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SARS-CoV-2 S-protein-mediated fusion is thought to involve the interaction of the membrane-distal or N-terminal heptad repeat (NHR) ("HR1") of the cleaved S2 segment of the protein and the membrane-proximal or C-terminal heptad repeat (CHR) ("HR2") regions of the protein. We examined the fusion inhibitory activity of a PEGylated HR2-derived peptide and its palmitoylated derivative using a pseudovirus infection assay. The latter peptide caused a 76% reduction in fusion activity at 10 µM. Our results suggest that small variations in peptide derivatization and differences in the membrane composition of pseudovirus preparations may affect the inhibitory potency of HR2-derived peptides. We suggest that future studies on the inhibition of infectivity of SARS-CoV-2 in both in vitro and in vivo systems consider the need for higher concentrations of peptide inhibitors.
Assuntos
Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Peptídeos/farmacologia , Peptídeos/química , Ácido Palmítico/farmacologia , Ácido Palmítico/química , Internalização do Vírus/efeitos dos fármacos , COVID-19/virologia , COVID-19/metabolismo , Antivirais/farmacologia , Antivirais/químicaRESUMO
Ferrocene is useful in modern organometallic chemistry due to its versatile applications in material sciences, catalysis, medicinal chemistry, and diagnostic applications. The ferrocene moiety can potentially serve many purposes in therapeutics and diagnostics. In the course of this study, (6-bromo-1-oxohexyl)ferrocene was combined with dimercaptomaleonitrile sodium salt to yield a novel maleonitrile derivative. Subsequently, this compound was subjected to an autocyclotetramerization reaction using the Linstead conditions in order to obtain an octaferrocenyl-substituted magnesium(II) sulfanyl porphyrazine. Following that, both compounds-the maleonitrile derivative and the porphyrazine derivative-were subjected to physicochemical characterization using UV-Vis, ES-TOF, MALDI-TOF, and one-dimensional and two-dimensional NMR spectroscopy. Moreover, the sulfanyl porphyrazine was subjected to various photophysical studies, including optical absorption and emission measurements, as well as the evaluation of its photochemical properties. Values of singlet oxygen generation quantum yields were obtained in different organic solvents. The electrochemical properties of the synthesized compounds were studied using cyclic voltammetry. According to the electrochemical results, the presence of electron-withdrawing oxohexyl groups attached to ferrocene afforded significantly more positive oxidation potentials of the ferrocene-based redox process up to 0.34 V vs. Fc+/Fc.
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Oxigênio Singlete , Metalocenos , Oxirredução , Oxigênio Singlete/química , SolventesRESUMO
An important subgroup within the porphyrazine (Pz) family constitutes seco-porphyrazines, in the chemical structure of which one pyrrole unit is opened in the oxidative process. So far, there are only limited data on N-seco- and C-seco-Pzs. Here, the synthesis of a novel member of the Pzs seco-family, represented by an S-seco-tribenzoporphyrazine analogue, 22,23-bis(4-(3,5-dibutoxycarbonylphenoxy)butylsulfanyl)tribenzo[b,g,l]-22,23-dioxo-22,23-seco-porphyrazinato magnesium(II), is reported, with moderate 34% yield. The new derivative was characterized using NMR spectroscopy, UV-Vis spectroscopy, and mass spectrometry. In the photochemical study performed following the indirect chemical method with 1,3-diphenylisobenzofuran, S-seco-Pz revealed a high singlet oxygen quantum yield of 0.27 in DMF. Potential photocytotoxicity of S-seco-Pz was assessed in vitro on three cancer cell lines - two oral squamous cell carcinoma cell lines derived from the tongue (CAL 27, HSC-3) and human cervical epithelial adenocarcinoma cells (HeLa). In the biological study, the macrocycle was tested in its free form and after loading into liposomes. It is worth noting that S-seco-Pz was found to be non-toxic in the dark, with cell viability levels over 80%. The photocytotoxic IC50 values for free S-seco-Pz were 0.61, 0.18, and 4.1 µM for CAL 27, HSC-3 and HeLa cells, respectively. Four different liposomal compositions were analyzed, and the cationic liposomes revealed the highest photokilling efficacy, with the IC50 values for CAL 27, HSC-3, and HeLa cells at 0.24, 0.25, and 0.31 µM, respectively. The results of the photocytotoxicity study indicate that the new S-seco-tribenzoporphyrazine can be considered as a potential photosensitizer in photodynamic therapy of cancer, along with the developed cationic liposomal nanocarrier.
