pathway of Programmed Cell Death (PCD). Gas Chromatography-Mass Spectrophotometry (GC-MS) analysis of HRE suggested twenty compounds that when docked in silico with Tubulin (1SA0) and Epidermal Growth Factor Receptor/ EGFR (1XKK) showed very intimate binding with the original ligands. Our results provided significant evidence of the toxicity mechanisms of HRE that may be beneficial for more rational applications of drug discovery for slowing down cancer progression. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

PubMed Central

Alexaline, Maia M.; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T.; Doucet, Christelle; Fortunel, Nicolas O.

2015-01-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. Significance This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. PMID:25848122

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

PubMed

Alexaline, Maia M; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T; Doucet, Christelle; Fortunel, Nicolas O; Lataillade, Jean-Jacques

2015-06-01

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. ©AlphaMed Press.

Mast cells are dispensable in a genetic mouse model of chronic dermatitis.

PubMed

Sulcova, Jitka; Meyer, Michael; Guiducci, Eva; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Werner, Sabine

2015-06-01

Chronic inflammatory skin diseases, such as atopic dermatitis, affect a large percentage of the population, but the role of different immune cells in the pathogenesis of these disorders is largely unknown. Recently, we found that mice lacking fibroblast growth factor receptor 1 (Fgfr1) and Fgfr2 (K5-R1/R2 mice) in the epidermis have a severe impairment in the epidermal barrier, which leads to the development of a chronic inflammatory skin disease that shares many features with human atopic dermatitis. Using Fgfr1-/Fgfr2-deficient mice, we analyzed the consequences of the loss of mast cells. Mast cells accumulated and degranulated in the skin of young Fgfr1-/Fgfr2-deficient mice, most likely as a consequence of increased expression of the mast cell chemokine Ccl2. The increase in mast cells occurred before the development of histological abnormalities, indicating a functional role of these cells in the inflammatory skin phenotype. To test this hypothesis, we mated the Fgfr1-/Fgfr2-deficient mice with mast cell-deficient CreMaster mice. Surprisingly, loss of mast cells did not or only mildly affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, accumulation and activation of different immune cells, or expression of different proinflammatory cytokines in the skin. These results reveal that mast cells are dispensable for the development of chronic inflammation in response to a defect in the epidermal barrier. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase Gene during Fruit Ripening Modifies Epidermal Cells and Gravitropism.

PubMed

Wang, Cuicui; Fu, Daqi

2018-03-21

Eggplant ( Solanum melongena L.) fruits accumulate flavonoids in their cuticle and epidermal cells during ripening. Although many mutants available in model plant species, such as Arabidopsis thaliana and Medicago truncatula, are enabling the intricacies of flavonoid-related physiology to be deduced, the mechanisms whereby flavonoids influence eggplant fruit physiology are unknown. Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoid function in fruit, and in this study, we successfully applied this technique to downregulate S. melongena chalcone synthase gene ( SmCHS) expression during eggplant fruit ripening. In addition to the expected change in fruit color attributable to a lack of anthocyanins, several other modifications, including differences in epidermal cell size and shape, were observed in the different sectors. We also found that silencing of CHS gene expression was associated with a negative gravitropic response in eggplant fruits. These observations indicate that epidermal cell expansion during ripening is dependent upon CHS expression and that there may be a relationship between CHS expression and gravitropism during eggplant fruit ripening.

Transient elevation of cytoplasmic calcium ion concentration at a single cell level precedes morphological changes of epidermal keratinocytes during cornification.

PubMed

Murata, Teruasa; Honda, Tetsuya; Egawa, Gyohei; Yamamoto, Yasuo; Ichijo, Ryo; Toyoshima, Fumiko; Dainichi, Teruki; Kabashima, Kenji

2018-04-26

Epidermal keratinocytes achieve sequential differentiation from basal to granular layers, and undergo a specific programmed cell death, cornification, to form an indispensable barrier of the body. Although elevation of the cytoplasmic calcium ion concentration ([Ca 2+ ] i ) is one of the factors predicted to regulate cornification, the dynamics of [Ca 2+ ] i in epidermal keratinocytes is largely unknown. Here using intravital imaging, we captured the dynamics of [Ca 2+ ] i in mouse skin. [Ca 2+ ] i was elevated in basal cells on the second time scale in three spatiotemporally distinct patterns. The transient elevation of [Ca 2+ ] i also occurred at the most apical granular layer at a single cell level, and lasted for approximately 40 min. The transient elevation of [Ca 2+ ] i at the granular layer was followed by cornification, which was completed within 10 min. This study demonstrates the tightly regulated elevation of [Ca 2+ ] i preceding the cornification of epidermal keratinocytes, providing possible clues to the mechanisms of cornification.

A new Gsdma3 mutation affecting anagen phase of first hair cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Shigekazu; Department of Genetics, School of Life Science, Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka 411-8540; Tamura, Masaru

2007-08-10

Recombination-induced mutation 3 (Rim3) is a spontaneous mouse mutation that exhibits dominant phenotype of hyperkeratosis and hair loss. Fine linkage analysis of Rim3 and sequencing revealed a novel single point mutation, G1124A leading to Ala348Thr, in Gsdma3 in chromosome 11. Transgenesis with BAC DNA harboring the Rim3-type Gsdma3 recaptured the Rim3 phenotype, providing direct evidence that Gsdma3 is the causative gene of Rim3. We examined the spatial expression of Gsdma3 and characterized the Rim3 phenotype in detail. Gsdma3 is expressed in differentiated epidermal cells in the skin, but not in the proliferating epidermal cells. Histological analysis of Rim3 mutant showedmore » hyperplasia of the epidermal cells in the upper hair follicles and abnormal anagen phase at the first hair cycle. Furthermore, immunohistochemical analysis revealed hyperproliferation and misdifferentiation of the upper follicular epidermis in Rim3 mutant. These results suggest that Gsdma3 is involved in the proliferation and differentiation of epidermal stem cells.« less

The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module.

PubMed

Su, Zhenxia; Zhao, Lihua; Zhao, Yuanyuan; Li, Shaofang; Won, SoYoun; Cai, Hanyang; Wang, Lulu; Li, Zhenfang; Chen, Piaojuan; Qin, Yuan; Chen, Xuemei

2017-06-05

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeted therapies in non-small cell lung carcinoma: what have we achieved so far?

PubMed Central

Houhou, Wissam

2013-01-01

The search for innovative therapeutic agents in non-small cell lung cancer (NSCLC) has witnessed a swift evolution. The number of targeted drugs that can improve patient outcomes with an acceptable safety profile is steadily increasing. In this review, we highlight current drugs that have already been approved or are under evaluation for the treatment of patients with NSCLC, either in monotherapy or combined therapy for both the first- and second-line settings. Experience with drugs targeting the vascular endothelial growth factor and its receptor, as well as the epidermal growth factor receptor is summarized. Moreover, we provide an overview of more novel targets in NSCLC and initial experience with the respective therapeutic agents. PMID:23858333

Exfoliation of the epidermal cells and defecation by amphibian larvae in response to coelomic fluid and lysenin from the earthworm Eisenia foetida.

PubMed

Kobayashi, Hideshi; Suzuki, Hirohumi; Ohta, Naoshi

2006-08-01

Coelomic fluid (CF) and lysenin from the earthworm Eisenia foetida induced heavy epidermal exfoliation in the larvae of Bufo japonicus formosus at developmental stages from hatching (stage 22) to operculum completion (stage 34). In experiments with Xenopus laevis, we observed that exfoliated cells were not stained by trypan blue. Thus, it appeared that these cells were still alive. It is likely, therefore, that both CF and lysenin might disrupt the adhesion between epidermal cells of larvae prior to stage 34. Since it is known that lysenin exerts its toxic effects through its specific binding to sphingomyelin (SM), SM might be involved in such adhesion. This hypothesis was supported by the observations that CF and lysenin which had been incubated with SM-liposomes lost their exfoliative activity. In larvae after stage 34, the mechanism of adhesion between epidermal cells seemed to change and the adhesion was no longer disrupted by CF and lysenin. In larvae at around stage 34, a collagen layer started to form beneath the basement membrane of the epidermis. Furthermore, larvae at around this stage started to eat solid food. The developing collagen layer and food intake might be related indirectly to the chemical change in epidermal adhesion. The induction of exfoliation by CF and lysenin was also observed in other amphibian species. In Bufo larvae, defecation was induced both by CF and by lysenin but this effect was independent of exfoliation.

Leaf Epidermis of the Rheophyte Dyckia brevifolia Baker (Bromeliaceae)

PubMed Central

Lobo, Ghislaine Maria; de Souza, Thaysi Ventura; Voltolini, Caroline Heinig; Reis, Ademir

2013-01-01

Some species of Dyckia Schult. f., including Dyckia brevifolia Baker, are rheophytes that live in the fast-moving water currents of streams and rivers which are subject to frequent flooding, but also period of low water. This study aimed to analyze the leaf epidermis of D. brevifolia in the context of epidermal adaptation to this aquatic plant's rheophytic habitat. The epidermis is uniseriate, and the cuticle is thickened. The inner periclinal and anticlinal walls of the epidermal cells are thickened and lignified. Stomata are tetracytic, located in the depressions in relation to the surrounding epidermal cells, and covered by peltate trichomes. While the epidermal characteristics of D. brevifolia are similar to those of Bromeliaceae species, this species has made particular adaptations of leaf epidermis in response to its rheophytic environment. PMID:23864825

Epidermal growth factor and tumor necrosis factor α cooperatively promote the motility of hepatocellular carcinoma cell lines via synergistic induction of fibronectin by NF-κB/p65.

PubMed

Liu, Zong-Cai; Ning, Fen; Wang, Hai-Fang; Chen, Dan-Yang; Cai, Yan-Na; Sheng, Hui-Ying; Lash, Gendie E; Liu, Li; Du, Jun

2017-11-01

The interaction between hepatocellular carcinoma (HCC) cells and their microenvironment plays a fundamental role in tumor metastasis. The HCC microenvironment is rich in epidermal growth factor (EGF) and tumor necrosis factor α (TNFα), which may cooperatively, rather than individually, interact with tumor cells to influence their biological behavior. Immunohistochemistry was performed to study the expression of EGF and TNFα in HCCs. Western blotting, immunofluorescence, qRT-PCR, wound healing scratch and invasion assay, and chromatin immunoprecipitation assays were used to study the combined roles of EGF and TNFα in the motility of HCC cells in vitro. We demonstrated that both EGF and TNFα were highly expressed in HCCs, and HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. In vitro, EGF and TNFα cooperatively promoted the motility of HCC cells mainly via synergistic induction of an extracellular matrix glycoprotein fibronectin (FN). Mechanistically, EGF and TNFα jointly increased the nuclear translocation and PKC mediated phosphorylation of NF-κB/p65 which could bind to the -356bp to -259bp fragment of the FN promoter, leading to a markedly increased activity of the FN promoter in HCC cells. HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. EGF and TNFα cooperatively promoted the motility of HCC cells mainly through NF-κB/p65 mediated synergistic induction of FN in vitro. These findings highlight the crosstalk between EGF and TNFα in promoting HCC, and provide potential targets for HCC prevention and treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Metformin targets multiple signaling pathways in cancer.

PubMed

Lei, Yong; Yi, Yanhua; Liu, Yang; Liu, Xia; Keller, Evan T; Qian, Chao-Nan; Zhang, Jian; Lu, Yi

2017-01-26

Metformin, an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes, has become the focus of intense research as a candidate anticancer agent. Here, we discuss the potential of metformin in cancer therapeutics, particularly its functions in multiple signaling pathways, including AMP-activated protein kinase, mammalian target of rapamycin, insulin-like growth factor, c-Jun N-terminal kinase/mitogen-activated protein kinase (p38 MAPK), human epidermal growth factor receptor-2, and nuclear factor kappaB pathways. In addition, cutting-edge targeting of cancer stem cells by metformin is summarized.

Safety assessment of azelaic acid and its derivatives entrapped in nanovesicles.

