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BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.
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Neoplasias da Mama , Proteínas Monoméricas de Ligação ao GTP , Animais , Feminino , Humanos , Neoplasias da Mama/genética , Proliferação de Células , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.
Assuntos
Melanoma , Talina , Humanos , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/genética , Integrinas/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Isoformas de Proteínas/metabolismo , Talina/genética , Talina/química , Talina/metabolismo , Linhagem Celular Tumoral/metabolismoRESUMO
AIM: To assess the humoral immunity to COVID-19 in nursing home residents six months after vaccination. METHODS: This seroepidemiological research enrolled 118 residents of one nursing home in Zagreb. All participants received two doses of BioNTech/Pfizer COVID-19 and had no previously detected SARS-CoV-2 infection. The samples were tested for the presence of neutralizing antibodies using a virus neutralization test. A SARS-CoV-2 strain isolated in Vero E6 cells from a Croatian COVID-19 patient was used as a stock virus. Neutralizing antibody titer was defined as the reciprocal of the highest serum dilution that showed at least 50% neutralization. Neutralizing antibody titer ≥8 was considered positive. RESULTS: Sixty-four (54%) participants had a positive neutralizing antibody titer, 27 (23%) had a low positive titer (titer 8), and 27 (23%) had a negative titer. Women had a significantly higher median titer than men (16 [interquartile range, IQR 24] vs 8 [IQR 12], Mann-Whitney U=1033, P=0.003). Age was negatively but not significantly correlated with neutralizing antibody titer (Spearman's rho -0.132, P=0.155). CONCLUSION: Almost half of the participants (46%) had a negative or low positive titer six months after having been fully vaccinated. This study suggests that humoral immunity among nursing home residents considerably wanes six months after BioNTech/Pfizer COVID-19 vaccination. Our results could contribute to the discussion about the need for a booster dose.
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Vacinas contra COVID-19 , COVID-19 , Croácia/epidemiologia , Feminino , Humanos , Masculino , Casas de Saúde , SARS-CoV-2 , VacinaçãoRESUMO
Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.
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Proteínas Adaptadoras de Transporte Vesicular/metabolismo , HIV-1/metabolismo , Herpesviridae/metabolismo , SARS-CoV-2/metabolismo , Ebolavirus/metabolismo , Endocitose , Humanos , Vírus Nipah/metabolismo , Transporte Proteico , Liberação de Vírus , Replicação ViralRESUMO
Low survival rates of patients with metastatic triple-negative breast cancer (TNBC) and melanoma, in which current therapies are ineffective, emphasize the need for new therapeutic approaches. Integrin ß1 appears to be a promising target when combined with chemotherapy, but recent data have shown that its inactivation increases metastatic potential owing to the compensatory upregulation of other integrin subunits. Consequently, we analyzed the potential of integrin subunits αv, α3, or α4 as targets for improved therapy in seven TNBC and melanoma cell lines. Experiments performed in an integrin αvß1-negative melanoma cell line, MDA-MB-435S, showed that knockdown of integrin subunit αv increased sensitivity to microtubule poisons vincristine or paclitaxel and decreased migration and invasion. In the MDA-MB-435S cell line, we also identified a phenomenon in which change in the expression of one integrin subunit changes the expression of other integrins, leading to an unpredictable influence on sensitivity to anticancer drugs and cell migration, referred to as the integrin switching effect. In a panel of six TNBCs and melanoma cell lines, the contribution of integrins αv versus integrins αvß3/ß5 was assessed by the combined action of αv-specific small interfering RNA or αvß3/ß5 inhibitor cilengitide with paclitaxel. Our results suggest that, for TNBC, knockdown of integrin αv in combination with paclitaxel presents a better therapeutic option than a combination of cilengitide with paclitaxel; however, in melanoma, neither of these combinations is advisable because a decreased sensitivity to paclitaxel was observed.
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Integrina alfaV/genética , Melanoma/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Venenos/farmacologia , Venenos de Serpentes/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Paclitaxel/farmacologia , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.