Assuntos
Metaloporfirinas/química , Metaloporfirinas/farmacologia , Neoplasias/tratamento farmacológico , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Células HeLa , Humanos , Metaloporfirinas/síntese química , Neoplasias/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/síntese química , Oxigênio Singlete/metabolismoRESUMO
BACKGROUND: Cervical cancer is the third most common cause of cancer in women. The 5-year survival rate in oropharyngeal squamous cell carcinomas is approximately 50% and this rate has not improved in recent decades. These cancers are accessible to direct intervention. We examined the ability of a highly efficient non-viral vector, TransfeX (ATCC, Manassas, VA, USA), to deliver the suicide gene HSV-tk to cervical, oral and pharyngeal cancer cells and to induce cytotoxicity following the administration of the prodrug, ganciclovir. METHODS: HeLa cervical carcinoma, HSC-3 and H357 oral squamous cell carcinoma and FaDu pharyngeal carcinoma cells were transfected with cytomegalovirus (CMV)- or enhanced episomal vector (EEV)-driven HSV-tk plasmids and treated with ganciclovir for 24-120 h. Cell viability was assessed by Alamar blue. RESULTS: The viability of HeLa cells was reduced to only 30-40%, despite the very high levels of transgene expression. By contrast, the viability of HSC-3 cells was reduced to 10%, although transgene expression was 18-fold lower than that in HeLa cells. An approximately five-fold higher transgene expression was obtained with the EEV-plasmid than from the CMV-plasmid. Nevertheless, HeLa cell viability after suicide gene + ganciclovir treatment was reduced by only 35% compared to 70% with the CMV-plasmid. For HSC-3 cells, the reduction was 40% for the EEV- and 80% for the CMV-plasmid. The lower efficiency of transfection with the EEV-plasmid may explain the lower cytotoxicity. CONCLUSIONS: TransfeX-mediated gene delivery to cervical, pharyngeal and oral cancer cells may be used for suicide gene therapy. The levels of transgene expression, however, do not translate directly to cytotoxicity.
Assuntos
Citomegalovirus/genética , Técnicas de Transferência de Genes , Genes Transgênicos Suicidas/genética , Plasmídeos/genética , Antivirais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Ganciclovir/farmacologia , Terapia Genética/métodos , Vetores Genéticos/genética , Células HeLa , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Neoplasias Faríngeas/genética , Neoplasias Faríngeas/patologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
An approach to curing HIV/AIDS is to specifically kill all infected cells. Because the lectins, Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), are potent inhibitors of HIV infection and bind the oligomannans on the HIV Env protein, we hypothesized that they would bind specifically to cells expressing the HIV Env protein on their plasma membrane. Flow cytometry experiments indicated, however, that these lectins bind equivalently to both Env-expressing and control cells without Env.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Lectinas de Ligação a Manose/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/uso terapêutico , Membrana Celular/metabolismo , Células Clonais , Meios de Cultura , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Infecções por HIV/prevenção & controle , Humanos , Liliaceae , Lectinas de Ligação a Manose/uso terapêutico , Lectinas de Plantas/metabolismo , Ligação Proteica , Receptores de HIV/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Linfócitos T/virologiaRESUMO