PubMed

Panyosak, A; Manosroi, J; Rojanasakul, Y; Manosroi, A

2009-06-01

The aim of this study was to determine the safety of azelaic acid (AA) and its derivatives in nanovesicles for pharmaceutical and cosmetic uses. The hydrophilic property of AA was modified by complexing AA with hydroxypropyl-beta-cyclodextrin (AACD). The lipophilic property of AA was improved to diethyl azelate (DA) by esterification with Fischer reaction. AA, AACD and DA were entrapped in liposomes and niosomes with the compositions of L-alpha-dipalmitoyl phosphatidylcholine/cholesterol = 7:3 and Tween 61/cholesterol = 1:1, respectively, by chloroform film method with sonication. The size of the vesicles ranged from 50 to 200 nm, indicating nanosize characteristics. The cytotoxicity of AA, AACD and DA entrapped nanovesicular formulations on mouse epidermal cell lines (JB6, normal cell lines) by the sulforhodamine B assay was modest when compared with cisplatin. Blank liposomes and niosomes gave no growth inhibitory effect. The irritation of AA, AACD and DA entrapped and not entrapped in nanovesicles on rabbit skin was examined according to the Environmental Protection Agency health effect test guidelines. The results showed no signs of erythema or edema within 72 h. AA and its derivatives were safe for topical use when entrapped in nanovesicles because of no toxicity to normal cell lines and no allergy on rabbit skin.

Protein shedding in urothelial bladder cancer: prognostic implications of soluble urinary EGFR and EpCAM.

PubMed

Bryan, R T; Regan, H L; Pirrie, S J; Devall, A J; Cheng, K K; Zeegers, M P; James, N D; Knowles, M A; Ward, D G

2015-03-17

Better biomarkers must be found to develop clinically useful urine tests for bladder cancer. Proteomics can be used to identify the proteins released by cancer cell lines and generate candidate markers for developing such tests. We used shotgun proteomics to identify proteins released into culture media by eight bladder cancer cell lines. These data were compared with protein expression data from the Human Protein Atlas. Epidermal growth factor receptor (EGFR) was identified as a candidate biomarker and measured by ELISA in urine from 60 noncancer control subjects and from 436 patients with bladder cancer and long-term clinical follow-up. Bladder cancer cell lines shed soluble EGFR ectodomain. Soluble EGFR is also detectable in urine and is highly elevated in some patients with high-grade bladder cancer. Urinary EGFR is an independent indicator of poor bladder cancer-specific survival with a hazard ratio of 2.89 (95% CI 1.81-4.62, P<0.001). In multivariable models including both urinary EGFR and EpCAM, both biomarkers are predictive of bladder cancer-specific survival and have prognostic value over and above that provided by standard clinical observations. Measuring urinary EGFR and EpCAM may represent a simple and useful approach for fast-tracking the investigation and treatment of patients with the most aggressive bladder cancers.

Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity

PubMed Central

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C. B.

2009-01-01

Background and Aims The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Methods Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. Key Results The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Conclusions Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established. PMID:19789174

Ultra-weak photon emission as a non-invasive tool for monitoring of oxidative processes in the epidermal cells of human skin: comparative study on the dorsal and the palm side of the hand.

PubMed

Rastogi, Anshu; Pospísil, Pavel

2010-08-01

All living organisms emit spontaneous ultra-weak photon emission as a result of cellular metabolic processes. Exposure of living organisms to exogenous factors results in oxidative processes and enhancement in ultra-weak photon emission. Here, hydrogen peroxide (H(2)O(2)), as a strongly oxidizing molecule, was used to induce oxidative processes and enhance ultra-weak photon emission in human hand skin. The presented work intends to compare both spontaneous and peroxide-induced ultra-weak photon emission from the epidermal cells on the dorsal and the palm side of the hand. A highly sensitive photomultiplier tube and a charge-coupled device camera were used to detect ultra-weak photon emission from human hand skin. Spontaneous ultra-weak photon emission from the epidermal cells on the dorsal side of the hand was 4 counts/s. Topical application of 500 mM H(2)O(2) to the dorsal side of the hand caused enhancement in ultra-weak photon emission to 40 counts/s. Interestingly, both spontaneous and peroxide-induced ultra-weak photon emission from the epidermal cells on the palm side of the hand were observed to increase twice their values, i.e. 8 and 80 counts/s, respectively. Similarly, the two-dimensional image of ultra-weak photon emission observed after topical application of H(2)O(2) to human skin reveals that photon emission from the palm side exceeds the photon emission from the dorsal side of the hand. The results presented indicate that the ultra-weak photon emission originating from the epidermal cells on the dorsal and the palm side of the hand is related to the histological structure of the human hand skin. Ultra-weak photon emission is shown as a non-destructive technique for monitoring of oxidative processes in the epidermal cells of the human hand skin and as a diagnostic tool for skin diseases.

A three-dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators.

PubMed

Cardoso, T C; Sakamoto, S S; Stockmann, D; Souza, T F B; Ferreira, H L; Gameiro, R; Vieira, F V; Louzada, M J Q; Andrade, A L; Flores, E F

2017-06-01

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model. © 2016 John Wiley & Sons Ltd.

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma*

PubMed Central

Makinoshima, Hideki; Takita, Masahiro; Saruwatari, Koichi; Umemura, Shigeki; Obata, Yuuki; Ishii, Genichiro; Matsumoto, Shingo; Sugiyama, Eri; Ochiai, Atsushi; Abe, Ryo; Goto, Koichi; Esumi, Hiroyasu; Tsuchihara, Katsuya

2015-01-01

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells. PMID:26023239

Regional Fluctuation in the Functional Consequence of LINE-1 Insertion in the Mitf Gene: The Black Spotting Phenotype Arisen from the Mitfmi-bw Mouse Lacking Melanocytes.

PubMed

Takeda, Kazuhisa; Hozumi, Hiroki; Ohba, Koji; Yamamoto, Hiroaki; Shibahara, Shigeki

2016-01-01

Microphthalmia-associated transcription factor (Mitf) is a key regulator for differentiation of melanoblasts, precursors to melanocytes. The mouse homozygous for the black-eyed white (Mitfmi-bw) allele is characterized by the white-coat color and deafness with black eyes due to the lack of melanocytes. The Mitfmi-bw allele carries LINE-1, a retrotransposable element, which results in the Mitf deficiency. Here, we have established the black spotting mouse that was spontaneously arisen from the homozygous Mitfmi-bw mouse lacking melanocytes. The black spotting mouse shows multiple black patches on the white coat, with age-related graying. Importantly, each black patch also contains hair follicles lacking melanocytes, whereas the white-coat area completely lacks melanocytes. RT-PCR analyses of the pigmented patches confirmed that the LINE-1 insertion is retained in the Mitf gene of the black spotting mouse, thereby excluding the possibility of the somatic reversion of the Mitfmi-bw allele. The immunohistochemical analysis revealed that the staining intensity for beta-catenin was noticeably lower in hair follicles lacking melanocytes of the homozygous Mitfmi-bw mouse and the black spotting mouse, compared to the control mouse. In contrast, the staining intensity for beta-catenin and cyclin D1 was higher in keratinocytes of the black spotting mouse, compared to keratinocytes of the control mouse and the Mitfmi-bw mouse. Moreover, the keratinocyte layer appears thicker in the Mitfmi-bw mouse, with the overexpression of Ki-67, a marker for cell proliferation. We also show that the presumptive black spots are formed by embryonic day 15.5. Thus, the black spotting mouse provides the unique model to explore the molecular basis for the survival and death of developing melanoblasts and melanocyte stem cells in the epidermis. These results indicate that follicular melanocytes are responsible for maintaining the epidermal homeostasis; namely, the present study has provided evidence for the link between melanocyte development and the epidermal microenvironment.

GATA3 expression in triple-negative breast cancers.

PubMed

Byrne, David J; Deb, Siddhartha; Takano, Elena A; Fox, Stephen B

2017-07-01

GATA-binding protein 3 (GATA3) is a well-studied transcription factor found to be essential in the development of luminal breast epithelium and has been identified in a variety of tumour types, including breast and urothelial carcinomas, making it a useful immunohistochemistry marker in the diagnosis of both primary and metastatic disease. We investigated GATA3 protein expression in a 106 primary triple-negative breast carcinomas (100 basal-like, six non-basal-like) using Cell Marque mouse monoclonal anti-GATA3 (L50-823). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to quantify mRNA expression in 22 triple-negative breast cancers (TNBCs) (20 primary and two cell lines), four luminal (three primary and one cell line) and five human epidermal growth factor receptor 2 (HER2) (four primary and one cell line) amplified tumours. In 98 TNBCs where IHC was assessable, 47 (48%) had a 1+ or greater staining with 20 (21%) having high GATA3 expression when using a weighted scoring. Our study has demonstrated that GATA3 expression is common in primary triple-negative breast carcinomas. It also suggests that although GATA3 is an oestrogen receptor (ER) regulated gene, it still proves useful in differentiating between primary and metastatic tumours in patients with a history of breast cancer regardless of its molecular subtype. © 2017 John Wiley & Sons Ltd.

Preparation of a Three-Dimensional Full Thickness Skin Equivalent.

PubMed

Reuter, Christian; Walles, Heike; Groeber, Florian

2017-01-01

In vitro test systems are a promising alternative to animal models. Due to the use of human cells in a three-dimensional arrangement that allows cell-cell or cell-matrix interactions these models may be more predictive for the human situation compared to animal models or two-dimensional cell culture systems. Especially for dermatological research, skin models such as epidermal or full-thickness skin equivalents (FTSE) are used for different applications. Although epidermal models provide highly standardized conditions for risk assessment, FTSE facilitate a cellular crosstalk between the dermal and epidermal layer and thus can be used as more complex models for the investigation of processes such as wound healing, skin development, or infectious diseases. In this chapter, we describe the generation and culture of an FTSE, based on a collagen type I matrix and provide troubleshooting tips for commonly encountered technical problems.

Estimating the Size of Onion Epidermal Cells from Diffraction Patterns

NASA Astrophysics Data System (ADS)

Groff, Jeffrey R.

2012-10-01

Bioscience and premedical profession students are a major demographic served by introductory physics courses at many colleges and universities. Exposing these students to biological applications of physical principles will help them to appreciate physics as a useful tool for their future professions. Here I describe an experiment suitable for introductory physics where principles of wave optics are applied to probe the size of onion epidermal cells. The epidermis tissue is composed of cells of relatively uniform size and shape (Fig. 1) so the tissue acts like a one-dimensional transmission diffraction grating. The diffraction patterns generated when a laser beam passes through the tissue (Fig. 2) are analyzed and an estimate of the average width of individual onion epidermal cells is calculated. The results are compared to direct measurements taken using a light microscope. The use of microscopes and plant-cell tissue slides creates opportunities for cross-discipline collaboration between physics and biology instructors.

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

NASA Astrophysics Data System (ADS)

Jin, Hong; Heller, Daniel A.; Kalbacova, Marie; Kim, Jong-Ho; Zhang, Jingqing; Boghossian, Ardemis A.; Maheshri, Narendra; Strano, Michael S.

2010-04-01

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

Human papillomavirus E6/E7 oncogenes promote mouse ear regeneration by increasing the rate of wound re-epithelization and epidermal growth.

PubMed

Valencia, Concepción; Bonilla-Delgado, José; Oktaba, Katarzyna; Ocádiz-Delgado, Rodolfo; Gariglio, Patricio; Covarrubias, Luis

2008-12-01

Mammals have limited regeneration capacity. We report here that, in transgenic mice (Tg(bK6-E6/E7)), the expression of the E6/E7 oncogenes of human papilloma virus type 16 (HPV16) under the control of the bovine keratin 6 promoter markedly improves the mouse's capacity to repair portions of the ear after being wounded. Increased repair capacity correlates with an increased number of epidermal proliferating cells. In concordance with the expected effects of the E6 and E7 oncogenes, levels of p53 decreased and those of p16 in epidermal cells increased. In addition, we observed that wound re-epithelization proceeded faster in transgenic than in wild-type animals. After the initial re-epithelization, epidermal cell migration from the intact surrounding tissue appears to be a major contributor to the growing epidermis, especially in the repairing tissue of transgenic mice. We also found that there is a significantly higher number of putative epidermal stem cells in Tg(bK6-E6/E7) than in wild-type mice. Remarkably, hair follicles and cartilage regenerated within the repaired ear tissue, without evidence of tumor formation. We propose that the ability to regenerate ear portions is limited by the capacity of the epidermis to repair itself and grow.

Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters.

PubMed

Kunimasa, Kei; Nagano, Tatsuya; Shimono, Yohei; Dokuni, Ryota; Kiriu, Tatsunori; Tokunaga, Shuntaro; Tamura, Daisuke; Yamamoto, Masatsugu; Tachihara, Motoko; Kobayashi, Kazuyuki; Satouchi, Miyako; Nishimura, Yoshihiro

2017-07-01

In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor-tyrosine kinase inhibitor-induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR-mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell-related markers by quantitative RT-PCR and the expression of the cellular senescence-associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. Drug-tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem-like cells (CSCs) and the other with the properties of therapy-induced senescent (TIS) cells. The CD133 high cell population had CSC properties and the CD133 low cell population had TIS properties. The CD133 low cell population containing TIS cells showed a senescence-associated secretory phenotype that supported the emergence of the CD133 high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133 low cell population. Withaferin A effectively eliminated the CD133 high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib-resistant tumor growth. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

[Comparison of the Efficacy and Safety of Icotinib with Standard Second-line 
Chemotherapy in Previously Treated Advanced Non-small Cell Lung Cancer].

PubMed

Yao, Shuyang; Qian, Kun; Wang, Ruotian; Li, Yuanbo; Zhang, Yi

2015-06-01

This study compared the efficacy and safety of icotinib with standard second-line chemotherapy (single-agent docetaxel or pemetrexed) in previously treated advanced non-small cell lung cancer (NSCLC). Thirty-two consecutive patients treated with icotinib and 33 consecutive patients treated with standard second-line chemotherapy in Xuanwu Hospital from January 2012 to July 2013 were enrolled in our retrospective research. The Response Evaluation Criteria in Solid Tumors were used to evaluate the tumor responses, and the progression-free survival (PFS) was evaluated by Kaplan-Meier method. Icotinib was comparable with standard second-line chemotherapy for advanced NSCLC in terms of overall response rate (ORR) (28.1% vs 18.2%, P=0.341), disease control rate (DFS)(43.8% vs 45.5%, P=0.890), and PFS (4.3 months vs 3.8 months, P=0.506). In the icotinib group, the ORR of epidermal growth factor receptor (EGFR) mutant was significantly higher than that of EGFR unknown or wild type (P=0.017). In multivariate analysis, age, gender, histology, and the optimum first-line treatment response were dependent prognostic factors based on the PFS of the icotinib group. The incidence of adverse events was significantly fewer in the icotinib group than in the chemotherapy group (P=0.001). Compared with the standard second-line chemotherapy, icotinib is active in the treatment of advanced NSCLC patients, especially with EGFR unknown in the second line, with an acceptable adverse event profile.

Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

PubMed Central

Miccichè, Francesca; Da Riva, Luca; Fabbi, Marina; Pilotti, Silvana; Mondellini, Piera; Ferrini, Silvano; Canevari, Silvana; Pierotti, Marco A.; Bongarzone, Italia

2011-01-01

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology. PMID:21364949

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

PubMed Central

Chen, Hongwei; Wang, Liya; Yu, Qiqi; Qian, Weiping; Tiwari, Diana; Yi, Hong; Wang, Andrew Y; Huang, Jing; Yang, Lily; Mao, Hui

2013-01-01

Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs. PMID:24124366

Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

PubMed Central

Guha, Gunjan; Li, Shan; Kyrylkova, Kateryna; Kioussi, Chrissa; Leid, Mark; Ganguli-Indra, Gitali; Indra, Arup K.

2012-01-01

Background Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice. Methodology/Principal Findings To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes. Conclusions/Significance Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2. PMID:23284675

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

PubMed

Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

2016-02-05

Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals a protein profile associated with NSCLC exosomes that suggests a role these vesicles have in the progression of lung carcinogenesis, as well as identifies several promising candidates that could be utilized as a multi-marker protein panel in a diagnostic platform for NSCLC. Copyright © 2015 Elsevier B.V. All rights reserved.

Live cumulative network meta-analysis: protocol for second-line treatments in advanced non-small-cell lung cancer with wild-type or unknown status for epidermal growth factor receptor.

PubMed

Créquit, Perrine; Trinquart, Ludovic; Ravaud, Philippe

2016-08-03

Many second-line treatments for advanced non-small-cell lung cancer (NSCLC) have been assessed in randomised controlled trials, but which treatments work the best remains unclear. Novel treatments are being rapidly developed. We need a comprehensive up-to-date evidence synthesis of all these treatments. We present the protocol for a live cumulative network meta-analysis (NMA) to address this need. We will consider trials of second-line treatments in patients with advanced NSCLC with wild-type or unknown epidermal growth factor receptor status. We will consider any single agent of cytotoxic chemotherapy, targeted therapy, combination of cytotoxic chemotherapy and targeted therapy and any combination of targeted therapies. The primary outcomes will be overall survival and progression-free survival. The live cumulative NMA will be initiated with a NMA and then iterations will be repeated at regular intervals to keep the NMA up-to-date over time. We have defined the update frequency as 4 months, based on an assessment of the pace of evidence production on this topic. Each iteration will consist of six methodological steps: adaptive search for treatments and trials, screening of reports and selection of trials, data extraction, assessment of risk of bias, update of the network of trials and synthesis, and dissemination. We will set up a research community in lung cancer, with different groups of contributors of different skills. We will distribute tasks through online crowdsourcing. This proof-of-concept study in second-line treatments of advanced NSCLC will allow one for assessing the feasibility of live cumulative NMA and opening the path for this new form of synthesis. Ethical approval is not required because our study will not include confidential participant data and interventions. The description of all the steps and the results of this live cumulative NMA will be available online. CRD42015017592. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

EPA Science Inventory

Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)
Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. Bromberg
Center fo...

Isolation of epidermal cells and cDNA cloning of TNF decoy receptor 3 of conger eel, Conger myriaster.

PubMed

Tsutsui, Shigeyuki; Yoshino, Yuko; Matsui, Saho; Nakamura, Osamu; Muramoto, Koji; Watanabe, Tasuku

2008-03-01

By using EDTA and a trypsin solution, we established a method for isolating the epidermal cells of the conger eel, Conger myriaster. We then identified TNF decoy receptor (DcR) cDNA in the species from a suppression subtractive hybridization library prepared from the epidermal cells stimulated with LPS. The full-length cDNA of conger TNF DcR (conDcR) consisted of 1479 base pairs, and the protein comprised 286 amino acid residues. Phylogenetic analysis indicated that conDcR was clustered into a DcR3 branch. ConDcR is likely to act as an important immune-regulating factor in inhibiting the apoptosis-inducing effect of TNF in the skin of conger eel.

Sulfur Mustard (SM) Lesions in Organ-Cultured Human Skin: Markers of Injury and Inflammatory Mediators

DTIC Science & Technology

1990-04-16

18. SUB3ECT TERMS (oont’d) epidermal injury organ culture •ranuaear vacuoles C-leucine incorpora’tion by full-thickness human akin explants hi stamine ...mast- cell degranulation prostaglandin E2 lysobomal enzymes: acid phosphatase, B-glucuronidase, 0-galactcsidase, lysozyme and lactic dehydrogenase...that histamline (from local mast cells ), and PA and POgk (probably from mast cells and epidermal cells ) are s3e of the early mediators of the inflmma

Cell water potential, osmotic potential, and turgor in the epidermis and mesophyll of transpiring leaves : Combined measurements with the cell pressure probe and nanoliter osmometer.

PubMed

Nonami, H; Schulze, E D

1989-01-01

Water potential, osmotic potential and turgor measurements obtained by using a cell pressure probe together with a nanoliter osmometer were compared with measurements obtained with an isopiestic psychrometer. Both types of measurements were conducted in the mature region of Tradescantia virginiana L. leaves under non-transpiring conditions in the dark, and gave similar values of all potentials. This finding indicates that the pressure probe and the osmometer provide accurate measurements of turgor, osmotic potentials and water potentials. Because the pressure probe does not require long equilibration times and can measure turgor of single cells in intact plants, the pressure probe together with the osmometer was used to determine in-situ cell water potentials, osmotic potentials and turgor of epidermal and mesophyll cells of transpiring leaves as functions of stomatal aperture and xylem water potential. When the xylem water potential was-0.1 MPa, the stomatal aperture was at its maximum, but turgor of both epidermal and mesophyll cells was relatively low. As the xylem water potential decreased, the stomatal aperture became gradually smaller, whereas turgor of both epidermal and mesophyll cells first increased and afterward decreased. Water potentials of the mesophyll cells were always lower than those of the epidermal cells. These findings indicate that evaporation of water is mainly occurring from mesophyll cells and that peristomatal transpiration could be less important than it has been proposed previously, although peristomatal transpiration may be directly related to regulation of turgor in the guard cells.

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

PubMed

Van Moerkercke, Alex; Galván-Ampudia, Carlos S; Verdonk, Julian C; Haring, Michel A; Schuurink, Robert C

2012-05-01

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved.

Epidermal Growth Factor Receptor (EGFR) Kinase Inhibitors and Non-Small Cell Lung Cancer (NSCLC) - Advances in Molecular Diagnostic Techniques to Facilitate Targeted Therapy.

PubMed

Ghafoor, Qamar; Baijal, Shobhit; Taniere, Phillipe; O'Sullivan, Brendan; Evans, Matthew; Middleton, Gary

2017-12-21

A subset of patients with non-small cell lung cancer (NSCLC) respond well to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), due to the presence of sensitising mutations in the gene encoding EGFR. Mutations associated with resistance to first generation EGFR TKIs have also been identified, which lead to therapeutic failure and the requirement for new drugs. Three generations of EGFR TKIs have been developed and either have been, or are being, evaluated as first and/or second line therapeutic agents. In this review, we consider the advances in molecular diagnostic techniques that are used, or are in development, to facilitate the targeted EGFR TKI therapy of patients with NSCLC. A literature search was conducted in May 2017 using PubMed, and spanning the period September 2005 (EU approval date of erlotinib) to May 2017. Search terms used were: EGFR TKI, NSCLC, clinical trial, erlotinib, gefitinib, afatinib, EGFR mutations, Exon 19 deletion, and Leu858Arg. The use of molecular data, in conjunction with other clinical and diagnostic information, will assist physicians to make the best therapeutic choice for each patient with advanced NSCLC. Personalized medicine and a rapidly developing therapy landscape will enable these patients to achieve optimal responses to EGFR TKIs.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Pharmacokinetic drug evaluation of osimertinib for the treatment of non-small cell lung cancer.

PubMed

Rossi, Antonio; Muscarella, Lucia Anna; Di Micco, Concetta; Carbonelli, Cristiano; D'alessandro, Vito; Notarangelo, Stefano; Palomba, Giuseppe; Sanpaolo, Gerardo; Taurchini, Marco; Graziano, Paolo; Maiello, Evaristo

2017-12-01

First- and second-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, erlotinib, icotinib, and afatinib are the standard-of-care for first-line therapy of non-small-cell lung cancer (NSCLC) harboring activating EGFR mutations. Unfortunately, after initial activity of an average 9-13 months, disease progression has been reported in the majority of patients. In about 50% of cases the progression is due to the onset of the T790M mutation in exon 20 of the EGFR gene. Third-generation EGFR-TKIs targeting this mutation were investigated, with osimertinib the only reaching clinical practice. Areas covered: A structured search of bibliographic databases for peer-reviewed research literature and of main meetings using a focused review question addressing osimertinib, was undertaken. Expert opinion: Osimertinib is the standard-of-care for EGFR-mutated patients progressing to first-line EGFR-TKIs due to the acquired EGFR T790M mutation. Results from the head-to-head first-line trial comparing osimertinib versus gefitinib or erlotinib in activating EGFR mutations might change the front-line approach. Osimertinib in combination regimens, such as immunotherapy, and in adjuvant setting are ongoing. Thus, the strategic approach for the management of EGFR-mutated NSCLC patients will change further in the next few years.

