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1.
Exp Dermatol ; 26(7): 598-606, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28423208

RESUMEN

The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF-mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K-AKT-mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib-resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti-apoptotic protein Mcl-1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock-down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Dacarbazina/análogos & derivados , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/tratamiento farmacológico , Sirolimus/análogos & derivados , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos Alquilantes/administración & dosificación , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Dacarbazina/administración & dosificación , Dacarbazina/uso terapéutico , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Indoles/administración & dosificación , Lentivirus , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Sirolimus/administración & dosificación , Sirolimus/uso terapéutico , Piel/metabolismo , Neoplasias Cutáneas/metabolismo , Sulfonamidas/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Temozolomida , Vemurafenib
2.
J Biol Chem ; 287(11): 8434-43, 2012 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-22262861

RESUMEN

Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn(2+). DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn(2+)-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.


Asunto(s)
Antiinfecciosos/química , Péptidos/química , Sudor/química , Antiinfecciosos/farmacología , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Péptidos/farmacología , Fosfolípidos/química , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo , Relación Estructura-Actividad , Sudor/metabolismo
3.
Exp Dermatol ; 22(2): 153-5, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23362876

RESUMEN

Experimental mouse models of bacterial skin infections that have been described show that pathogenic microorganisms can readily invade the epidermis and dermis to produce localized infections. We used an epicutaneous mouse skin infection model to determine how the level of barrier disruption by tape-stripping correlates with persistence of Staphylococcus aureus skin colonization, concomitant induction of cutaneous inflammation and infection. Furthermore, we investigated how murine skin responds to S. aureus colonization in a physiologic setting by analysing proinflammatory cytokines and antimicrobial peptides in mouse skin. We show that previous cutaneous damage allows skin inflammation to develop and favours S. aureus persistence leading to cutaneous colonization, suggesting an interdependence of cutaneous bacteria and skin. Our study suggests that skin barrier defects favour S. aureus skin colonization, which is associated with profound cutaneous inflammation.


Asunto(s)
Regulación Bacteriana de la Expresión Génica , Inflamación/microbiología , Piel/microbiología , Infecciones Cutáneas Estafilocócicas/inmunología , Animales , Biopsia , Ensayo de Unidades Formadoras de Colonias , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Queratinocitos/microbiología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Piel/inmunología , Piel/patología
4.
Clin Cancer Res ; 23(20): 6203-6214, 2017 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-28724666

RESUMEN

Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203-14. ©2017 AACR.


Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , GTP Fosfohidrolasas/genética , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Melanoma/genética , Melanoma/metabolismo , Proteínas de la Membrana/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Melanoma/patología , Modelos Biológicos , Fosforilación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos
5.
Toxicology ; 204(2-3): 141-54, 2004 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-15388240

RESUMEN

Insulin and dexamethasone are potent inhibitors of apoptosis induced by transforming growth factor-beta1 (TGF-beta) in hepatoma cells. Using FTO-2B rat hepatoma cells, we determined whether the anti-apoptotic effects of these agents result from interference within or upstream of the TGF-beta-induced caspase cascade. Activation of different initiator and effector caspases, Bax and Bcl-xL expression, mitochondrial cytochrome c release and activation of PKB/Akt were analyzed by use of synthetic caspase substrates and Western blotting, respectively. TGF-beta-induced apoptosis was characterized by release of cytochrome c from mitochondria and activation of caspases-3, -7, -8 and -9. These effects were observable as early as 8-12 h after start of treatment and increased with time of observation. Inhibition of TGF-beta-induced apoptosis by insulin and dexamethasone was paralleled by a strong reduction of caspase-3-like activity. Caspase-8 activation was almost completely suppressed by these agents, and caspase-9 activity was decreased to levels within or slightly above unstimulated control cells. In addition, cytochrome c release from mitochondria was efficiently repressed, which was associated with upregulation of Bcl-xL by dexamethasone and activation of PKB/Akt by insulin. Thus, both anti-apoptotic compounds exert their inhibitory effects through modulation of anti-apoptotic signalling pathways involved in regulation of cytochrome c release and activation of the caspase machinery.


Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Inhibidores de Caspasas , Dexametasona/farmacología , Insulina/farmacología , Neoplasias Hepáticas Experimentales/patología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/metabolismo , Ratas , Factor de Crecimiento Transformador beta/farmacología
6.
J Innate Immun ; 6(4): 467-84, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24458132

RESUMEN

Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin α chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin α3, α4 and α5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin α3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin α3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the α3, α4 and α5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection.


Asunto(s)
Antiinfecciosos/metabolismo , Infecciones Bacterianas/inmunología , Candidiasis/inmunología , Queratinocitos/fisiología , Laminina/metabolismo , Leucocitos Mononucleares/fisiología , Fragmentos de Péptidos/metabolismo , Piel/patología , Línea Celular Transformada , Supervivencia Celular , Quimiotaxis , Humanos , Inmunidad Innata , Queratinocitos/microbiología , Laminina/genética , Fragmentos de Péptidos/genética , Proteolisis , Piel/inmunología , Piel/microbiología , Cicatrización de Heridas
7.
J Invest Dermatol ; 131(2): 382-90, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21048787

RESUMEN

Little is known about the impact of different microbial signals on skin barrier organ function and the interdependency between resident microflora and pathogenic microorganisms. This study shows that commensal and pathogenic staphylococci differ in their ability to induce expression of antimicrobial peptides/proteins (AMPs) and activate different signaling pathways in human primary keratinocytes. Whereas secreted factors of skin commensals induce expression of the AMPs HBD-3 and RNase7 in primary human keratinocytes via Toll-like receptor (TLR)-2, EGFR, and NF-κB activation, those of pathogenic staphylococci activate the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways and suppress NF-κB activation. Interestingly, commensal bacteria are able to amplify the innate immune response of human keratinocytes to pathogens by increased induction of AMP expression and abrogation of NF-κB suppression, suggesting that the two activation pathways can act in a synergistic way. These data indicate that commensal and pathogenic microorganisms evolved specific mechanisms to modulate innate immunity of the skin.


Asunto(s)
Inmunidad Innata/fisiología , Transducción de Señal/fisiología , Piel/inmunología , Piel/microbiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Diferenciación Celular/fisiología , Células Cultivadas , Receptores ErbB/metabolismo , Humanos , FN-kappa B/metabolismo , Ribonucleasas/metabolismo , Piel/metabolismo , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/fisiología , Staphylococcus epidermidis/patogenicidad , Staphylococcus epidermidis/fisiología , Receptor Toll-Like 2/metabolismo , beta-Defensinas/metabolismo
8.
Int J Cancer ; 120(7): 1459-64, 2007 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-17205518

RESUMEN

The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UV-irradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H2O2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H2O2-exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Mutación/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Western Blotting , Carcinoma Hepatocelular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/genética , Ratones , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Rayos Ultravioleta
9.
Toxicol Pathol ; 31(1): 99-102, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12597453

RESUMEN

Tumor promoters are nonmutagenic chemicals that increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. The molecular mechanisms underlying this process are only partly understood but interference with signaling pathways regulating cell division and/or cell death is likely to be important. Ras- and beta-Catenin-dependent signaling is important for both of these processes and ras and beta-catenin genes are known mutational targets in mouse hepatocarcinogenesis. About 80% of liver tumors generated in mice by a promotional regimen including phenobarbital (PB) as tumor promoter and N-nitrosodiethylamine (DEN) as initiator showed beta-catenin mutations whereas Ha-ras mutations were not detected. By contrast, tumors from mice treated with DEN alone showed a approximately 30% Ha-ras mutation prevalence but no beta-catenin mutations. This result suggests that PB-mediated promotion in mouse liver consists in a positive selection for hepatocytes harboring mutations in beta-catenin. The gap junction protein connexin 32 (Cx32) was also found to be involved in tumor promotion by PB because Cx32 gene knockout mice were almost entirely resistent to the promotional effects of the barbiturate. The link between beta-catenin-signaling and Cx32-dependent gap junctional intercellular communication, if existent, remains obscure.


Asunto(s)
Carcinógenos/toxicidad , Cocarcinogénesis , Conexinas/genética , Proteínas del Citoesqueleto/genética , Neoplasias Hepáticas Experimentales/genética , Hígado/efectos de los fármacos , Transactivadores/genética , Animales , División Celular/efectos de los fármacos , División Celular/genética , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Noqueados , Mutación , beta Catenina , Proteína beta1 de Unión Comunicante
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