Infection of oral epithelial cells with periodontopathogenic bacteria results in the production of pro-inflammatory cytokines involved in the initiation and progression of periodontal disease. The purpose of this study was to examine the release of interleukin (IL)-6 and IL-8 by oral epithelial cells after exposure to Porphyromonas gingivalis. Non-tumor-derived, immortalized human GMSM-K cells, and human oral squamous cell carcinoma, HSC-3 and H413 cells, were co-cultured with live and heat-inactivated P. gingivalis 2561 (ATCC 33277) and W83 (ATCC BAA-308™). IL-6 and IL-8 were quantified in the culture supernatants after 6 and 24 h. The basal levels of both cytokines and the responses to P. gingivalis were strongly dependent on cell type. GMSM-K cells produced less IL-8 than HSC-3 and H413 cells. Live P. gingivalis induced significant IL-6 and IL-8 secretion in GMSM-K and HSC-3 cells, and heat-inactivation of bacteria enhanced greatly IL-6 and IL-8 stimulation in these cells. Uninfected H413 cells produced high levels of IL-6 and IL-8, but were not responsive to live P. gingivalis; heat-inactivated P. gingivalis up-regulated IL-6 and IL-8 secretion in these cells. Since base-line secretion of IL-6 and IL-8, and responses to P. gingivalis depend on the cell type, conclusions on the responses to P. gingivalis should not be based on studies with a single cell type.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Porphyromonas gingivalis/imunologia , Linhagem Celular , Técnicas de Cocultura , HumanosRESUMO
Photodynamic therapy exploits the light-activation of a photosensitizer to cause cytotoxicity. Liposomes can be used to deliver hydrophobic photosensitizers to bacteria. Positively charged dioleoyltrimethylammoniumpropane:palmitoyloleoylphosphatidylcholine (1:1) liposomes bound quantitatively to the periodontal pathogen, Porphyromonas gingivalis. Following illumination, free and liposomal zinc phthalocyanine reduced the colony-forming unit (CFU) to 65 percent and 23 percent of controls, respectively. Thus, localization of the photosensitizer at the surface of bacteria via liposome binding enhanced the photodynamic cytotoxicity of zinc phthalocyanine.
Assuntos
Indóis/farmacologia , Lipossomos/química , Compostos Organometálicos/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Aderência Bacteriana , Contagem de Colônia Microbiana , Ácidos Graxos Monoinsaturados/farmacologia , Humanos , Isoindóis , Proteínas de Membrana , Periodontite/tratamento farmacológico , Periodontite/microbiologia , Fosfatidilcolinas/farmacologia , Ligação Proteica , Compostos de Amônio Quaternário/farmacologia , Compostos de ZincoRESUMO
Cancer is a leading cause of death in the world today. In addition to the side effects of the chemotherapeutic drugs used to treat cancer, the development of resistance to the drugs renders the existing drugs ineffective. Therefore, there is an urgent need to develop novel anticancer agents. Medicinally important phytochemicals such as curcumin, naringenin, quercetin, epigallocatechin gallate, thymoquinone, kaempferol, resveratrol, genistein, and apigenin have some drawbacks, including low solubility in water, stability and bioavailability issues, despite having significant anticancer effects. Encapsulation of these natural compounds into polymer nanoparticles (NPs) is a novel technology that could overcome these constraints. In comparison to the free compounds, phytochemicals loaded into nanoparticles have greater activity and bioavailability against many cancer types. In this review, we describe the preparation and characterization of natural phytochemical-loaded polymer NP formulations with significant antioxidant and anti-inflammatory effects, their in vitro and in vivo anticancer activities, as well as their possible cellular targets.
RESUMO
The ideal therapy for HIV infection requires a method to eliminate all HIV-harboring cells in the infected individual. The authors are developing an HIV-specific promoter to drive the expression of suicide genes that would induce cell death specifically in HIV-infected cells. The authors constructed a promoter that is 100-fold more responsive to the HIV transcriptional activator, Tat, than cellular transcription factors, using a plasmid expressing luciferase under the control of the mutated LTR promoter.