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Carcinoma de Células Escamosas/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Integrina alfaVbeta3/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Neoplasias da Língua/patologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cisplatino/farmacologia , Dasatinibe/farmacologia , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes src , Humanos , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Cadeias beta de Integrinas/fisiologia , Mitomicina/farmacologia , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Mutação Puntual , Multimerização Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Interferência de RNA , Neoplasias da Língua/genética , Neoplasias da Língua/metabolismoRESUMO
The serotype specificity of adenovirus ion-exchange chromatography has previously been studied using standard particle-based columns, and the hexon protein has been reported to determine retention time. In this study, we have submitted Adenovirus type 5 recombinants to anion-exchange chromatography using methacrylate monolithic supports. Our experiments with hexon-modified adenoviral vectors show more precisely that the retention time is affected by the substitution of amino acids in hypervariable region 5, which lies within the hexon DE1 loop. By exploring the recombinants modified in the fiber protein, we have proven the previously predicted chromatographic potential of this surface constituent. Modifications that preserve the net charge of the hexon protein, or those that cause only a small charge difference in the fiber protein, in addition to shortening the fiber shaft, did not change the chromatographic behavior of the adenovirus particles. However, modifications that include the deletion of just two negatively charged amino acids in the hexon protein, or the introduction of a heterologous fiber protein, derived from another serotype, revealed recognizable changes in anion-exchange chromatography. This could be useful in facilitating chromatography-approach purification by creating targeted capsid modifications, thereby shifting adenovirus particles away from particular interfering substances present in the crude lysate.
Assuntos
Adenoviridae , Proteínas do Capsídeo/química , Cromatografia por Troca Iônica , Vetores Genéticos , Substituição de Aminoácidos , Cromatografia Líquida de Alta PressãoRESUMO
BACKGROUND: In order to increase the effectiveness of cancer treatment, new compounds with potential anticancer activities are synthesized and screened. Here we present the screening of a new class of compounds, 1-(2-picolyl)-, 4-(2-picolyl)-, 1-(2-pyridyl)-, and 4-(2-pyridyl)-3-methyl-1,2,3-triazolium salts and 'parent' 1,2,3-triazole precursors. METHODS: Cytotoxic activity of new compounds was determined by spectrophotometric MTT assay on several tumour and one normal cell line. Effect of the selected compound to bind double stranded DNA (ds DNA) was examined by testing its influence on thermal stability of calf thymus DNA while its influence on cell cycle was determined by flow cytometric analysis. Generation of reactive oxygen species (ROS) was determined by addition of specific substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). RESULTS: Parent triazoles were largely inactive, while some of the triazolium salts were highly cytotoxic for HeLa cells. Triazolium salts exhibited high cell-type dependent cytotoxicity against different tumour cells. Selected compound (4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexafluorophosphate(V) (2b) was significantly more cytotoxic against tumour cells than to normal cells, with very high therapeutic index 7.69 for large cell lung carcinoma H460 cells. Additionally, this compound was similarly cytotoxic against parent laryngeal carcinoma HEp-2 cells and their drug resistant 7T subline, suggesting the potential of this compound in treatment of drug resistant cancers. Compound 2b arrested cells in the G1 phase of the cell cycle. It did not bind ds DNA, but induced ROS in treated cells, which further triggered cell death. CONCLUSIONS: Our results suggest that the 'click' triazolium salts are worthy of further investigation as anti-cancer agents.
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Previously, we described the synthesis and biological activity of a new class of anticancer molecules that preferentially target malignant cells and may serve as potential antitumor agents. Among several synthesized agents, we selected 3-acetyl-1,3-bis(2-chloro-4-nitrophenyl)-1-triazene (8b) as a representative of the group of 4-nitro-substituted 1,3-diaryltriazenes. The aim of this study was to further investigate the mechanism of cell response to the 8b compound. The HeLa human cervical carcinoma cell line was used as an experimental model to further investigate the mechanism of cell response to the 8b compound. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to address cell survival, and western blot (immunoblotting) was used for the expression of relevant proteins after 8b drug exposure. The pretreatment of HeLa cells with salubrinal, a specific inhibitor of endoplasmic reticulum (ER) stress, confirmed the importance of ER stress in apoptosis induced by 8b. We also demonstrate that 8b triggers the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in a time-dependent and dose-dependent manner. Moreover, the inhibition of SAPK/JNK activity by JNK II before 8b treatment increased the survival rate of HeLa cells relative to survival in the presence of 8b alone, indicating the importance of this kinase in cell death. The simultaneous inhibition of ER stress induction and SAPK/JNK activation increased the survival of HeLa cells upon 8b treatment more than inhibition of both pathways independently, suggesting the separate triggering of both signaling pathways. Our data indicate that cytotoxic activity of the novel compound 8b is based on its ability to induce ER stress and SAPK/JNK signaling pathways independently, driving cells to cell death.