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors.

PubMed

Ghosh, Gargi; Lian, Xiaojun; Kron, Stephen J; Palecek, Sean P

2012-03-20

Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown. An erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity in vitro were assessed in erlotinib resistant H1650-ER1 cells. The erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib. Our findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes.

PubMed

Klar, Agnes S; Biedermann, Thomas; Simmen-Meuli, Claudia; Reichmann, Ernst; Meuli, Martin

2017-03-01

Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model. Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy. All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b + , CD68 + ) and granulocytes (HIS48 + ) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b + , CD68 + ) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks. These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury.

PubMed

Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2011-07-15

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury

PubMed Central

Nakrieko, Kerry-Ann; Rudkouskaya, Alena; Irvine, Timothy S.; D'souza, Sudhir J. A.; Dagnino, Lina

2011-01-01

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15–expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury. PMID:21593206

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

PubMed

Pontiggia, Luca; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Oliveira, Carol; Braziulis, Erik; Klar, Agnieszka S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2014-06-01

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

Topical grape seed proanthocyandin extract reduces sunburn cells and mutant p53 positive epidermal cell formation, and prevents depletion of Langerhans cells in an acute sunburn model.

PubMed

Yuan, Xiao-Ying; Liu, Wei; Hao, Jian-Chun; Gu, Wei-Jie; Zhao, Yan-Shuang

2012-01-01

The purpose of this study was to investigate whether grape seed proanthocyanidin extract (GSPE) can provide photoprotection against ultraviolet (UV) irradiation. Study has shown that GSPE is a natural oxidant, and is used in many fields such as ischemia-reperfusion injury, chronic pancreatitis, and even cancer. However, the effect of GSPE on UV irradiation is as yet unknown. Cutaneous areas on the backs of normal volunteers were untreated or treated with GSPE solutions or vehicles 30 min before exposure to two minimal erythema doses (MED) of solar simulated radiation. Cutaneous areas at different sites were examined histologically for the number of sunburn cells, or immunohistochemically for Langerhans cells and mutant p53 epidermal cells. On histological and immunohistochemical examination, skin treated with GSPE before UV radiation showed fewer sunburn cells and mutant p53-positive epidermal cells and more Langerhans cells compared with skin treated with 2-MED UV radiation only (p<0.001, p<0.001, and p<0.01, respectively). GSPE may be a possible preventive agent for photoprotection.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

PubMed

Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

2012-11-01

This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3.

Cervi cornus Colla (deer antler glue) induce epidermal differentiation in the reconstruction of skin equivalents.

PubMed

Choi, H-R; Nam, K-M; Kim, D-S; Huh, C-H; Na, J-I; Park, K-C

2013-06-01

In the reconstruction of skin equivalents (SEs), keratinocyte differentiation is important because epidermal differentiation is closely related with barrier function. The aim of this study was to investigate the effects of Cervi cornus Colla (CCC) on the stem cell activity and epidermal differentiation in the reconstruction of skin equivalent. Four different models were constructed according to different composition of dermal substitute. Results showed similar morphologic findings when hyaluronic acid (HA) and/or CCC was added. But, immunohistochemical staining showed that p63 was significantly increased by addition of HA and/or CCC. Increased staining of integrin α6 and β1 was variably observed when HA and/or CCC was added to make dermal substitute. These finding showed that addition of HA and/or CCC may affect the stem cell activity in the reconstruction of skin. Furthermore, filaggrin expression was much increased when CCC was added. It showed that epidermal differentiation was significantly improved by addition of CCC. In conclusion, simultaneous presence of HA and CCC contributed to the stem cell activity and epidermal differentiation in the reconstruction of SE. Legislation in the EU prohibits marketing cosmetics and personal care products that contain constituents that have been examined through animal experiments. To avoid these limitations, SEs can be used for testing the safety or the efficacy of cosmetic ingredients. Therefore, our results showed that combined use of HA and CCC can be helpful for the reconstruction of SE with good stem cell activity and epidermal differentiation. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes.

PubMed

Hsieh, Wan-Ling; Lin, Yin-Ku; Tsai, Chi-Neu; Wang, Ta-Min; Chen, Tzu-Ya; Pang, Jong-Hwei S

2012-08-01

Topical indigo naturalis ointment is clinically proved to be an effective therapy for plaque-type psoriasis. Indirubin, as the active component of indigo naturalis, inhibits cell proliferation of epidermal keratinocytes. However, the detailed underlying mechanism is not fully understood. To further investigate the anti-proliferating effects of indigo naturalis and indirubin on epidermal keratinocytes. The decreased expression of CDC25B in indigo naturalis- or indirubin-treated epidermal keratinocytes, as revealed by cDNA microarray analysis, was studied. The CDC25B expression was examined under different serum concentrations and compared between primary and immortalized keratinocytes. The activation of EGFR and the effect of EGF on the cell proliferation and CDC25B expression were also investigated in epidermal keratinocytes. RT/real-time PCR and western blot method were used to analyze the CDC25B expression at the mRNA and protein levels, respectively. Indigo naturalis and indirubin were confirmed to down-regulate CDC25B expression significantly at both the mRNA and protein levels. The growth-dependent expression of CDC25B was demonstrated by the increased expression in serum-stimulated and immortalized keratinocytes. The activation of EGF receptor, known to be highly expressed in psoriatic lesions, was inhibited by indigo naturalis or indirubin. The cell proliferation and CDC25B expression of epidermal keratinocytes were induced by EGF alone and confirmed to be inhibited by indigo naturalis or indirubin. Except being a common therapeutic target in various cancers, CDC25B also plays an important role in the hyper-proliferation of epidermal keratinocytes which can be suppressed by anti-psoriatic drug indigo naturalis and its component, indirubin. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Delphinidin, a dietary antioxidant, induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three-dimensional epidermal equivalent models.

PubMed

Chamcheu, Jean Christopher; Afaq, Farrukh; Syed, Deeba N; Siddiqui, Imtiaz A; Adhami, Vaqar M; Khan, Naghma; Singh, Sohinderjit; Boylan, Brendan T; Wood, Gary S; Mukhtar, Hasan

2013-05-01

Delphinidin (Del), [3,5,7,3'-,4'-,5'-hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three-dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10-40 μm; 24-48 h) significantly enhanced keratinocyte differentiation. In Del-treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase-14 and transglutaminase-1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis-related proteins including Bcl-2 family of proteins and poly(ADP-ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase-14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses. © 2013 John Wiley & Sons A/S.

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

PubMed

Chang, Wing Y; Bryce, Dawn M; D'Souza, Sudhir J A; Dagnino, Lina

2004-12-03

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.

Transgenic expression of human amphiregulin in mouse skin: inflammatory epidermal hyperplasia and enlarged sebaceous glands.

PubMed

Li, Yong; Stoll, Stefan W; Sekhon, Sahil; Talsma, Caroline; Camhi, Maya I; Jones, Jennifer L; Lambert, Sylviane; Marley, Hue; Rittié, Laure; Grachtchouk, Marina; Fritz, Yi; Ward, Nicole L; Elder, James T

2016-03-01

To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5' and 3' untranslated region sequences (AREG-UTR) led to a >10-fold increase in hAREG expression compared to an otherwise-identical construct containing only the coding region (AREG-CDR). AREG-UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG-CDR mice. Histologically, AREG-UTR mice presented with marked epidermal hyperplasia of tail skin (2.1-fold increase in epidermal thickness with a 9.5-fold increase in Ki-67(+) cells) accompanied by significantly increased CD4+ T-cell infiltration. Dorsal skin of AREG-UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG-UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki-67(+) cells. To determine the response of AREG-UTR animals to a pro-inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG-UTR and wild type mice (1.7- and 2.2-fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue

PubMed Central

2017-01-01

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 μm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity. PMID:28926847

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

Tissue Equivalents Based on Cell-Seeded Biodegradable Microfluidic Constructs

PubMed Central

Borenstein, Jeffrey T.; Megley, Katie; Wall, Kimberly; Pritchard, Eleanor M.; Truong, David; Kaplan, David L.; Tao, Sarah L.; Herman, Ira M.

2010-01-01

One of the principal challenges in the field of tissue engineering and regenerative medicine is the formation of functional microvascular networks capable of sustaining tissue constructs. Complex tissues and vital organs require a means to support oxygen and nutrient transport during the development of constructs both prior to and after host integration, and current approaches have not demonstrated robust solutions to this challenge. Here, we present a technology platform encompassing the design, construction, cell seeding and functional evaluation of tissue equivalents for wound healing and other clinical applications. These tissue equivalents are comprised of biodegradable microfluidic scaffolds lined with microvascular cells and designed to replicate microenvironmental cues necessary to generate and sustain cell populations to replace dermal and/or epidermal tissues lost due to trauma or disease. Initial results demonstrate that these biodegradable microfluidic devices promote cell adherence and support basic cell functions. These systems represent a promising pathway towards highly integrated three-dimensional engineered tissue constructs for a wide range of clinical applications.

Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors

PubMed Central

Akbay, Esra A; Koyama, Shohei; Carretero, Julian; Altabef, Abigail; Tchaicha, Jeremy H; Christensen, Camilla L; Mikse, Oliver R; Cherniack, Andrew D; Beauchamp, Ellen M; Pugh, Trevor J; Wilkerson, Matthew D; Fecci, Peter E; Butaney, Mohit; Reibel, Jacob B; Soucheray, Margaret; Cohoon, Travis J; Janne, Pasi A; Meyerson, Matthew; Hayes, D. Neil; Shapiro, Geoffrey I; Shimamura, Takeshi; Sholl, Lynette M; Rodig, Scott J; Freeman, Gordon J; Hammerman, Peter S; Dranoff, Glenn; Wong, Kwok-Kin

2013-01-01

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition. PMID:24078774

ROLE OF GRB2-ASSOCIATED BINDER 1 (GAB1) IN EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)-INDUCED SIGNALING IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

PubMed Central

Hoeben, A.; Martin, D.; Clement, P. M.; Cools, J.; Gutkind, J. S.

2012-01-01

The Epidermal Growth Factor Receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents. PMID:22865653

Insufficiency of pro-heparin-binding epidermal growth factor-like growth factor shedding enhances hypoxic cell death in H9c2 cardiomyoblasts via the activation of caspase-3 and c-Jun N-terminal kinase.

PubMed

Uetani, Teruyoshi; Nakayama, Hironao; Okayama, Hideki; Okura, Takafumi; Higaki, Jitsuo; Inoue, Hirofumi; Higashiyama, Shigeki

2009-05-01

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.

The development of a 3D immunocompetent model of human skin.

PubMed

Chau, David Y S; Johnson, Claire; MacNeil, Sheila; Haycock, John W; Ghaemmaghami, Amir M

2013-09-01

As the first line of defence, skin is regularly exposed to a variety of biological, physical and chemical insults. Therefore, determining the skin sensitization potential of new chemicals is of paramount importance from the safety assessment and regulatory point of view. Given the questionable biological relevance of animal models to human as well as ethical and regulatory pressure to limit or stop the use of animal models for safety testing, there is a need for developing simple yet physiologically relevant models of human skin. Herein, we describe the construction of a novel immunocompetent 3D human skin model comprising of dendritic cells co-cultured with keratinocytes and fibroblasts. This model culture system is simple to assemble with readily-available components and importantly, can be separated into its constitutive individual layers to allow further insight into cell-cell interactions and detailed studies of the mechanisms of skin sensitization. In this study, using non-degradable microfibre scaffolds and a cell-laden gel, we have engineered a multilayer 3D immunocompetent model comprised of keratinocytes and fibroblasts that are interspersed with dendritic cells. We have characterized this model using a combination of confocal microscopy, immuno-histochemistry and scanning electron microscopy and have shown differentiation of the epidermal layer and formation of an epidermal barrier. Crucially the immune cells in the model are able to migrate and remain responsive to stimulation with skin sensitizers even at low concentrations. We therefore suggest this new biologically relevant skin model will prove valuable in investigating the mechanisms of allergic contact dermatitis and other skin pathologies in human. Once fully optimized, this model can also be used as a platform for testing the allergenic potential of new chemicals and drug leads.