Assuntos
Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/genética , Linfócitos T/virologia , Morte Celular/genética , Regulação Viral da Expressão Gênica/genética , Genes Transgênicos Suicidas/genética , Genes tat/genética , Repetição Terminal Longa de HIV/genética , Células HeLa , Humanos , Luciferases , Substâncias Luminescentes , Mutação/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , TransfecçãoRESUMO
The very low success rates of grant applications to the National Institutes of Health (NIH) and the National Science Foundation (NSF) are highly detrimental to the progress of science and the careers of scientists. The peer review process that evaluates proposals has been claimed arbitrarily to be the best there is. This consensus system, however, has never been evaluated scientifically against an alternative. Here we delineate the 15 major problems with the peer review process. We challenge the Science Advisor to the President, and the leadership of NIH, NSF, the U.S. National Academy of Sciences and other funding agencies throughout the world to refute each of these criticisms. We call for the implementation of more equitable alternatives that will not constrain the progress of science. We propose a system that will fund at least 80,000 principal investigators, including young scientists, with about half the current NIH budget, seven-times as many as the current number of NIH "research project grants," and that will forego the cumbersome, expensive, and counterproductive "peer" review stage. Further, we propose that the success of the two systems over 5-10 years be compared scientifically.
Assuntos
Pesquisa Biomédica , Criatividade , National Institutes of Health (U.S.) , Apoio à Pesquisa como Assunto , Pesquisa Biomédica/economia , Estados Unidos , National Institutes of Health (U.S.)/economia , Apoio à Pesquisa como Assunto/economia , Humanos , Consenso , Revisão da Pesquisa por ParesRESUMO
Fusion of lipid-enveloped viruses with the cellular plasma membrane or the endosome membrane is mediated by viral envelope proteins that undergo large conformational changes following binding to receptors. The HIV-1 fusion protein gp41 undergoes a transition into a "six-helix bundle" after binding of the surface protein gp120 to the CD4 receptor and a co-receptor. Synthetic peptides that mimic part of this structure interfere with the formation of the helix structure and inhibit membrane fusion. This approach also works with the S spike protein of SARS-CoV-2. Here we review the peptide inhibitors of membrane fusion involved in infection by influenza virus, HIV-1, MERS and SARS coronaviruses, hepatitis viruses, paramyxoviruses, flaviviruses, herpesviruses and filoviruses. We also describe recent computational methods used for the identification of peptide sequences that can interact strongly with protein interfaces, with special emphasis on SARS-CoV-2, using the PePI-Covid19 database.
RESUMO
Antibiotic-resistant infections present a serious health concern worldwide. It is estimated that there are 2.8 million antibiotic-resistant infections and 35,000 deaths in the United States every year. Such microorganisms include Acinetobacter, Enterobacterioceae, Pseudomonas, Staphylococcus and Mycobacterium. Alternative treatment methods are, thus, necessary to treat such infections. Bacteriophages are viruses of bacteria. In a lytic infection, the newly formed phage particles lyse the bacterium and continue to infect other bacteria. In the early 20th century, d'Herelle, Bruynoghe and Maisin used bacterium-specific phages to treat bacterial infections. Bacteriophages are being identified, purified and developed as pharmaceutically acceptable macromolecular "drugs," undergoing strict quality control. Phages can be applied topically or delivered by inhalation, orally or parenterally. Some of the major drug-resistant infections that are potential targets of pharmaceutically prepared phages are Pseudomonas aeruginosa, Mycobacterium tuberculosis and Acinetobacter baumannii.
RESUMO
BACKGROUND: Inhaled budesonide benefits patients with COVID-19. ProLung™-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung™-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it's effect on SARS-CoV-2 replication is unknown. OBJECTIVE: To determine the efficacy of ProLung™-budesonide against SARS-CoV-2-infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation. METHODS: SARS-CoV-2-infected Vero 76 cells were treated with ProLung™-budesonide ([0.03-100 µg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung™-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung™ carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL). RESULTS: ProLung™-budesonide showed significant inhibition of viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value >24. Weekly ProLung™-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung™-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challenge. CONCLUSIONS: ProLung™-budesonide significantly inhibited viral replication in SARS-CoV-2-infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.