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Antineoplásicos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Triazenos/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática , Glutationa/metabolismo , Células HeLa , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Radiotherapy of prostate cancer (PC) can lead to the acquisition of radioresistance through molecular mechanisms that involve, in part, cell adhesion-mediated signaling. To define these mechanisms, we employed a DU145 PC model to conduct a comparative mass spectrometry-based proteomic analysis of the purified integrin nexus, i.e., the cell-matrix junction where integrins bridge assembled extracellular matrix (matrisome components) to adhesion signaling complexes (adhesome components). When parental and radioresistant cells were compared, the expression of integrins was not changed, but cell radioresistance was associated with extensive matrix remodeling and changes in the complement of adhesion signaling proteins. Out of 72 proteins differentially expressed in the parental and radioresistant cells, four proteins were selected for functional validation based on their correlation with biochemical recurrence-free survival. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) and lysyl-like oxidase-like 2 (LOXL2) were upregulated, while sushi repeat-containing protein X-linked (SRPX) and laminin subunit beta 3 (LAMB3) were downregulated in radioresistant DU145 cells. Knockdown of perlecan/HSPG2 sensitized radioresistant DU145 RR cells to irradiation while the sensitivity of DU145 parental cells did not change, indicating a potential role for perlecan/HSPG2 and its associated proteins in suppressing tumor radioresistance. Validation in androgen-sensitive parental and radioresistant LNCaP cells further supported perlecan/HSPG2 as a regulator of cell radiosensitivity. These findings extend our understanding of the interplay between extracellular matrix remodeling and PC radioresistance and signpost perlecan/HSPG2 as a potential therapeutic target and biomarker for PC.
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The major histocompatibility complex (MHC) contains genes important for immune response in mammals, and these genes exhibit high polymorphism and diversity. The DRA gene, a member of the MHC class II family, is highly conserved across a large number of mammalian species, but it displays exceptionally rich sequence variations in Equidae members. We analyzed allelic polymorphism of the DRA locus in 248 donkeys sampled across the Balkan Peninsula (Albania, Bulgaria, Croatia, Macedonia, Greece and Montenegro). Five known alleles and two new alleles were identified. The new allele Eqas-DRA*0601 was found to carry a synonymous mutation, and new allele Eqas-DRA*0701, a non-synonymous mutation. We further analyzed the historical selection and allele genealogy at the DRA locus in equids. Signals of positive selection obtained by various tests were ambiguous. A conservative conclusion is that DRA polymorphism occurred relatively recently and that positive selection has been acting on the DRA locus for a relatively brief period.
Assuntos
Alelos , Antiportadores de Cloreto-Bicarbonato/genética , Equidae/genética , Genes MHC da Classe II/genética , Polimorfismo Genético/genética , Animais , Península Balcânica , Sequência de Bases , Frequência do Gene/genética , Genética Populacional , Funções Verossimilhança , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , Seleção Genética , Análise de Sequência de DNA/veterináriaRESUMO
Construction of a library of structurally diverse diazenecarboxamide-extended cis-[Pt(2-picolyl-1,2,3-triazole)Cl2,] and cis-[Pt(propan-1,3-diamine)CBDCA] (CBDCA = 1,1 -cyclobutanedicarboxylate) complexes 1-4 is described. These compounds retain oxidative properties of parent diazenecarboxamides against glutathione as demonstrated by NMR spectroscopy and high resolution mass spectrometry experiments. Cytotoxic activity of 1-4 was investigated against human cervical carcinoma HeLa cells. Four library members were found to possess moderate cytotoxic activity. Some model compounds were also examined, returning [PtCl2L2] (L = 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole) as the most potent under this investigation with IC50 of 19.05 microM, comparable to that of cisplatin (IC50 = 16.3 microM).
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Amidas/química , Antineoplásicos/química , Carboplatina/análogos & derivados , Cisplatino/análogos & derivados , Desenho de Fármacos , Amidas/farmacologia , Antineoplásicos/farmacologia , Carboplatina/química , Carboplatina/farmacologia , Cromatografia Líquida , Cisplatino/química , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvß3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.
Assuntos
Adenovírus Humanos , Humanos , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismoRESUMO
BACKGROUND: The α(v) -integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie and adenovirus receptor. However, until now, it has been unclear whether disulfide bonds in the RGD4C motif influence the retargeting potential of RGD4C-modified Ad5. METHODS: Replication deficient Ad5 bearing wild-type fiber (Ad5wt) or RGD4G, RGD4C and RGD2C2G insertions within the HI loop of the fiber protein (Ad5RGD4G, Ad5RGD4C and Ad5RGD2C2G, respectively) were used to transduce a panel of cancer cell lines, with or without previous treatment of these Ad5s with the reducing agent dithiothreitol (DTT). In parallel, native and DTT-treated fiber proteins isolated from purified Ad5RGD4C were compared by mass spectrometry. RESULTS: Ad5RGD4C transduced all studied cell lines much more efficiently than Ad5wt, whereas Ad5RGD4G transduced cells only slightly more efficiently than Ad5wt. DTT treatment had no effect on cell transduction by wild-type Ad5wt and Ad5RGD4G but abolished the increased transduction efficacy of Ad5RGD4C in a dose-dependent manner. The mass spectra of native and DTT-reduced tryptic digests of the Ad5RGD4C fiber protein are consistent with the presence of a C(547) -C(549) linkage in the C(547) DC(549) RGDC(553) FC(555) motif. Finally, the high transduction efficacy of Ad5RGD4C is conserved in Ad5RGD2C2G. CONCLUSIONS: We provide genetic and biochemical data strongly suggesting that cysteines C(547) and C(549) from the C(547) DC(549) RGDC(553) FC(555) motif inserted in the HI loop of the Ad5 fiber form a single disulfide bond, with this disulfide bond being crucial for Ad5RGD4C retargeting to av-integrins.