Heterogeneous fates and dynamic rearrangement of regenerative epidermis-derived cells during zebrafish fin regeneration.

PubMed

Shibata, Eri; Ando, Kazunori; Murase, Emiko; Kawakami, Atsushi

2018-04-13

The regenerative epidermis (RE) is a specialized tissue that plays an essential role in tissue regeneration. However, the fate of the RE during and after regeneration is unknown. In this study, we performed Cre- loxP -mediated cell fate tracking and revealed the fates of a major population of the RE cells that express fibronectin 1b ( fn1b ) during zebrafish fin regeneration. Our study showed that these RE cells are mainly recruited from the inter-ray epidermis, and that they follow heterogeneous cell fates. Early recruited cells contribute to initial wound healing and soon disappear by apoptosis, while the later recruited cells contribute to the regenerated epidermis. Intriguingly, many of these cells are also expelled from the regenerated tissue by a dynamic caudal movement of the epidermis over time, and in turn the loss of epidermal cells is replenished by a global self-replication of basal and suprabasal cells in fin. De-differentiation of non-basal epidermal cells into the basal epidermal cells did not occur during regeneration. Overall, our study reveals the heterogeneous fates of RE cells and a dynamic rearrangement of the epidermis during and after regeneration. © 2018. Published by The Company of Biologists Ltd.

Epidermal growth factor in alkali-burned corneal epithelial wound healing.

PubMed

Singh, G; Foster, C S

1987-06-15

We conducted a double-masked study to evaluate the effect of epidermal growth factor on epithelial wound healing and recurrent erosions in alkali-burned rabbit corneas. Epithelial wounds 10 mm in diameter healed completely under the influence of topical epidermal growth factor, whereas the control corneas did not resurface in the center. On reversal of treatment, the previously nonhealing epithelial defects healed when treated with topical epidermal growth factor eyedrops. Conversely, the epidermal growth factor-treated and resurfaced corneas developed epithelial defects when treatment was discontinued. Histopathologic examination disclosed hyperplastic epithelium growing over the damaged stroma laden with polymorphonuclear leukocytes when treated with epidermal growth factor eyedrops, but it did not adhere to the underlying tissue. Hydropic changes were seen intracellularly as well as between the epithelial cells and the stroma.

The mysterious human epidermal cell cycle, or an oncogene-induced differentiation checkpoint

PubMed Central

Gandarillas, Alberto

2012-01-01

Fifteen years ago, we reported that proto-oncogene MYC promoted differentiation of human epidermal stem cells, a finding that was surprising to the MYC and the skin research communities. MYC was one of the first human oncogenes identified, and it had been strongly associated with proliferation. However, it was later shown that MYC could induce apoptosis under low survival conditions. Currently, the notion that MYC promotes epidermal differentiation is widely accepted, but the cell cycle mechanisms that elicit this function remain unresolved. We have recently reported that keratinocytes respond to cell cycle deregulation and DNA damage by triggering terminal differentiation. This mechanism might constitute a homeostatic protection face to cell cycle insults. Here, I discuss recent and not-so-recent evidence suggesting the existence of a largely unexplored oncogene-induced differentiation response (OID) analogous to oncogene-induced apoptosis (OIA) or senescence (OIS). In addition, I propose a model for the role of the cell cycle in skin homeostasis maintenance and for the dual role of MYC in differentiation. PMID:23114621

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

PubMed Central

Laplante, Caroline

2011-01-01

During Drosophila melanogaster dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. We show that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties. PMID:21263031

Periostin contributes to epidermal hyperplasia in psoriasis common to atopic dermatitis

PubMed Central

Arima, Kazuhiko; Ohta, Shoichiro; Takagi, Atsushi; Shiraishi, Hiroshi; Masuoka, Miho; Ontsuka, Kanako; Suto, Hajime; Suzuki, Shoichi; Yamamoto, Ken-ichi; Ogawa, Masahiro; Simmons, Olga; Yamaguchi, Yukie; Toda, Shuji; Aihara, Michiko; Conway, Simon J.; Ikeda, Shigaku; Izuhara, Kenji

2016-01-01

Background Epidermal hyperplasia is a histological hallmark observed in both atopic dermatitis (AD) and psoriasis, although the clinical features and the underlying immunological disorders of these diseases are different. We previously showed that periostin, a matricellular protein, plays a critical role in epidermal hyperplasia in AD, using a mouse model and a 3-dimensional organotypic coculture system. In this study, we explore the hypothesis that periostin is involved in epidermal hyperplasia in psoriasis. Methods To examine expression of periostin in psoriasis patients, we performed immunohistochemical analysis on skin biopsies from six such patients. To investigate periostin’s role in the pathogenesis of psoriasis, we evaluated periostin-deficient mice in a psoriasis mouse model induced by topical treatment with imiquimod (IMQ). Results Periostin was substantially expressed in the dermis of all investigated psoriasis patients. Epidermal hyperplasia induced by IMQ treatment was impaired in periostin-deficient mice, along with decreased skin swelling. However, upon treatment with IMQ, periostin deficiency did not alter infiltration of inflammatory cells such as neutrophils; production of IL-17, –22, or –23; or induction/expansion of IL-17– and IL-22–producing group 3 innate lymphoid cells. Conclusions Periostin plays an important role during epidermal hyperplasia in IMQ-induced skin inflammation, independently of the IL-23–IL-17/IL-22 axis. Periostin appears to be a mediator for epidermal hyperplasia that is common to AD and psoriasis. PMID:25572557

Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand–dependent terminal keratinocyte differentiation

PubMed Central

Cobzaru, Cristina; Triantafyllopoulou, Antigoni; Löffek, Stefanie; Horiuchi, Keisuke; Threadgill, David W.; Kurz, Thomas; van Rooijen, Nico; Bruckner-Tuderman, Leena

2012-01-01

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17ΔKC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17ΔKC skin, and topical treatment of A17ΔKC mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (EgfrΔKC) closely resembled A17ΔKC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17–EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects. PMID:22565824

Docetaxel immunonanocarriers as targeted delivery systems for HER 2-positive tumor cells: preparation, characterization, and cytotoxicity studies.

PubMed

Koopaei, Mona Noori; Dinarvand, Rassoul; Amini, Mohsen; Rabbani, Hojatollah; Emami, Shaghayegh; Ostad, Seyed Nasser; Atyabi, Fatemeh

2011-01-01

The objective of this study was to develop pegylated poly lactide-co-glycolide acid (PLGA) immunonanocarriers for targeting delivery of docetaxel to human breast cancer cells. The polyethylene glycol (PEG) groups on the surface of the PLGA nanoparticles were functionalized using maleimide groups. Trastuzumab, a monoclonal antibody against human epidermal growth factor receptor 2 (HER2) antigens of cancer cells, used as the targeting moiety, was attached to the maleimide groups on the surface of pegylated PLGA nanoparticles. Nanoparticles prepared by a nanoprecipitation method were characterized for their size, size distribution, surface charge, surface morphology, drug-loading, and in vitro drug release profile. The average size of the trastuzumab-decorated nanoparticles was 254 ± 16.4 nm and their zeta potential was -11.5 ± 1.4 mV. The average size of the nontargeted PLGA nanoparticles was 183 ± 22 nm and their zeta potential was -2.6 ± 0.34 mV. The cellular uptake of nanoparticles was studied using both HER2-positive (SKBR3 and BT-474) and HER2-negative (Calu-6) cell lines. The cytotoxicity of the immunonanocarriers against HER2-positive cell lines was significantly higher than that of nontargeted PLGA nanoparticles and free docetaxel.

Therapeutic approach to mite-induced intractable dermatitis using novel immunomodulator FTY720 ointment (fingolimod) in NC/Nga mice.

PubMed

Tsuji, Takumi; Okuno, Satoshi; Kuroda, Ayano; Hamazaki, Junya; Chikami, Takuma; Sakurai, Sakura; Yoshida, Yuya; Banno, Rie; Fujita, Tetsuro; Kohno, Takeyuki

2016-04-01

The increasing incidence and prevalence of atopic dermatitis (AD) demands new therapeutic approaches for treating the disease. We investigated the therapeutic efficacy of immunomodulator FTY720 ointment (fingolimod) for mite-induced intractable AD using an NC/Nga mouse model. Female NC/Nga mice that developed severe AD were divided into four groups: (1) FTY720 (0.001% FTY720 ointment), (2) tacrolimus (tacrolimus hydrate ointment) (3) betamethasone (betamethasone ointment), and (4) ointment base (hydrophilic petrolatum), all of which received treatment six times per week. Therapeutic efficacy after two weeks was evaluated in terms of AD severity, histochemical observations (epidermal hypertrophy, mast cell accumulation, and CD3(+) T cell infiltration), transepidermal water loss (TEWL), and epidermal barrier function (filaggrin expression). Betamethasone treatment showed little effect, confirming that the AD was intractable. In the FTY720 group, AD improved significantly compared with the ointment base group, as did epidermal hypertrophy, mast cell accumulation, and CD3(+) T cell infiltration. In contrast, AD in the tacrolimus and betamethasone groups did not improve significantly, nor did epidermal hypertrophy or mast cell accumulation. Furthermore, in the FTY720 group, TEWL decreased significantly compared with the ointment base group, and filaggrin expression significantly increased compared with the betamethasone and ointment base groups. FTY720 ointment is a promising candidate for treatment of intractable AD. These findings also provide the first evidence that FTY720 ointment ameliorates epidermal barrier function. Copyright © 2015 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Zebrafish grainyhead-like1 is a common marker of different non-keratinocyte epidermal cell lineages, which segregate from each other in a Foxi3-dependent manner

PubMed Central

JÄNICKE, MARTINA; RENISCH, BJÖRN; HAMMERSCHMIDT, MATTHIAS

2012-01-01

Grainyhead/CP2 transcription factor family members are widely conserved among the animal kingdom and have been implicated in different developmental processes. Thus far, nothing has been known about their roles in zebrafish. Here we identify seven zebrafish grainyhead-like (grhl) / cp2 genes, with focus on grhl1, which is expressed in the periderm and in epidermal ionocyte progenitors, but downregulated when ionocytes differentiate. In addition, expression was detected in other “non-keratinocyte” cell types of the epidermis, such as pvalb8-expressing cells, which according to our lineage tracing experiments are derived from the same pool of progenitor cells like keratinocytes and ionocytes. Antisense morpholino oligonucleotide-based loss-of-function analysis revealed that grhl1 is dispensable for the development and function of all investigated epidermal cell types, but required as a negative regulator of its own transcription during ionocyte differentiation. Knockdown of the transcription factor Foxi3a, which is expressed in a subset of the grhl1 population, caused a loss of ionocytes and a corresponding increase in the number of pvalb8-expressing cells, while leaving the number of grhl1-positive cells unaltered. We propose that grhl1 is a novel common marker of all or most “non-keratinocyte” epidermal progenitors, and that the sub-functionalisation of these cells is regulated by differential positive and negative effects of Foxi3 factors. PMID:19757382

Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100.

PubMed

Upeniece, Ilze; Groma, Valerie; Skuja, Sandra; Cauce, Vinita

The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3%) for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p < 0.001). The greatly varying apoptotic index was the highest in the lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

Combined inhibition of EMMPRIN and epidermal growth factor receptor prevents the growth and migration of head and neck squamous cell carcinoma cells.

PubMed

Suzuki, Shinsuke; Ishikawa, Kazuo

2014-03-01

It has been reported that the epidermal growth factor receptor (EGFR) expression is associated with the extracellular matrix metalloproteinase inducer (EMMPRIN) in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in head and neck squamous cell carcinoma (HNSCC), HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. EMMPRIN expression in HNSCC cells was upregulated by EGF. In addition, EGF stimulation induced HNSCC cell invasion and MMP-9 expression. This increase in invasion and MMP-9 expression was abrogated by downmodulation of EMMPRIN. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in HNSCC, HNSCC cells were treated with an EMMPRIN function-blocking antibody and the EGFR inhibitor AG1478. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced tumorigenicity and that combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

PubMed

Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

2012-05-30

Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation might be crucial in gastric cancer invasion. HB-EGF-C nuclear translocation may offer a prognostic marker and a new molecular target for gastric cancer therapy.