RESUMO
Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01-100 microM) or fluconazole (1-200 microM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 +/- 2.2, 6.9 +/- 3.7 and 1.7 +/- 1.5 microM, and IC(50) (planktonic cells) of 3.0 +/- 0.5, 2.6 +/- 0.1 and 4.8 +/- 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 +/- 4.8 and 62.5 +/- 0.7 microM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC(50) > 100 microM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1-200 microM; e.g. at 100 microM only approximately 20% inhibition was observed for C. albicans, and approximately 30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Fluconazol/farmacologia , Histatinas/farmacologia , Candida albicans/fisiologia , Candida glabrata/fisiologia , Contagem de Colônia Microbiana , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacosRESUMO
In this work we have examined the ability of various lipopolyplexes to deliver genes into liver cancer cells. We evaluated different parameters such as the protocol of preparation, the lipid/DNA molar ratio, and the molecular weight and type of PEI, to optimize the formulation to achieve high transfection activity. Our hypothesis was that the association of PEI with cationic liposomes (lipopolyplexes) would increase luciferase expression compared to lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA) alone.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Lipossomos/farmacologia , Polietilenoimina/farmacologia , Cátions , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/genética , Regulação Enzimológica da Expressão Gênica , Células Hep G2 , Humanos , Lipídeos/química , Lipídeos/genética , Lipossomos/química , Luciferases/genética , Luciferases/metabolismo , Peso Molecular , Polietilenoimina/químicaRESUMO
Aim: Photodynamic therapy utilizes a light-sensitive molecule that produces reactive oxygen species following irradiation. Photodynamic activities of free Zn phthalocyanine (ZnPc) and its liposomal formulations on human oral squamous cell carcinoma and pharyngeal carcinoma cells were assessed. Materials & methods: ZnPc was incorporated in extruded and nonextruded liposomes composed of palmitoyloleoylphosphatidylglycerol (POPG):palmitoyloleoylphosphatidylcholine (POPC) or POPG:dioleoylphosphatidylethanolamine liposomes and incubated with CAL 27 or FaDu cells. Cell viability was assessed following illumination and further incubation. Results: ZnPc incorporated in extruded POPG:POPC liposomes caused extensive cytotoxicity, while ZnPc in extruded or nonextruded POPG:dioleoylphosphatidylethanolamine liposomes or in multilamellar POPG:POPC liposomes were not effective. Conclusion: Extruded POPG:POPC liposomes are a useful delivery vehicle for ZnPc in photodynamic therapy of oral and pharyngeal cancers.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias Faríngeas , Fotoquimioterapia , Carcinoma de Células Escamosas/tratamento farmacológico , Humanos , Indóis , Isoindóis , Lipossomos , Neoplasias Bucais/tratamento farmacológico , Compostos Organometálicos , Neoplasias Faríngeas/tratamento farmacológico , Fármacos Fotossensibilizantes , Carcinoma de Células Escamosas de Cabeça e Pescoço , Compostos de ZincoRESUMO
The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
Assuntos
Pichia/química , Pichia/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/biossíntese , Inibidor Secretado de Peptidases Leucocitárias/química , Técnicas de Cultura de Células , Fermentação , Glicosilação , Humanos , Pichia/genética , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Inibidor Secretado de Peptidases Leucocitárias/genética , Inibidor Secretado de Peptidases Leucocitárias/isolamento & purificação , Transfecção , Tripsina/metabolismoRESUMO
Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is associated with malignant transformation and is over-expressed in most human tumors. Using lipoplex-mediated transfection, we evaluated the activity of the reporter enzyme, luciferase, expressed from plasmids encoding the enzyme under the control of either the cytomegalovirus (CMV) or survivin promoters, in tumor- and non-tumor-derived human and murine cells. We also examined whether there is a correlation between the survivin promoter-driven expression of luciferase and the level of endogenous survivin. Human cancer cells (HeLa, KB, HSC-3, H357, H376, H413), oral keratinocytes, GMSM-K, and chemically immortalized human mammary cells, 184A-1, were transfected with Metafectene at 2 microl/1 microg DNA. Murine squamous cell carcinoma cells, SCCVII, mouse embryonic fibroblasts, NIH-3T3, and murine immortalized mammary cells, NMuMG, were transfected with Metafectene PRO at 2 microl/1 microg DNA. The expression of luciferase was driven by the CMV promoter (pCMV.Luc), the human survivin promoter (pSRVN.Luc-1430), or the murine survivin promoters (pSRVN.Luc-1342 and pSRVN.Luc-194). Luciferase activity was measured, using the Luciferase Assay System and expressed as relative light units (RLU) per ml of cell lysate or per mg of protein. The level of survivin in the lysates of human cells was determined by ELISA and expressed as ng survivin/mg protein. In all cell lines, significantly higher luciferase activity was driven by the CMV promoter than by survivin promoters. The expression of luciferase driven by the CMV and survivin promoters in murine cells was much higher than that in human cells. The cells displayed very different susceptibilities to transfection; nevertheless, high CMV-driven luciferase activity appeared to correlate with high survivin-promoter driven luciferase expression. The survivin concentration in lysates of cancer cells ranged from 5.8 +/- 2.3 to 24.3 +/- 2.9 ng/mg protein (mean, 13.7 ng/mg). Surprisingly, elevated survivin protein was determined in lysates of non-tumor-derived cells. Survivin levels for GMSM-K and 184A-1 cells, were 16.7 +/- 8.7 and 13.5 +/- 6.2 ng/mg protein, respectively. The expression of endogenous survivin did not correlate with the level of survivin promoter-driven transgene activity in the same cells. The expression of survivin by non-tumorigenic, transformed cell lines may be necessary for their proliferative activity. The level of survivin promoter-driven gene expression achieved via liposomal vectors in OSCC cells was too low to be useful in cancer-cell specific gene therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/genética , Regiões Promotoras Genéticas , Animais , Morte Celular , Linhagem Celular Tumoral , Citomegalovirus/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Inibidoras de Apoptose , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Survivina , Transfecção , TransgenesRESUMO
BACKGROUND: Candida biofilms with reduced susceptibility to conventional antifungals are sensitive to lipid formulations of amphotericin B (AMB). We examined the effect of the liposomal AMB formulation, AmBisome, and free AMB on the adherence of C. albicans to HeLa cervical carcinoma and HSC-3 oral squamous cell carcinoma cells. MATERIAL/METHODS: HeLa and HSC-3 cells were incubated with three oral isolates of C. albicans either in the presence of AmBisome or AMB, or pre-incubated with yeasts and subsequently exposed to the drug. The effect of the drugs on the viability of HeLa and HSC-3 cells was determined by an Alamar Blue assay. RESULTS: Following a 1-h incubation in the presence of AmBisome, at 1-256 microg/ml, the adherence of C. albicans to HeLa and HSC-3 cells was reduced considerably. For example at 16 microg/ml, adherence was diminished by approximately 66% in HeLa and by approximately 36% in HSC-3 cells. The susceptibility of cell-associated Candida to antifungals was decreased markedly. The reduction in adherence was between 3.3 and 13.7%, when compared to the drug-free controls. AmBisome was not toxic in the range 1-256 microg/ml, while free AMB was not toxic at 1 and 4 microg/ml to HeLa cells and at 1, 4 and 16 microg/ml to HSC-3 cells. CONCLUSIONS: AmBisome inhibited candidal attachment when present during the "adherence phase" but did not cause detachment of cell-associated yeasts. The effect of AmBisome on candidal adherence to HSC-3 cells was less inhibitory than that observed with HeLa cells. Candidal adherence to epithelial cells is significantly reduced when antifungal polyenes are present during the "adherence phase", while cell-associated Candida is resistant to antifungals in terms of adherence.