Assuntos
Adenoviridae/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dissulfetos , Integrina alfaV/química , Integrina alfaV/metabolismo , Domínios e Motivos de Interação entre Proteínas , Adenoviridae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/metabolismo , Linhagem Celular Tumoral , Vetores Genéticos/genética , Humanos , Integrina alfaV/imunologia , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transdução GenéticaRESUMO
AIMS: Adenoviruses that have CNGRCVSGCAGRC peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cysteines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. METHODS: Transduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13. KEY FINDINGS: AdFNGR and AdHNGR attach better to CD13 and/or αvß3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of αvß3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on αvß3 integrin expression. SIGNIFICANCE: Insertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors.
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Adenoviridae/genética , Adenoviridae/metabolismo , Integrina alfaVbeta3/fisiologia , Linhagem Celular Tumoral , Dissulfetos/química , Células Endoteliais/metabolismo , Vetores Genéticos/genética , Células HEK293 , Humanos , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/farmacologia , Transdução Genética/métodos , Transfecção/métodosRESUMO
The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was the first adenovirus-based vector to receive marketing authorization from European Commission. HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such, represent a highly valuable tool for fighting infectious diseases. Despite well-described immunogenicity in vivo, the basic biology of HAdV26 still needs some refinement. The aim of this study was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and then investigate the underlying molecular mechanism. The expression of studied genes and proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines and chemokines, namely IL-6, IL-8, IL-1ß, and TNF-α, with the most pronounced difference shown for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced IL-6 gene expression is αvß3 integrin dependent and NF-κB mediated. Our findings provide new data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained within this research increases our current knowledge of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-based vaccine vectors.
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Adenovírus Humanos , Integrina alfaVbeta3 , Interleucina-6 , NF-kappa B , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Células Cultivadas , Quimiocinas/genética , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Humanos , Integrina alfaVbeta3/metabolismo , Interleucina-6/genética , NF-kappa B/metabolismoRESUMO
The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell-cycle arrest while Kank3 was identified as hypoxia-inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Adesões Focais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Adesões Focais/patologia , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Paclitaxel/uso terapêutico , Domínios Proteicos , Transdução de Sinais , Resultado do Tratamento , Vincristina/uso terapêuticoRESUMO
Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVß3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVß3 expressing Cal27-derived cell clone 2B1, αVß5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6ß4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6ß4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6ß4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVß3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6ß4 not occurred. Taken together, our results identify a key role for α6ß4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.
RESUMO
Cisplatin (cDDP) is an anticancer agent that is widely used in the treatment of many solid tumors. A major obstacle to successful cDDP-based chemotherapy, however, is the intrinsic and acquired resistance of tumor cells to this drug. Greater insight into the molecular mechanisms underlying the modulation of cellular responses to cDDP will aid in the development and optimization of new therapeutic strategies. Apart from induction of DNA damage, recent data have suggested that cDDP also induces the formation of reactive oxygen species that can trigger cell death. Cell death occurs as the result of several simultaneously activated signaling pathways. The specific pathway responsible for cell death depends on the cell type and the treatment conditions. This review focuses on the relationship between glutathione and BCL-2 and their protective role in cDDP-induced reactive oxygen species formation and cDDP resistance.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Dano ao DNA , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , HumanosRESUMO
Nascent adhesions are submicron transient structures promoting the early adhesion of cells to the extracellular matrix. Nascent adhesions typically consist of several tens of integrins, and serve as platforms for the recruitment and activation of proteins to build mature focal adhesions. They are also associated with early stage signaling and the mechanoresponse. Despite their crucial role in sampling the local extracellular matrix, very little is known about the mechanism of their formation. Consequently, there is a strong scientific activity focused on elucidating the physical and biochemical foundation of their development and function. Precisely the results of this effort will be summarized in this article.