Intratumoral heterogeneity in EGFR mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition

PubMed Central

Soucheray, Margaret; Capelletti, Marzia; Pulido, Inés; Kuang, Yanan; Paweletz, Cloud P.; Becker, Jeffrey H.; Kikuchi, Eiki; Xu, Chunxiao; Patel, Tarun B.; Al-shahrour, Fatima; Carretero, Julián; Wong, Kwok-Kin; Jänne, Pasi A.; Shapiro, Geoffrey I.; Shimamura, Takeshi

2015-01-01

Non-small cell lung cancers (NSCLC) that have developed resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated-EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT, but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared to cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly develops EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele is lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. PMID:26282169

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

PubMed Central

2012-01-01

Background G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. Methods The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Results Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. Conclusion The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression. PMID:23273253

Anticancer activity of silver nanoparticles from Panax ginseng fresh leaves in human cancer cells.

PubMed

Castro-Aceituno, Verónica; Ahn, Sungeun; Simu, Shakina Yesmin; Singh, Priyanka; Mathiyalagan, Ramya; Lee, Hyun A; Yang, Deok Chun

2016-12-01

The pharmaceutical role of silver nanoparticles has been increased over the last decades, especially those synthesized through herbal medicinal plants, due to their variety of pharmacological importance. Panax ginseng Meyer (P. ginseng) has been widely used as a therapeutic herbal medicine for a long time in cancer treatment. In this study, the cytotoxic and oxidative effect of a novel silver nanoparticles synthesized from P. ginseng fresh leaves (P.g AgNPs) were evaluated in different human cancer cell lines. In addition, the effect of P.g AgNPs on cell migration, apoptosis and the determination of the mechanism involve was determinate by the use of A549 lung cancer cell line. It was found that P.g AgNPs treatment inhibited cell viability and induced oxidative stress in A549, MCF7 and HepG2 cancer cell lines. Likewise, P.g AgNPs treatment inhibited the epidermal growth factor (EGF)-enhanced migration, as well as decreased the mRNA levels and phosphorylation of EGF receptors in A549 cells. Moreover, P.g AgNPs modified the morphology of the cell nucleus and increase apoptosis percentage; this effect was linked to the stimulation of p38 MAPK/p53 pathway. Taken together, our results showed that P.g AgNPs exhibited anti-cancer activity in A549 and the regulation of EGFR/p38 MAPK/p53 pathway might be the possible mechanism of its anti-activity. Further experiments are suggested to determinate the mechanism by which P.g AgNPs induce cytotoxicity and ROS generation in MCF-7 and HepG2 cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer.

PubMed

Jala, Venkatakrishna Rao; Radde, Brandie N; Haribabu, Bodduluri; Klinge, Carolyn M

2012-12-28

G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.

SOX9 regulates ERBB signalling in pancreatic cancer development.

PubMed

Grimont, Adrien; Pinho, Andreia V; Cowley, Mark J; Augereau, Cécile; Mawson, Amanda; Giry-Laterrière, Marc; Van den Steen, Géraldine; Waddell, Nicola; Pajic, Marina; Sempoux, Christine; Wu, Jianmin; Grimmond, Sean M; Biankin, Andrew V; Lemaigre, Frédéric P; Rooman, Ilse; Jacquemin, Patrick

2015-11-01

The transcription factor SOX9 was recently shown to stimulate ductal gene expression in pancreatic acinar-to-ductal metaplasia and to accelerate development of premalignant lesions preceding pancreatic ductal adenocarcinoma (PDAC). Here, we investigate how SOX9 operates in pancreatic tumourigenesis. We analysed genomic and transcriptomic data from surgically resected PDAC and extended the expression analysis to xenografts from PDAC samples and to PDAC cell lines. SOX9 expression was manipulated in human cell lines and mouse models developing PDAC. We found genetic aberrations in the SOX9 gene in about 15% of patient tumours. Most PDAC samples strongly express SOX9 protein, and SOX9 levels are higher in classical PDAC. This tumour subtype is associated with better patient outcome, and cell lines of this subtype respond to therapy targeting epidermal growth factor receptor (EGFR/ERBB1) signalling, a pathway essential for pancreatic tumourigenesis. In human PDAC, high expression of SOX9 correlates with expression of genes belonging to the ERBB pathway. In particular, ERBB2 expression in PDAC cell lines is stimulated by SOX9. Inactivating Sox9 expression in mice confirmed its role in PDAC initiation; it demonstrated that Sox9 stimulates expression of several members of the ERBB pathway and is required for ERBB signalling activity. By integrating data from patient samples and mouse models, we found that SOX9 regulates the ERBB pathway throughout pancreatic tumourigenesis. Our work opens perspectives for therapy targeting tumourigenic mechanisms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Reduced growth factor requirement of keloid-derived fibroblasts may account for tumor growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, S.B.; Trupin, K.M.; Rodriguez-Eaton, S.

Keloids are benign dermal tumors that form during an abnormal wound-healing process is genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid culture grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloidmore » fibroblasts responded differently than normal adult fibroblasts to transforming growth factor ..beta... Whereas transforming growth factor ..beta.. reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of growth-control mechanism that is developmentally regulated.« less

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

PubMed Central

Tao, Jessica J.; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S.; Carey, Lisa A.; Perou, Charles M.; Baselga, José; Scaltriti, Maurizio

2014-01-01

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting. PMID:24667376

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

PubMed

Tao, Jessica J; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S; Carey, Lisa A; Perou, Charles M; Baselga, José; Scaltriti, Maurizio

2014-03-25

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway

PubMed Central

Chen, Xu; Li, Min; Li, Li; Xu, Song; Huang, Dan; Ju, Mei; Huang, Ju; Chen, Kun; Gu, Heng

2016-01-01

Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process which mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. Trehalose-induced autophagy was also observed in primary keratinocytes and the A431 epidermal cancer cell line. mTOR signalling was not affected by trehalose treatment, suggesting that trehalose induced autophagy through an mTOR-independent pathway. mTOR-independent autophagy induction was also observed in HaCaT and HeLa cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had a cytoprotective effect on cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes. PMID:27328819

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells.

PubMed

Massa, Fabienne; Devader, Christelle; Béraud-Dufour, Sophie; Brau, Frédéric; Coppola, Thierry; Mazella, Jean

2013-05-01

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolation and functional assessment of cutaneous stem cells.

PubMed

Doucet, Yanne S; Owens, David M

2015-01-01

The epidermis and associated appendages of the skin represent a multi-lineage tissue that is maintained by perpetual rounds of renewal. During homeostasis, turnover of epidermal lineages is achieved by input from regionalized keratinocytes stem or progenitor populations with little overlap from neighboring niches. Over the last decade, molecular markers selectively expressed by a number of these stem or progenitor pools have been identified, allowing for the isolation and functional assessment of stem cells and genetic lineage tracing analysis within intact skin. These advancements have led to many fundamental observations about epidermal stem cell function such as the identification of their progeny, their role in maintenance of skin homeostasis, or their contribution to wound healing. In this chapter, we provide a methodology to identify and isolate epidermal stem cells and to assess their functional role in their respective niche. Furthermore, recent evidence has shown that the microenvironment also plays a crucial role in stem cell function. Indeed, epidermal cells are under the influence of surrounding fibroblasts, adipocytes, and sensory neurons that provide extrinsic signals and mechanical cues to the niche and contribute to skin morphogenesis and homeostasis. A better understanding of these microenvironmental cues will help engineer in vitro experimental models with more relevance to in vivo skin biology. New approaches to address and study these environmental cues in vitro will also be addressed.

Intraepidermal but not dermal T lymphocytes are positive for a cell-cycle-associated antigen (Ki-67) in mycosis fungoides.

PubMed Central

Nickoloff, B. J.; Griffiths, C. E.

1990-01-01

The Ki-67 antibody, which reacts with nuclei of actively proliferating cells, was used in an immunohistochemical study to determine if there was any difference between T cells located in the epidermis rather than the dermis, in mycosis fungoides. In 12 of 14 cases of patch/plaque stage mycosis fungoides, the epidermal T cells were Ki-67 positive, while the dermal T cells were Ki-67 negative in all cases. Both epidermal and dermal T cells belonged primarily to the memory-versus-naive subset. The intraepidermal Ki-67-positive T cells were slightly larger than the dermal Ki-67-negative cells and could be easily distinguished from occasional basal keratinocytes that were also Ki-67 positive. We conclude that dermal T cells, despite expressing HLA-DR and a memory phenotype, are essentially in a resting (Go or noncycling state) in mycosis fungoides. Furthermore, it appears that the movement of T cells into the epidermal compartment is associated with activation and entry into the cell cycle. Such intraepidermal activation may lead to lymphokine release, and play an important pathophysiologic role in mycosis fungoides. Images Figure 1 Figure 5 PMID:1968314

Inhibition of STAT-3 Results in Radiosensitization of Human Squamous Cell Carcinoma

PubMed Central

Bonner, James A.; Trummell, Hoa Q.; Willey, Christopher D.; Plants, Brian A.; Raisch, Kevin P.

2009-01-01

Background Signal Transducer and Activator of Transcription – 3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein may produce radiosensitization. Methods/Results A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31 h as compared to 18-24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. Conclusion A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by inhibition of EGFr. PMID:19616333

Epidermal Viral Immunity Induced by CD8α+ Dendritic Cells But Not by Langerhans Cells

NASA Astrophysics Data System (ADS)

Allan, Rhys S.; Smith, Chris M.; Belz, Gabrielle T.; van Lint, Allison L.; Wakim, Linda M.; Heath, William R.; Carbone, Francis R.

2003-09-01

The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8α+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.

Her2/neu extracellular domain shedding in uterine serous carcinoma: implications for immunotherapy with trastuzumab.

PubMed

Todeschini, P; Cocco, E; Bellone, S; Varughese, J; Lin, K; Carrara, L; Guzzo, F; Buza, N; Hui, P; Silasi, D-A; Ratner, E; Azodi, M; Schwartz, P E; Rutherford, T J; Pecorelli, S; Santin, A D

2011-10-11

We evaluated shedding of epidermal growth factor type II receptor (Her2/neu) extracellular domain (ECD) in primary uterine serous carcinoma (USC) cell lines and in the serum of USC patients and its biological effects in experiments of trastuzumab-induced cytotoxicity in vitro. Her2/neu expression was evaluated by immunohistochemistry (IHC), real-time PCR and flow cytometry, while c-erbB2 gene amplification was assessed using fluorescent in situ hybridisation (FISH). Her2/neu ECD levels in the supernatants of USC cell lines and in the serum of 38 USC patients and 19 controls were tested using ELISA. The biologic effect of Her2/neu ECD on trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in 5-h chromium-release assays. Five out of ten USC cell lines overexpressed Her2/neu by IHC and showed amplification of the c-erbB2 gene. High levels of Her2/neu ECD were found in supernatants of all FISH-positive tumours. In contrast, FISH-negative USC was negative for Her2/neu ECD shedding. Serum Her2/neu ECD levels in patients harbouring 3+Her2/neu tumours were higher than those found in healthy women (P=0.02) or USC patients with 2+ or 1+/negative Her2/neu expression (P=0.02). In cytotoxicity experiments, trastuzumab-mediated ADCC was significantly decreased by the addition of Her2/neu ECD-containing supernatants (P=0.01). FISH-positive c-erbB2 USC cell lines shed high levels of Her2/neu ECD. High levels of Her2/neu ECD in USC patients may reduce trastuzumab-mediated ADCC in vitro and potentially neutralise its therapeutic effect in vivo.

Development of cassava periclinal chimera may boost production.

PubMed

Bomfim, N; Nassar, N M A

2014-02-10

Plant periclinal chimeras are genotypic mosaics arranged concentrically. Trials to produce them to combine different species have been done, but pratical results have not been achieved. We report for the second time the development of a very productive interspecific periclinal chimera in cassava. It has very large edible roots up to 14 kg per plant at one year old compared to 2-3 kg in common varieties. The epidermal tissue formed was from Manihot esculenta cultivar UnB 032, and the subepidermal and internal tissue from the wild species, Manihot fortalezensis. We determined the origin of tissues by meiotic and mitotic chromosome counts, plant anatomy and morphology. Epidermal features displayed useful traits to deduce tissue origin: cell shape and size, trichome density and stomatal length. Chimera roots had a wholly tuberous and edible constitution with smaller starch granule size and similar distribution compared to cassava. Root size enlargement might have been due to an epigenetic effect. These results suggest a new line of improved crop based on the development of interspecific chimeras composed of different combinations of wild and cultivated species. It promises boosting cassava production through exceptional root enlargement.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

PubMed Central

Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

2016-01-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

Light-Emitting Photon-Upconversion Nanoparticles in the Generation of Transdermal Reactive-Oxygen Species.

PubMed

Prieto, Martin; Rwei, Alina Y; Alejo, Teresa; Wei, Tuo; Lopez-Franco, Maria Teresa; Mendoza, Gracia; Sebastian, Victor; Kohane, Daniel S; Arruebo, Manuel

2017-12-06

Common photosensitizers used in photodynamic therapy do not penetrate the skin effectively. In addition, the visible blue and red lights used to excite such photosensitizers have shallow penetration depths through tissue. To overcome these limitations, we have synthesized ultraviolet- and visible-light-emitting, energy-transfer-based upconversion nanoparticles and coencapsulated them inside PLGA-PEG (methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid)) nanoparticles with the photosensitizer protoporphyrin IX. Nd 3+ has been introduced as a sensitizer in the upconversion nanostructure to allow its excitation at 808 nm. The subcytotoxic doses of the hybrid nanoparticles have been evaluated on different cell lines (i.e., fibroblasts, HaCaT, THP-1 monocytic cell line, U251MG (glioblastoma cell line), and mMSCs (murine mesenchymal stem cells). Upon NIR (near infrared)-light excitation, the upconversion nanoparticles emitted UV and VIS light, which consequently activated the generation of reactive-oxygen species (ROS). In addition, after irradiating at 808 nm, the resulting hybrid nanoparticles containing both upconversion nanoparticles and protoporphyrin IX generated 3.4 times more ROS than PLGA-PEG nanoparticles containing just the same dose of protoporphyrin IX. Their photodynamic effect was also assayed on different cell cultures, demonstrating their efficacy in selectively killing treated and irradiated cells. Compared to the topical application of the free photosensitizer, enhanced skin permeation and penetration were observed for the nanoparticulate formulation, using an ex vivo human-skin-permeation experiment. Whereas free protoporphyrin IX remained located at the outer layer of the skin, nanoparticle-encapsulated protoporphyrin IX was able to penetrate through the epidermal layer slightly into the dermis.

AXL is a logical molecular target in head and neck squamous cell carcinoma

PubMed Central

Brand, Toni M.; Iida, Mari; Stein, Andrew P.; Corrigan, Kelsey L.; Braverman, Cara M.; Coan, John; Pearson, Hannah E.; Bahrar, Harsh; Fowler, Tyler L.; Bednarz, Bryan P.; Saha, Sandeep; Yang, David; Gill, Parkash S.; Lingen, Mark W.; Saloura, Vassiliki; Villaflor, Victoria M.; Salgia, Ravi; Kimple, Randall J.; Wheeler, Deric L.

2016-01-01

Purpose Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard of care treatments for HNSCC patients include surgery, radiation and chemotherapy. Additionally, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult to treat malignancy. Thus, identification of new molecular targets is critical. Experimental Design In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient derived xenografts (PDXs), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard of care treatment regimes used in HNSCC. Results AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusion Collectively, this study provides evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC. PMID:25767293

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals

PubMed Central

Van Moerkercke, Alex; Galván-Ampudia, Carlos S.; Verdonk, Julian C.; Haring, Michel A.; Schuurink, Robert C.

2012-01-01

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved. PMID:22345641

Osimertinib making a breakthrough in lung cancer targeted therapy

PubMed Central

Zhang, Haijun

2016-01-01

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the evidence-based first-line treatment for advanced non-small-cell lung cancer that harbors sensitizing EGFR mutations (EGFRm+) such as exon 19 deletions and L858R substitutions in exon 21. However, acquired resistance to EGFR TKIs is mostly driven by a second-site EGFR T790M mutation, which negates their inhibitory activity. Osimertinib (AZD9291, Tagrisso™), an oral, third-generation EGFR TKI, has been designed to target the EGFR T790M mutation, while sparing wild-type EGFR. In this up-to-date review, focus is not only on the structure, mechanisms, and pharmacokinetics of osimertinib but also on summarizing clinical trials and making recommendations of osimertinib for patients with non-small-cell lung cancer. PMID:27660466

Durable response to osimertinib in EGFR mutated T790M wildtype non-small cell lung cancer with leptomeningeal metastases: A case report.

PubMed

Chalmers, Anna; Jensen, Leif; Akerley, Wallace

2017-12-01

In patients with non-small cell lung cancer (NSCLC) progression with leptomeningeal (LM) metastases is a catastrophic event with limited treatment options. We report a patient who developed leptomeningeal disease while on front-line erlotinib. High-dose tyrosine kinase inhibitor was started but ineffective. She was transitioned to third-generation TKI osimertinib, despite lacking a T790M mutation, and responded with complete resolution of symptoms and malignant cytology in the cerebrospinal fluid (CSF). Recent phase one data and our case indicate osimertinib should be viewed as a best practice for treatment of LM disease in epidermal growth factor receptor (EGFR) mutated NSCLC regardless of T790M status. Copyright © 2017 Elsevier B.V. All rights reserved.

“String of pearls pattern”: report of three cases of non clear-cell acanthoma*

PubMed Central

Espinosa, Ana Elena Domínguez; Akay, Bengu Nisa; González-Ramírez, Roger Adrian

2017-01-01

The coiled and dotted vessels in a serpiginous arrangement or “string of pearls” is considered a classical vascular pattern associated with clear cell acanthoma. We present three cases of epidermal tumors different from clear cell acanthoma that have the same “string of pearls” vascular pattern. Even though most authors keep considering the “string of pearls” vascular pattern an almost pathognomonic sign of clear-cell acanthoma, the cases presented here suggest that some other epidermal tumors can also show this pattern. PMID:29267474

dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

PubMed

Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

2015-08-11

The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of floral cells, thus, this technique may have application in floriculture biotechnology.

Epidermal 'alarm substance' cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation.

PubMed

Chivers, Douglas P; Wisenden, Brian D; Hindman, Carrie J; Michalak, Tracy A; Kusch, Robin C; Kaminskyj, Susan G W; Jack, Kristin L; Ferrari, Maud C O; Pollock, Robyn J; Halbgewachs, Colin F; Pollock, Michael S; Alemadi, Shireen; James, Clayton T; Savaloja, Rachel K; Goater, Cameron P; Corwin, Amber; Mirza, Reehan S; Kiesecker, Joseph M; Brown, Grant E; Adrian, James C; Krone, Patrick H; Blaustein, Andrew R; Mathis, Alicia

2007-10-22

Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway

PubMed Central

Kanda, Yasunari; Mizuno, Katsushige; Kuroki, Yasutomi; Watanabe, Yasuhiro

2001-01-01

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC.In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478.In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC. PMID:11309236

SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells.

PubMed

Rybak, Adrian P; Tang, Damu

2013-12-01

SOX2 is an essential transcription factor for stem cells and plays a role in tumorigenesis, however its role in prostate cancer stem cells (PCSCs) remains unclear. We report here a significant upregulation of SOX2 at both mRNA and protein levels in DU145 PCSCs propagated as suspension spheres in vitro. The expression of SOX2 in DU145 PCSCs is positively regulated by epidermal growth factor receptor (EGFR) signaling. Activation of EGFR signaling, following the addition of epidermal growth factor (EGF) or ectopic expression of a constitutively-active EGFR mutant (EGFRvIII), increased SOX2 expression and the self-renewal of DU145 PCSCs. Conversely, a small molecule EGFR inhibitor (AG1478) blocked EGFR activation, reduced SOX2 expression and inhibited PCSC self-renewal activity, implicating SOX2 in mediating EGFR-dependent self-renewal of PCSCs. In line with this notion, ectopic SOX2 expression enhanced EGF-induced self-renewal of DU145 PCSCs, while SOX2 knockdown reduced PCSC self-renewal with EGF treatment no longer capable of enhancing their propagation. Furthermore, SOX2 knockdown reduced the capacity of DU145 PCSCs to grow under anchorage-independent conditions. Finally, DU145 PCSCs generated xenograft tumors more aggressively with elevated levels of SOX2 expression compared to xenograft tumors derived from non-PCSCs. Collectively, we provide evidence that SOX2 plays a critical role in EGFR-mediated self-renewal of DU145 PCSCs. © 2013.

Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit1

PubMed Central

Giménez, Estela; Dominguez, Eva; Pineda, Benito; Heredia, Antonio; Moreno, Vicente; Angosto, Trinidad

2015-01-01

Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene ARLEQUIN/TOMATO AGAMOUS-LIKE1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato. PMID:26019301

Multiphoton imaging the disruptive nature of sulfur mustard lesions

NASA Astrophysics Data System (ADS)

Werrlein, Robert J.; Braue, Catherine R.; Dillman, James F.

2005-03-01

Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α6β4 integrin, laminin 5 and α3β1 integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the "clinical latent phase" that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.

Mimicking the extracellular matrix with functionalized, metal-assembled collagen peptide scaffolds.

PubMed

Hernandez-Gordillo, Victor; Chmielewski, Jean

2014-08-01

Natural and synthetic three-dimensional (3-D) scaffolds that mimic the microenvironment of the extracellular matrix (ECM), with growth factor storage/release and the display of cell adhesion signals, offer numerous advantages for regenerative medicine and in vitro morphogenesis and oncogenesis modeling. Here we report the design of collagen mimetic peptides (CMPs) that assemble into a highly crosslinked 3-D matrix in response to metal ion stimuli, that may be functionalized with His-tagged cargoes, such as green fluorescent protein (GFP-His8) and human epidermal growth factor (hEGF-His6). The bound hEGF-His6 was found to gradually release from the matrix in vitro and induce cell proliferation in the EGF-dependent cell line MCF10A. The additional incorporation of a cell adhesion sequence (RGDS) at the N-terminus of the CMP creates an environment that facilitated the organization of matrix-encapsulated MCF10A cells into spheroid structures, thus mimicking the ECM environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Optimization of Dosing for EGFR-Mutant Non–Small Cell Lung Cancer with Evolutionary Cancer Modeling

PubMed Central

Chmielecki, Juliann; Foo, Jasmine; Oxnard, Geoffrey R.; Hutchinson, Katherine; Ohashi, Kadoaki; Somwar, Romel; Wang, Lu; Amato, Katherine R.; Arcila, Maria; Sos, Martin L.; Socci, Nicholas D.; Viale, Agnes; de Stanchina, Elisa; Ginsberg, Michelle S.; Thomas, Roman K.; Kris, Mark G.; Inoue, Akira; Ladanyi, Marc; Miller, Vincent A.; Michor, Franziska; Pao, William

2012-01-01

Non–small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance. PMID:21734175

γδ T cells in homeostasis and host defence of epithelial barrier tissues

PubMed Central

Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

2018-01-01

Epithelial surfaces line the body and provide a critical interface between the body and the external environment which is essential to maintaining the symbiotic relationship between the host and the microbiome. Tissue-resident epithelial γδ T cells represent a major T cell population in epithelia and are ideally positioned to perform barrier surveillance and aid in tissue homeostasis and repair. In this review we focus on the intraepithelial γδ compartment in the two largest epithelial tissues in the body, namely the epidermis and intestine, and provide a comprehensive overview of the crucial contributions of intraepithelial γδ cells at these sites to tissue integrity and repair, host homeostasis and host protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we address epithelia-specific butyrophilin-like molecules and touch upon their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires. PMID:28920588

The ABC of BTC: structural properties and biological roles of betacellulin.

PubMed

Dahlhoff, Maik; Wolf, Eckhard; Schneider, Marlon R

2014-04-01

Betacellulin was initially detected as a growth-promoting factor in the conditioned medium of a mouse pancreatic β-cell tumor cell line. Sequencing of the purified protein and of the cloned cDNA supported the assumption that betacellulin is a new ligand of the epidermal growth factor receptor (EGFR), which was later confirmed experimentally. As a typical EGFR ligand, betacellulin is expressed by a variety of cell types and tissues, and the soluble growth factor is proteolytically cleaved from a larger membrane-anchored precursor. Importantly, BTC can - in addition to the EGFR - bind and activate all possible heterodimeric combinations of the related ERBB receptors including the highly oncogenic ERBB2/3 dimer, as well as homodimers of ERBB4. While a large number of studies attest a role for betacellulin in the differentiation of pancreatic β-cells, the last decade witnessed the association of betacellulin with a large number of additional biological processes, ranging from reproduction to the control of neural stem cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tecoma sambucifolia: anti-inflammatory and antinociceptive activities, and 'in vitro' toxicity of extracts of the 'huarumo' of peruvian incas.

PubMed

Alguacil, L F; Galán de Mera, A; Gómez, J; Llinares, F; Morales, L; Muñoz-Mingarro, M D; Pozuelo, J M; Vicente Orellana, J A

2000-06-01

Aqueous and alcoholic extracts of pods and flowers of Tecoma sambucifolia H.B.K. (Bignoniaceae) ('huarumo') were analysed to determine their anti-inflammatory activity (carrageenan-induced edema test), antinociceptive activity (acetic acid writhing test) and 'in vitro' toxicity in Chinese hamster ovary cells, human hepatome cells and human larynx epidermal carcinoma cells. The cytotoxic effects of both extracts were evaluated by two endpoint systems: neutral red uptake assay and tetrazolium assay. The results showed that all extracts have anti-inflammatory and antinociceptive activity, but the highest potency is that of the alcoholic extracts. There were significant differences in cytotoxicity between extracts and among the response of cells to them. The highest cytotoxicity was noted with the alcoholic extract, and the human hepatome cell line was the most sensitive, especially to the alcoholic extract of flowers. The aqueous pod extract appeared to have the best pharmaco-toxicological profile, since it provided a significant reduction of both pain and inflammation together with the lowest cytotoxicity.

Preparation of Rhodium(III) complexes with 2(1H)-quinolinone derivatives and evaluation of their in vitro and in vivo antitumor activity.

PubMed

Lu, Xing; Wu, Yi-Ming; Yang, Jing-Mei; Ma, Feng-E; Li, Liang-Ping; Chen, Sheng; Zhang, Ye; Ni, Qing-Ling; Pan, Ying-Ming; Hong, Xue; Peng, Yan

2018-05-10

A series of 2(1H)-quinolinone derivatives and their rhodium (III) complexes were designed and synthesized. All the rhodium (III) complexes exhibited higher in vitro cytotoxicity for Hep G2, HeLa 229, MGC80-3, and NCI-H460 human tumor cell lines than their ligands and cisplatin, and among them complex 9 was found to be selectively cytotoxic to tumor cells. Further investigation revealed that complex 9 caused cell cycle arrest at the G2/M phase and induced apoptosis, and inhibited the proliferation of Hep G2 cells by impeding the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream enzymes. Complex 9 also up-regulated the proapoptotic proteins Bak, Bax, and Bim, which altogether activated caspase-3/9 to initiate cell apoptosis. Notably, complex 9 effectively inhibited tumor growth in the NCI-H460 xenograft mouse model with less adverse effect than cisplatin. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Cell-based capacitance sensor for analysis of EGFR expression on cell membrane

NASA Astrophysics Data System (ADS)

Shin, Dong-Myeong; Shin, Yong-Cheol; Ha, Ji Hye; Lee, Jong-Ho; Han, Dong-Wook; Kim, Jong-Man; Kim, Hyung Kook; Hwang, Yoon-Hwae

2013-02-01

Cancer cells have many kinds of cancer biomarkers. Among them, the epidermal growth factor (EGF) receptors can show a possibility for a cancer marker because the over-expression of EGF receptor is related with fibrous, colorectal, cervical and gastric tumorigenesis. We fabricated the capacitance sensor with a gap area of 50 μm × 200 μm by using photolithography and lift-off method. Using the capacitance sensor, we investigated the time dependent capacitance changes of different kinds of fibrous cells, such as HT1080 fibrosarcoma, L-929 fibroblast cell line and nHDF dermal fibroblast primary cell. We found that when we put the EGF, the capacitance decreased due to the immobilization of EGF to EGF receptor on the cell membrane. The quantitative determination of EGF receptor level for various fibrous cells was carried out and the results showed good correlation with conventional method. Based on our results, we suggest that the capacitance sensor can measure the expression level of the EGF receptor on cell membrane and be a good candidate as a cancer diagnosis.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as amore » plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.« less

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

PubMed

Sirinian, Chaido; Papanastasiou, Anastasios D; Schizas, Michail; Spella, Magda; Stathopoulos, Georgios T; Repanti, Maria; Zarkadis, Ioannis K; King, Tari A; Kalofonos, Haralabos P

2018-05-29

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

Genetic Correction of Stem Cells in the Treatment of Inherited Diseases and Focus on Xeroderma Pigmentosum

PubMed Central

Rouanet, Sophie; Warrick, Emilie; Gache, Yannick; Scarzello, Sabine; Avril, Marie-Françoise; Bernerd, Françoise; Magnaldo, Thierry

2013-01-01

Somatic stem cells ensure tissue renewal along life and healing of injuries. Their safe isolation, genetic manipulation ex vivo and reinfusion in patients suffering from life threatening immune deficiencies (for example, severe combined immunodeficiency (SCID)) have demonstrated the efficacy of ex vivo gene therapy. Similarly, adult epidermal stem cells have the capacity to renew epidermis, the fully differentiated, protective envelope of our body. Stable skin replacement of severely burned patients have proven life saving. Xeroderma pigmentosum (XP) is a devastating disease due to severe defects in the repair of mutagenic DNA lesions introduced upon exposure to solar radiations. Most patients die from the consequences of budding hundreds of skin cancers in the absence of photoprotection. We have developed a safe procedure of genetic correction of epidermal stem cells isolated from XP patients. Preclinical and safety assessments indicate successful correction of XP epidermal stem cells in the long term and their capacity to regenerate a normal skin with full capacities of DNA repair. PMID:24113582

Integrin Beta 1 Suppresses Multilayering of a Simple Epithelium

PubMed Central

Chen, Jichao; Krasnow, Mark A.

2012-01-01

Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program. PMID:23285215

Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

PubMed

Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

2017-04-15

Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug delivery. However these nanocarriers' interactions with epidermal epithelial barrier are yet unknown. Unveiling the mechanisms involved in NLCs transport across the epidermal epithelial monolayers will contribute with valuable information to achieve enhanced skin permeability, superior bioavailability and consequently improved therapeutic effect. With our present work we could certainly provide researchers and clinicians guidance for the design of optimized transdermal delivery systems, based on the nanomaterials and biological interactions. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Expression and analysis of exogenous proteins in epidermal cells.

PubMed

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

Increase in epidermal planar cell density accompanies decreased russeting of “Golden Delicious” apples treated with gibberellin A4+7

USDA-ARS?s Scientific Manuscript database

A two-year study was conducted in a “Golden Delicious” (Malus Xdomestica Borkh.) orchard having a high historical incidence of physiological fruit russeting, to examine the effect of gibberellin A4+7 (GA4+7) on apple epidermal cell size. Beginning at petal fall, four sequential applications of GA4+7...

Histological study of some Echium vulgare, Pulmonaria officinalis and Symphytum officinale populations.

PubMed

Papp, Nóra; Bencsik, Tímea; Németh, Kitti; Gyergyák, Kinga; Sulc, Alexandra; Farkas, Agnes

2011-10-01

Plants living in different ecological habitats can show significant variability in their histological and phytochemical characters. The main histological features of various populations of three medicinal plants from the Boraginaceae family were studied. Stems, petioles and leaves were investigated by light microscopy in vertical and transverse sections. The outline of the epidermal cells, as well as the shape and cell number of trichomes was studied in leaf surface casts. Differences were measured among the populations of Echium vulgare in the width and height of epidermis cells in the stem, petiole and leaf, as well as in the size of palisade cells in the leaves. Among the populations of Pulmonaria officinalis significant differences were found in the length of trichomes and in the slightly or strongly wavy outline of epidermal radial cell walls. Populations of Symphytum officinale showed variance in the height of epidermal cells in leaves and stems, length of palisade cells and number of intercellular spaces in leaves, and the size of the central cavity in the stem. Boraginaceae bristles were found to be longer in plants in windy/shady habitats as opposed to sunny habitats, both in the leaves and stems ofP. officinalis and S. officinale, which might be connected to varying levels of exposure to wind. Longer epidermal cells were detected in the leaves and stems of both E. vulgare and S. officinale plants living in shady habitats, compared with shorter cells in sunny habitats. Leaf mesophyll cells were shorter in shady habitats as opposed to longer cells in sunny habitats, both in E. vulgare and S. officinale. This combination of histological characters may contribute to the plant's adaptation to various amounts of sunshine. The reported data prove the polymorphism of the studied taxa, as well as their ability to adapt to various ecological circumstances.

Pigment Production Analysis in Human Melanoma Cells.

PubMed

Hopkin, Amelia Soto; Paterson, Elyse K; Ruiz, Rolando; Ganesan, Anand K

2016-05-25

The human epidermal melanocyte is a highly specialized pigmented cell that serves to protect the epidermis from ultraviolet (UV) damage through the production of melanin, or melanogenesis. Misregulation in melanogenesis leading to either hyper- or hypo-pigmentation is found in human diseases such as malasma and vitiligo. Current therapies for these diseases are largely unsuccessful and the need for new therapies is necessary. In order to identify genes and or compounds that can alter melanogenesis, methods are required that can detect changes in pigment production as well as expression of key melanogenesis transcription factors and enzymes. Here we describe methods to detect changes in melanogenesis in a human melanoma cell line, MNT-1, by (1) analyzing pigment production by measuring the absorbance of melanin present by spectrophotometry, (2) analyzing transcript expression of potent regulators of melanogenesis by qunatitative reverse-transcription (RT)PCR and (3) analyzing protein expression of potent regulators of melanogenesis by Western blot (WB).

Glucose transporter member 1 is involved in UVB-induced epidermal hyperplasia by enhancing proliferation in epidermal keratinocytes.

PubMed

Tochio, Takumi; Tanaka, Hiroshi; Nakata, Satoru

2013-03-01

Glucose transporter member 1 (GLUT-1) is one of the major facilitated glucose transporters and contributes to the promotion of keratinocyte proliferation in psoriasis and carcinogenic lesions. In this study, we postulate that GLUT-1 is involved in ultraviolet B (UVB)-induced epidermal hyperplasia. The purpose of this study is to investigate the possible role of GLUT-1 in UVB-induced hyperplasia. The effects of UVB on GLUT-1 expression levels were investigated in in vitro and in vivo studies. In addition, the involvement of epidermal growth factor (EGF) and hypoxia inducible factor-1 alpha (HIF-1α), transcriptional factors for GLUT-1, in GLUT-1-related events were investigated. GLUT-1 mRNA and its protein levels were markedly increased by UVB irradiation in HaCaT cells. In in vivo studies, a strong immunofluorescence signal of GLUT-1 was clearly observed around the basal layer of the epidermis, which proliferated excessively by UVB irradiation. In HaCaT cells, EGF mRNA and its protein levels were markedly increased by UVB irradiation, and then the GLUT-1 mRNA level was significantly increased by treatment with EGF. Additionally, the upregulation of GLUT-1 by both UVB irradiation and treatment with EGF was significantly suppressed by transfection with HIF-1α siRNA. We conclude that GLUT-1 is involved in UVB-induced epidermal hyperplasia by enhancing proliferation of epidermal basal cells, and the GLUT-1-related event might be regulated by an increase in HIF-1α stimulated by EGF. © 2013 The International Society of Dermatology.